CN116023455A - Method for creating early maturing rice - Google Patents

Abstract

The invention discloses a method for editing and modifying rice hd2 gene and/or hd4 gene to obtain early maturing rice. Specifically, using a CRISPR/Cas9 system to edit hd2 genes and/or hd4 genes in the starting rice, and enabling the hd2 genes and/or hd4 genes to mutate to cause early termination of translation proteins, so as to obtain transgenic rice; realizes the edition of hd2 gene and/or hd4 gene in rice. The invention uses CRISPR/Cas9 mediated gene editing technology to knock out rice hd2 gene and/or hd4 gene at specific target points to obtain rice mutant materials with earlier maturity and heading period than comparison, thereby providing new materials for breeding early maturing rice varieties.

Description

Translated fromChinese

创制早熟水稻的方法Method for creating early-maturing rice

技术领域technical field

本发明涉及植物生物技术领域，具体涉及基因编辑修饰hd2和hd4获得早熟水稻的方法。The invention relates to the field of plant biotechnology, in particular to a method for gene editing and modifying hd2 and hd4 to obtain early-maturing rice.

背景技术Background technique

0667水稻为牡丹江地域优质品种，生育期较长，约140天，当地插秧季节在五月下旬，而北方地区“十一”前的早霜对期充分成熟将成为考验。为提高0667水稻的地域适应性，可考虑对其进行改良，提早其成熟期。0667 rice is a high-quality variety in the Mudanjiang area, with a long growth period of about 140 days. The local transplanting season is in late May, and the early frost in the northern region before the "October" will become a test for full maturity. In order to improve the regional adaptability of 0667 rice, it can be considered to improve it and advance its maturity period.

CRISPR/Cas9系统是新近发展起来的基因编辑技术，将CRISPR/Cas9系统导入细胞后，通过对特定靶序列识别，可实现对细胞基因组的高效率编辑和改造。The CRISPR/Cas9 system is a newly developed gene editing technology. After the CRISPR/Cas9 system is introduced into cells, the efficient editing and transformation of the cell genome can be realized by identifying specific target sequences.

发明内容Contents of the invention

本发明所要解决的技术问题是如何提早水稻的成熟期或如何缩短水稻的生育期。The technical problem to be solved by the invention is how to advance the maturity period of rice or how to shorten the growth period of rice.

为了解决上述技术问题，本发明首先提供了调控蛋白质A和/或蛋白质B活性或含量的物质，或调控所述蛋白质A的编码基因和/或所述蛋白质B的编码基因的表达物质的应用，所述应用为下述任一种：In order to solve the above technical problems, the present invention firstly provides substances for regulating the activity or content of protein A and/or protein B, or the application of substances for regulating the expression of the gene encoding protein A and/or the gene encoding protein B, The application is any of the following:

P1、在调控水稻成熟期或促进水稻早熟中的应用；P1, the application in regulating the maturity stage of rice or promoting the early maturity of rice;

P2、在调控水稻抽穗期或促进水稻早抽穗中的应用；P2. Application in regulating rice heading stage or promoting early heading of rice;

P3、在调控水稻株高或矮化水稻中的应用；P3. Application in regulating rice plant height or dwarfing rice;

P4、在水稻育种中的应用；P4. Application in rice breeding;

所述蛋白质A可为如下A1)、A2)或A3)的蛋白质：The protein A may be a protein of the following A1), A2) or A3):

A1)氨基酸序列是序列表中序列3的蛋白质；A1) The amino acid sequence is the protein of sequence 3 in the sequence listing;

A2)将A1)所示的氨基酸序列经过一个以上氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的由A1)衍生的或与A1)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质；A2) The amino acid sequence shown in A1) is obtained by substituting and/or deleting and/or adding more than one amino acid residue and has the same function derived from A1) or has more than 80% of the protein shown in A1) Proteins that are identical and have the same function;

A3)在A1)、A2)或A3)的N末端或/和C末端连接蛋白标签得到的融合蛋白质；A3) A fusion protein obtained by linking protein tags at the N-terminus or/and C-terminus of A1), A2) or A3);

所述蛋白质B可为如下B1)、B2)或B3)的蛋白质：The protein B can be the following B1), B2) or B3) protein:

B1)氨基酸序列是序列表中序列6的蛋白质；B1) The amino acid sequence is the protein of sequence 6 in the sequence listing;

B2)将B1)所示的氨基酸序列经过一个以上氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的由B1)衍生的或与B1)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质；B2) The amino acid sequence shown in B1) is obtained by substituting and/or deleting and/or adding one or more amino acid residues and has the same function, which is derived from B1) or has more than 80% of the protein shown in B1) Proteins that are identical and have the same function;

B3)在B1)、B2)或B3)的N末端或/和C末端连接蛋白标签得到的融合蛋白质。B3) A fusion protein obtained by linking protein tags at the N-terminus or/and C-terminus of B1), B2) or B3).

上述应用中，所述蛋白质A、所述蛋白质B均可来源于水稻。In the above application, both the protein A and the protein B can be derived from rice.

上述应用中，序列表中序列3由742个氨基酸残基组成，序列表中序列6由287个氨基酸残基组成。In the above application, sequence 3 in the sequence listing consists of 742 amino acid residues, and sequence 6 in the sequence listing consists of 287 amino acid residues.

上文所述一个以上氨基酸残基具体可为十个以内的氨基酸残基。The one or more amino acid residues mentioned above may specifically be within ten amino acid residues.

上述应用中，所述80％以上的同一性可为至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、98％或99％的同一性。In the above application, the above 80% identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 98% or 99% identity.

上述应用中，所述蛋白质A的编码基因可为如下a1)或a2)或a3)所示的DNA分子：In the above application, the gene encoding protein A may be a DNA molecule as shown in a1) or a2) or a3) as follows:

a1)编码序列是序列表中序列2所示的DNA分子；a1) the coding sequence is the DNA molecule shown in sequence 2 in the sequence listing;

a2)与a1)限定的核苷酸序列具有90％或90％以上同一性，且编码上文所述蛋白质A的DNA分子；a2) A DNA molecule that has 90% or more identity to the nucleotide sequence defined in a1) and encodes the above-mentioned protein A;

a3)在严格条件下与a1)或a2)限定的核苷酸序列杂交，且编码上文所述蛋白质A的DNA分子。a3) A DNA molecule that hybridizes to the nucleotide sequence defined in a1) or a2) under stringent conditions and encodes the protein A described above.

上述应用中，所述蛋白质B的编码基因可为如下b1)或b2)或b3)所示的DNA分子：In the above application, the gene encoding the protein B can be a DNA molecule as shown in b1) or b2) or b3) as follows:

b1)编码序列是序列表中序列5所示的DNA分子；b1) the coding sequence is the DNA molecule shown insequence 5 in the sequence listing;

b2)与b1)限定的核苷酸序列具有90％或90％以上同一性，且编码上文所述蛋白质B的DNA分子；b2) A DNA molecule that has 90% or more identity to the nucleotide sequence defined in b1) and encodes the above-mentioned protein B;

b3)在严格条件下与b1)或b2)限定的核苷酸序列杂交，且编码上文所述蛋白质B的DNA分子。b3) A DNA molecule that hybridizes to the nucleotide sequence defined in b1) or b2) under stringent conditions and encodes the protein B described above.

上述应用中，所述调控所述蛋白质A的编码基因和蛋白质B的编码基因表达的物质可为进行如下6种调控中至少一种调控的物质：1)在所述基因转录水平上进行的调控；2)在所述基因转录后进行的调控(也就是对所述基因的初级转录物的剪接或加工进行的调控)；3)对所述基因的RNA转运进行的调控(也就是对所述基因的mRNA由细胞核向细胞质转运进行的调控)；4)对所述基因的翻译进行的调控；5)对所述基因的mRNA降解进行的调控；6)对所述基因的翻译后的调控(也就是对所述基因翻译的蛋白质的活性进行调控)。In the above application, the substance that regulates the expression of the gene encoding the protein A and the gene encoding the protein B can be a substance that performs at least one of the following six types of regulation: 1) regulation at the level of gene transcription 2) regulation performed after transcription of the gene (i.e. regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e. regulation of the 4) regulation of the translation of the gene; 5) regulation of the mRNA degradation of the gene; 6) post-translational regulation of the gene ( That is, the activity of the protein translated by the gene is regulated).

上述应用中，调控蛋白质A和/或蛋白质B活性或含量的物质可为敲除所述蛋白质A的编码基因和/或蛋白质B的编码基因的物质，和/或调控所述蛋白质A的编码基因和/或蛋白质B的编码基因表达的物质。In the above application, the substance that regulates the activity or content of protein A and/or protein B can be a substance that knocks out the gene encoding protein A and/or the gene encoding protein B, and/or regulates the gene encoding protein A and/or the substance expressed by the gene encoding protein B.

上述应用中，所述调控所述蛋白质A的编码基因和/或蛋白质B的编码基因表达的物质可为抑制或降低所述基因表达，所述抑制或降低所述基因表达可通过基因敲除实现或通过基因沉默实现。In the above application, the substance that regulates the expression of the gene encoding the protein A and/or the gene encoding the protein B can be to inhibit or reduce the expression of the gene, and the inhibition or reduction of the expression of the gene can be achieved by gene knockout Or through gene silencing.

所述基因敲除(geneknockout)是指通过同源重组使特定靶基因失活的现象。基因敲除是通过DNA序列的改变使特定靶基因失活。The gene knockout (geneknockout) refers to a phenomenon in which a specific target gene is inactivated by homologous recombination. Gene knockout is the inactivation of a specific target gene by changing the DNA sequence.

所述基因沉默是指在不损伤原有DNA的情况下使基因不表达或低表达的现象。基因沉默以不改变DNA序列为前提，使基因不表达或低表达。基因沉默可发生在两种水平上，一种是由于DNA甲基化、异染色质化以及位置效应等引起的转录水平的基因沉默,另一种是转录后基因沉默，即在基因转录后的水平上通过对靶标RNA进行特异性抑制而使基因失活,包括反义RNA、共抑制(co-suppression)、基因压抑(quelling)、RNA干扰(RNAi)和微小RNA(miRNA)介导的翻译抑制等。The gene silencing refers to the phenomenon of non-expression or low expression of genes without damaging the original DNA. Gene silencing is based on the premise of not changing the DNA sequence, so that the gene is not expressed or expressed at a low level. Gene silencing can occur at two levels, one is gene silencing at the transcription level caused by DNA methylation, heterochromatinization, and position effects, and the other is post-transcriptional gene silencing, that is, after gene transcription Gene inactivation at the horizontal level through specific inhibition of target RNAs, including antisense RNAs, co-suppression, quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translation Inhibition etc.

上述应用中，所述调控所述蛋白质A的编码基因和/或蛋白质B的编码基因表达的物质可为抑制或降低所述基因表达的试剂。所述抑制或降低所述基因表达的试剂可为敲除所述基因的试剂，如通过同源重组敲除所述基因的试剂，或通过CRISPR-Cas9敲除所述基因的试剂。所述抑制或降低所述基因表达的试剂可以包含靶向所述基因的多核苷酸，例如siRNA、shRNA、sgRNA、miRNA或反义RNA。In the above application, the substance that regulates the expression of the gene encoding the protein A and/or the gene encoding the protein B may be a reagent that inhibits or reduces the expression of the gene. The reagent for inhibiting or reducing the expression of the gene can be a reagent for knocking out the gene, such as a reagent for knocking out the gene through homologous recombination, or a reagent for knocking out the gene through CRISPR-Cas9. The agent for inhibiting or reducing the expression of the gene may comprise a polynucleotide targeting the gene, such as siRNA, shRNA, sgRNA, miRNA or antisense RNA.

上述应用中，调控蛋白质A和/或蛋白质B活性或含量的物质，或调控所述蛋白质A的编码基因和/或所述蛋白质B的编码基因的表达物质，可为如下c1)-c4)任一种物质：In the above application, the substance that regulates the activity or content of protein A and/or protein B, or the substance that regulates the expression of the gene encoding the protein A and/or the gene encoding the protein B can be any of the following c1)-c4) a substance:

c1)抑制或降低上文所述蛋白质A编码基因和/或蛋白质B编码基因表达的核酸分子；c1) A nucleic acid molecule that inhibits or reduces the expression of the protein A coding gene and/or the protein B coding gene described above;

c2)含有c1)所述核酸分子的表达盒；c2) an expression cassette containing the nucleic acid molecule of c1);

c3)含有c1)所述核酸分子的重组载体、或含有c2)所述表达盒的重组载体；c3) a recombinant vector containing the nucleic acid molecule of c1), or a recombinant vector containing the expression cassette of c2);

c4)含有c1)所述核酸分子的重组微生物、或含有c2)所述表达盒的重组微生物、或含有c3)所述重组载体的重组微生物。c4) A recombinant microorganism containing the nucleic acid molecule of c1), or a recombinant microorganism containing the expression cassette of c2), or a recombinant microorganism containing the recombinant vector of c3).

c1)所述的核酸分子可为表达靶向上文所述所述蛋白质A编码基因和/或蛋白质B编码基因表达的sgRNA或表达所述sgRNA的DNA分子。The nucleic acid molecule in c1) can be an sgRNA targeting the expression of the protein A-encoding gene and/or protein B-encoding gene described above or a DNA molecule expressing the sgRNA.

所述sgRNA为名称为sgRNA1的sgRNA和名称为sgRNA2的sgRNA，sgRNA1的靶序列为序列1的第200-222位，所述sgRNA2的靶序列为序列表序列4的第387-409位。The sgRNA is sgRNA named sgRNA1 and sgRNA named sgRNA2, the target sequence of sgRNA1 is 200-222 of sequence 1, and the target sequence of sgRNA2 is 387-409 of sequence 4 in the sequence listing.

术语“同一性”指与天然核酸序列的序列相似性。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。所述具有90％或90％以上同一性可为至少具有90％、至少具有95％、至少具有96％、至少具有97％、至少具有98％或至少具有99％的同一性。The term "identity" refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed visually or with computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. The identity of 90% or more may be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

为了解决上述技术问题，本发明还提供了一种提早水稻成熟期和/或提早水稻抽穗期水稻和/或缩短水稻生育期的方法，包括通过抑制或降低水稻基因组中上文所述的蛋白质A编码基因和/或蛋白质B编码基因的表达量来提早水稻成熟期和/或提早水稻抽穗期水稻和/或缩短水稻生育期。In order to solve the above-mentioned technical problems, the present invention also provides a method for early rice maturity and/or early rice heading stage rice and/or shortening rice growth period, including by inhibiting or reducing the above-mentioned protein A in the rice genome The expression level of the coding gene and/or the protein B coding gene is used to advance the maturation period of rice and/or to advance the heading stage of rice and/or shorten the growth period of rice.

上述降低或抑制水稻中上文所述蛋白质A编码基因和蛋白质B编码基因的表达量可采用现有技术中的任何方式实现，以使基因产生缺失突变、插入突变或碱基变换突变，进而实现基因功能降低或丧失，具体可为化学诱变、物理诱变、RNAi、基因组定点编辑或同源重组等。The above-mentioned reduction or suppression of the expression of the above-mentioned protein A coding gene and protein B coding gene in rice can be realized by any means in the prior art, so that the gene produces deletion mutation, insertion mutation or base conversion mutation, and then realizes Gene function reduction or loss, specifically chemical mutagenesis, physical mutagenesis, RNAi, genome-directed editing or homologous recombination, etc.

上述基因组定点编辑方法中，可采用锌指核酸酶(Zinc finger nuclease,ZFN)技术、类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease,TALEN)技术或成簇的规律间隔的短回文重复序列及其相关系统(Clustered regularlyinterspaced shortpalindromic repeats/CRISPR associated,CRISPR/Cas9 system)技术，以及其它能实现基因组定点编辑的技术。无论采取哪种方法，既可对上文所述蛋白的整个编码基因作为靶标，又可将调控上文所述蛋白编码基因表达的各个元件作为靶标，只要能实现基因功能丧失或降低即可。如可以将上文所述蛋白的编码基因的外显子或5’UTR等作为靶标。Among the above-mentioned targeted genome editing methods, zinc finger nuclease (Zinc finger nuclease, ZFN) technology, transcription activator-like effector nuclease (Transcription activator-like effector nuclease, TALEN) technology or clustered regularly spaced short loops can be used. CRISPR/Cas9 system (Clustered regularly interspaced shortpalindromic repeats/CRISPR associated, CRISPR/Cas9 system) technology, and other technologies that can realize genome-specific editing. No matter which method is adopted, the entire coding gene of the above-mentioned protein can be used as a target, and each element that regulates the expression of the above-mentioned protein-coding gene can also be targeted, as long as the loss or reduction of gene function can be achieved. For example, the exon or 5'UTR of the gene encoding the above-mentioned protein can be used as a target.

上文所述方法可包括向所述水稻中导入降低或抑制上文所述的蛋白质A和/或蛋白质B的活性的物质，或导入降低或抑制所述蛋白质A编码基因和/或蛋白质B编码基因表达的物质。所述降低或抑制上文所述的降低或抑制上文所述的蛋白质A和/或蛋白质B的活性，或降低或抑制所述蛋白质A编码基因和/或蛋白质B编码基因表达的物质可为如下c1)-c4)任一种物质：The method described above may include introducing into the rice a substance that reduces or inhibits the activity of the protein A and/or protein B described above, or introducing a substance that reduces or inhibits the gene encoding the protein A and/or the protein B encoding Substances of gene expression. The substance that reduces or inhibits the above-mentioned reduction or inhibition of the above-mentioned protein A and/or protein B activity, or reduces or inhibits the expression of the protein A coding gene and/or protein B coding gene can be Any of the following c1)-c4) substances:

c1)抑制或降低上文所述蛋白质A编码基因和/或蛋白质B编码基因表达的核酸分子；c1) A nucleic acid molecule that inhibits or reduces the expression of the protein A coding gene and/or the protein B coding gene described above;

c2)含有c1)所述核酸分子的表达盒；c2) an expression cassette containing the nucleic acid molecule of c1);

c3)含有c1)所述核酸分子的重组载体、或含有c2)所述表达盒的重组载体；c3) a recombinant vector containing the nucleic acid molecule of c1), or a recombinant vector containing the expression cassette of c2);

c4)含有c1)所述核酸分子的重组微生物、或含有c2)所述表达盒的重组微生物、或含有c3)所述重组载体的重组微生物。c4) A recombinant microorganism containing the nucleic acid molecule of c1), or a recombinant microorganism containing the expression cassette of c2), or a recombinant microorganism containing the recombinant vector of c3).

c1)所述的核酸分子为靶向上文所述蛋白质A编码基因和/或上文所述蛋白质B编码基因的sgRNA或表达所述sgRNA的DNA分子。The nucleic acid molecule in c1) is the sgRNA targeting the protein A coding gene mentioned above and/or the protein B coding gene mentioned above or a DNA molecule expressing the sgRNA.

所述sgRNA为名称为sgRNA1的sgRNA和名称为sgRNA2的sgRNA，sgRNA1的靶序列为序列1的第200-222位，所述sgRNA2的靶序列为序列表序列4的第387-409位。The sgRNA is sgRNA named sgRNA1 and sgRNA named sgRNA2, the target sequence of sgRNA1 is 200-222 of sequence 1, and the target sequence of sgRNA2 is 387-409 of sequence 4 in the sequence listing.

上文所述抑制或降低水稻基因组中所述的蛋白质A编码基因和/或所述蛋白质B编码基因的表达量为将水稻基因组中的序列1所示的所述蛋白质A编码基因替换为hd2-1基因，并将序列4所示的所述蛋白质B编码基因替换为得到hd4-1基因，基因，所述hd2-1基因是将序列表中序列1的第216位和217位之间插入核苷酸T，保持序列表中序列1的其它核苷酸不变得到的DNA分子；所述hd4-1基因是将序列表中序列4的第第403位和第404位之间插入核苷酸T，保持序列表中序列4的其它核苷酸不变得到的DNA分子。Inhibiting or reducing the expression of the protein A coding gene and/or the protein B coding gene in the rice genome described above is to replace the protein A coding gene shown in sequence 1 in the rice genome with hd2- 1 gene, and the protein B coding gene shown in sequence 4 is replaced by hd4-1 gene, gene, the hd2-1 gene is inserted into the nucleus between the 216th and 217th positions of sequence 1 in the sequence table Nucleotide T, a DNA molecule obtained by keeping other nucleotides of sequence 1 in the sequence listing unchanged; the hd4-1 gene is a nucleotide inserted between the 403rd and 404th positions of sequence 4 in the sequence listing T, the DNA molecule obtained by keeping other nucleotides of sequence 4 in the sequence listing unchanged.

本发明公开了对水稻hd2基因和hd4基因进行编辑修饰，获得早熟水稻的方法。具体利用CRISPR/Cas9系统对出发水稻中hd2基因和hd4基因进行编辑，且使所述hd2基因和/或hd4基因发生突变导致翻译蛋白提前终止，得到转基因水稻；实现出发水稻中hd2基因和/或hd4基因编辑。本发明利用CRISPR/Cas9介导的基因编辑技术对水稻hd2基因和/或hd4基因进行特定靶点的定点敲除，获得抽穗期、成熟期较对照早的水稻突变体材料，为早熟水稻品种选育提供新材料。The invention discloses a method for editing rice hd2 gene and hd4 gene to obtain early-maturing rice. Specifically, the CRISPR/Cas9 system is used to edit the hd2 gene and hd4 gene in the starting rice, and the hd2 gene and/or hd4 gene is mutated to cause the translation protein to terminate prematurely, and the transgenic rice is obtained; the hd2 gene and/or the starting rice are realized. hd4 gene editing. The invention utilizes CRISPR/Cas9-mediated gene editing technology to knock out rice hd2 gene and/or hd4 gene at a specific target point, and obtain rice mutant materials with earlier heading stage and maturity stage than the control, and are selected for early-maturing rice varieties. provide new materials.

附图说明Description of drawings

图1为基因编辑植株和受体对照8月上旬的田间抽穗表现对比照片，其中图1的左侧为基因编辑植株Mu-5，图1的右侧为野生型水稻0667(WT)。Figure 1 is a comparison photo of the field heading performance of the gene-edited plants and the recipient control in early August. The left side of Figure 1 is the gene-edited plant Mu-5, and the right side of Figure 1 is the wild-type rice 0667 (WT).

图2为2020年10月8日收割的田间材料表现对比照片，其中左侧为野生型水稻0667(WT)，图3的右侧为基因编辑植株Mu-5。Figure 2 is a comparison photo of the performance of field materials harvested on October 8, 2020. The left side is wild-type rice 0667 (WT), and the right side of Figure 3 is the gene-edited plant Mu-5.

图3为2020年10月8日收割的田间材料表现对比照片，其中右侧为野生型水稻0667(WT)，图3的左侧为基因编辑植株Mu-5。Figure 3 is a comparison photo of the performance of field materials harvested on October 8, 2020. The right side is the wild-type rice 0667 (WT), and the left side of Figure 3 is the gene-edited plant Mu-5.

具体实施方式Detailed ways

下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

下述实施方法和实施例中所使用的术语，除非特殊说明，一般具有本领域的普通技术人员通常理解的含义。The terms used in the following implementation methods and examples, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

以下实施例中，水稻品种0667是牡丹江当地优质的粳稻品种，记载于非专利文献“黄静娴.0667水稻OsWaxy基因编辑纯合体鉴定.牡丹江师范学院本科生毕业论文”，公众可以从申请人牡丹江师范学院处获得，以重复本申请实验。In the following examples, therice variety 0667 is a local high-quality japonica rice variety in Mudanjiang, which is recorded in the non-patent document "Huang Jingxian. Homozygous Identification of 0667 Rice OsWaxy Gene Editing. Graduation Thesis for Undergraduates of Mudanjiang Normal University". The public can learn from the applicant Mudanjiang Normal University Obtained from the College to repeat the experiment for this application.

以下实施例中，pYLsgRNA-OsU3质粒、pYLsgRNA-OsU6质粒和pYLCRISPR/Cas9-MH载体记载于非专利文献“柯焕春.基于CRISPR/Cas9系统的水稻品种穗颈长等性状的遗传改良。2019年，华南农业大学硕士毕业论文”，公众可以从申请人牡丹江师范学院处获得，以重复本申请实验。In the following examples, the pYLsgRNA-OsU3 plasmid, pYLsgRNA-OsU6 plasmid and pYLCRISPR/Cas9-MH vector are described in the non-patent literature "Ke Huanchun. Genetic improvement of traits such as panicle neck length of rice varieties based on CRISPR/Cas9 system. 2019, South China Agricultural University Master's Graduation Thesis", the public can obtain from the applicant Mudanjiang Normal University to repeat the application experiment.

以下实施例中，PCR Mix为百泰克生物技术有限公司产品；限制酶Eco31Ⅰ为Thermos公司产品；T4 DNA连接酶为NEB公司产品；植物基因组DNA提取试剂盒、DNA纯化回收试剂盒、质粒提取试剂盒为天根生化科技有限产品；头孢曲松钠为Sigma公司产品；潮霉素、乙酰丁香酮为Amresco公司产品。In the following examples, PCR Mix is a product of Biotech Biotechnology Co., Ltd.; restriction enzyme Eco31I is a product of Thermos; T4 DNA ligase is a product of NEB; plant genomic DNA extraction kit, DNA purification and recovery kit, and plasmid extraction kit It is a product of Tiangen Biochemical Technology Co., Ltd.; ceftriaxone sodium is a product of Sigma Company; hygromycin and acetosyringone are products of Amresco Company.

以下实施例中，水稻遗传转化相关培养基配方见表1：In the following examples, the formulation of the medium for genetic transformation of rice is shown in Table 1:

表1培养基配方Table 1 medium formula

表2氨基酸3号悬浮液配方Table 2 Amino Acid No. 3 Suspension Formula

名称name10×(g/L)10×(g/L)谷氨酰胺Glutamine8.778.77天冬氨酸aspartic acid2.662.66L-精氨酸L-Arginine2.282.28甘氨酸Glycine0.750.75

实施例1Example 1

一、构建敲除水稻hd2和hd4基因的载体1. Construction of vectors for knocking out rice hd2 and hd4 genes

1、sgRNA的获得1. Obtaining sgRNA

参考水稻基因组中目的基因序列，锚定两个抽穗抑制因子编码基因hd2、hd4，选择hd2(LOC_Os07g49460)的第一外显子和hd4(LOC_Os07g15770)的第二外显子作为靶点设计区域。Referring to the target gene sequence in the rice genome, two genes encoding heading inhibitory factors hd2 and hd4 were anchored, and the first exon of hd2 (LOC_Os07g49460) and the second exon of hd4 (LOC_Os07g15770) were selected as target design regions.

PCR扩增水稻品种0667的hd2基因并测序，其核苷酸序列如序列表中序列1所示(序列1中第1-262位为第一外显子、5725-5886位为第二外显子、5997-6130位为第三外显子、6226-6433位为第四外显子、8966-9152位为第五外显子、8966-9152位为第六外显子、9247-9658位为第七外显子、9740-10517位为第八外显子、11046-11171位为第九外显子，编码基因序列为序列2，其编码氨基酸序列为序列3的蛋白质HD2)，选择位于hd2(LOC_Os07g49460)的第一外显子上的靶点Hd2，序列如下：The hd2 gene ofrice variety 0667 was amplified by PCR and sequenced, and its nucleotide sequence was shown in sequence 1 in the sequence table (positions 1-262 in sequence 1 are the first exon, and positions 5725-5886 are the second exon 5997-6130 is the third exon, 6226-6433 is the fourth exon, 8966-9152 is the fifth exon, 8966-9152 is the sixth exon, 9247-9658 is the seventh exon, 9740-10517 is the eighth exon, 11046-11171 is the ninth exon, the coding gene sequence is sequence 2, and its encoded amino acid sequence is the protein HD2 of sequence 3). The target Hd2 on the first exon of hd2 (LOC_Os07g49460), the sequence is as follows:

Hd2：5’-TTGATAGCGATGACTCCACCAGG-3’(位于序列表中序列1的第200-222位，同序列2的第200-222位)。Hd2: 5'-TTGATAGCGATGACTCCACCAGG-3' (located at No. 200-222 of Sequence 1 in the Sequence Listing, same as No. 200-222 of Sequence 2).

PCR扩增水稻品种0667的hd4基因并测序，其核苷酸序列如序列表中序列4所示(序列4中第1-64位为第一外显子、340-809位为第二外显子、2455-2784位为第三外显子，编码基因序列为序列5，其编码氨基酸序列为序列6的蛋白质HD4)选择位于hd4(LOC_Os07g15770)的第二外显子上的靶点Hd4，序列如下：The hd4 gene ofrice variety 0667 was amplified by PCR and sequenced, and its nucleotide sequence was shown in sequence 4 in the sequence table (positions 1-64 in sequence 4 are the first exon, positions 340-809 are the second exon 2455-2784 is the third exon, the coding gene sequence issequence 5, and its coding amino acid sequence is the protein HD4 of sequence 6) select the target Hd4 located on the second exon of hd4 (LOC_Os07g15770), the sequence as follows:

Hd4：5’-GGAGAAGGATGTGGCCTGTGCGG-3’(位于序列表中序列4的第387-409位，同序列5的第112-134位)。Hd4: 5'-GGAGAAGGATGTGGCCTGTGCGG-3' (located at No. 387-409 of Sequence 4 in the Sequence Listing, same as No. 112-134 of Sequence 5).

针对Hd2和Hd4构建双靶点基因编辑表达载体Construction of dual-target gene editing expression vector targeting Hd2 and Hd4

采用Golden Gate cloning方法，通过重组DNA操作分别将两个靶点构建至基因编辑表达载体pYLCRISPR/Cas9Pubi-H中，具体步骤如下：Using the Golden Gate cloning method, the two targets were respectively constructed into the gene editing expression vector pYLCRISPR/Cas9Pubi-H through recombinant DNA manipulation. The specific steps are as follows:

根据Hd2和Hd4靶点设计引物序列如下：The primer sequences were designed according to the Hd2 and Hd4 targets as follows:

U-F:GGCGCGCgGTctcGCTCGACTAGTGCTCCGTTTTACCTGTGGAATCGU-F: GGCGCGCgGTctcGCTCGACTAGTGCTCCGTTTTACCTGTGGAATCG

OsU3T-Hd2:GGTGGAGTCATCGCTATCAACGGCAGCCAAGCCAGCAOsU3T-Hd2:GGTGGAGTCATCGCTATCAACGGCAGCCAAGCCAGCA

OsgRT-Hd2:TTGATAGCGATGACTCCACCGTTTTAGAGCTAGAAATOsgRT-Hd2:TTGATAGCGATGACTCCACCGTTTTGAGCTAGAAAT

OsU6T-Hd4:CACAGGCCACATCCTTCTCCCGGCAGCCAAGCCAGCAOsU6T-Hd4:CACAGGCCACATCCTTCTCCCGGCAGCCAAGCCAGCA

OsgRT-Hd4:GGAGAAGGATGTGGCCTGTGGTTTTAGAGCTAGAAATOsgRT-Hd4:GGAGAAGGATGTGGCCTGTGGTTTTAGAGCTAGAAAT

gR-R:GCGCGCggtctcTACCGACGCGTCCACGGAGGAAAATTCCATCCACgR-R:GCGCGCggtctcTACCGACGCGTCCACGGAGGAAAATTCCATCCAC

以pYLsgRNA-OsU3质粒或pYLsgRNA-OsU6a质粒为模板分别经过两轮PCR构建两个基因的sgRNA表达盒。具体如下：Using the pYLsgRNA-OsU3 plasmid or pYLsgRNA-OsU6a plasmid as a template, the sgRNA expression cassettes of the two genes were constructed through two rounds of PCR. details as follows:

1)构建针对靶点Hd2的sgRNA1表达盒：1) Construct the sgRNA1 expression cassette targeting Hd2:

首轮PCR：The first round of PCR:

以1μL pYLsgRNA-OsU3质粒为模板，以U-F和OsU3T-Hd2组成的组合引物1建立PCR反应，扩增得到产物1。Using 1 μL pYLsgRNA-OsU3 plasmid as a template, a PCR reaction was established with the combined primer 1 consisting of U-F and OsU3T-Hd2, and product 1 was amplified.

以1μL pYLsgRNA-OsU3质粒为模板，以OsgRT-Hd2和gR-R组成的组合引物2建立PCR反应，扩增得到产物2。Using 1 μL pYLsgRNA-OsU3 plasmid as a template, a PCR reaction was established with the combined primer 2 composed of OsgRT-Hd2 and gR-R, and product 2 was amplified.

次轮PCR：Second round of PCR:

首轮PCR获得的产物1和产物2各取0.5μL作为混合模板，以U-F和gR-R组成的组合引物建立PCR反应，得到构建针对靶点Hd2的sgRNA1表达盒扩增产物。0.5 μL of product 1 and product 2 obtained in the first round of PCR were used as mixed templates, and a PCR reaction was established with a combination of primers consisting of U-F and gR-R to obtain the amplified product of the sgRNA1 expression cassette targeting Hd2.

2)构建针对靶点Hd4的sgRNA2表达盒：2) Construct the sgRNA2 expression cassette targeting Hd4:

首轮PCR：The first round of PCR:

以1μL pYLsgRNA-OsU6a质粒为模板，以U-F和OsU6T-Hd4组成的组合引物3建立PCR反应，扩增得到产物3。Using 1 μL pYLsgRNA-OsU6a plasmid as a template, a PCR reaction was established with the combined primer 3 composed of U-F and OsU6T-Hd4, and product 3 was amplified.

以1μL pYLsgRNA-OsU6a质粒为模板，以OsgRT-Hd4和gR-R组成的组合引物3建立PCR反应，扩增得到产物4。Using 1 μL pYLsgRNA-OsU6a plasmid as a template, a PCR reaction was established with the combined primer 3 composed of OsgRT-Hd4 and gR-R, and product 4 was amplified.

次轮PCR：Second round of PCR:

首轮PCR获得的产物3和产物4各取0.5μL作为混合模板，以U-F和gR-R组成的组合引物建立PCR反应，得到构建针对靶点Hd4的sgRNA2表达盒扩增产物。0.5 μL each of product 3 and product 4 obtained in the first round of PCR was used as a mixed template, and a PCR reaction was established with a combination of primers consisting of U-F and gR-R to obtain the amplified product of the sgRNA2 expression cassette targeting Hd4.

2、表达sgRNA的载体的制备2. Preparation of vectors expressing sgRNA

以LB液体培养基(含卡那霉素，50mg/L)活化pYLCRISPR/Cas9Pubi-H载体菌株，依照质粒提取试剂盒使用说明提取质粒pYLCRISPR/Cas9Pubi-H。利用Eco31Ⅰ分别酶切载体pYLCRISPR/Cas9Pubi-H及前述针对靶点Hd2的sgRNA1表达盒扩增产物和针对靶点Hd4的sgRNA2表达盒扩增产物，回收酶切的线性化载体pYLCRISPR/Cas9Pubi-H及sgRNA1表达盒、sgRNA2表达盒，备用。Activate the pYLCRISPR/Cas9Pubi-H vector strain with LB liquid medium (containing kanamycin, 50mg/L), and extract the plasmid pYLCRISPR/Cas9Pubi-H according to the instructions of the plasmid extraction kit. Eco31Ⅰ was used to digest the vector pYLCRISPR/Cas9Pubi-H and the amplified product of the sgRNA1 expression cassette targeting Hd2 and the amplified product of the sgRNA2 expression cassette targeting Hd4 respectively, and recovered the digested linearized vector pYLCRISPR/Cas9Pubi-H and sgRNA1 expression cassette, sgRNA2 expression cassette, spare.

将sgRNA1表达盒、sgRNA2表达盒同线性化载体pYLCRISPR/Cas9Pubi-H，完成最终载体pYLCRISPR/Cas9Pubi-H-sgRNA1-sgRNA2的构建。pYLCRISPR/Cas9Pubi-H-sgRNA1-sgRNA2表达靶向hd2基因上靶点Hd2的sgRNA1和靶向hd4基因上靶点Hd4的sgRNA2。Synchronize the sgRNA1 expression cassette and sgRNA2 expression cassette with the vector pYLCRISPR/Cas9Pubi-H to complete the construction of the final vector pYLCRISPR/Cas9Pubi-H-sgRNA1-sgRNA2. pYLCRISPR/Cas9Pubi-H-sgRNA1-sgRNA2 expresses sgRNA1 targeting Hd2 on the hd2 gene and sgRNA2 targeting Hd4 on the hd4 gene.

二、水稻hd2和/或hd4突变体的获得2. Obtaining rice hd2 and/or hd4 mutants

1、将步骤一中获得的pYLCRISPR/Cas9Pubi-H-sgRNA1-sgRNA2载体通过电击法转化农杆菌感受态EHA105，PCR鉴定，测序引物如下：1. The pYLCRISPR/Cas9Pubi-H-sgRNA1-sgRNA2 vector obtained in step 1 was transformed into Agrobacterium competent EHA105 by electric shock method, and identified by PCR. The sequencing primers were as follows:

SP1：5’-CCCGACATAGATGCAATAACTTC-3’；SP1: 5'-CCCGACATAGATGCAATAACTTC-3';

SP2：5’-GCGCGGTGTCATCTATGTTACT-3’。SP2: 5'-GCGCGGTGTCATCTATGTTACT-3'.

将测序验证正确(含有sgRNA1基因和sgRNA2基因)的重组菌株命名EHA105-pYLCRISPR/Cas9Pubi-H-sgRNA1-sgRNA2，-80℃保存备用。The recombinant strain verified correctly by sequencing (containing sgRNA1 gene and sgRNA2 gene) was named EHA105-pYLCRISPR/Cas9Pubi-H-sgRNA1-sgRNA2, and stored at -80°C for future use.

2、0667水稻的遗传转化：2. Genetic transformation of 0667 rice:

以水稻栽培品种0667为受体材料，进行农杆菌介导的水稻遗传转化，具体步骤如下：Usingrice cultivar 0667 as the recipient material, Agrobacterium-mediated genetic transformation of rice was carried out, and the specific steps were as follows:

将步骤1保存的重组菌株EHA105-pYLCRISPR/Cas9Pubi-H-sgRNA1-sgRNA2在YEP平板上进行三级划线，28℃，倒置培养48h，备用。Streak the recombinant strain EHA105-pYLCRISPR/Cas9Pubi-H-sgRNA1-sgRNA2 saved in step 1 on the YEP plate for tertiary streaking, and culture it upside down at 28°C for 48 hours, and set it aside.

水稻栽培品种0667的种子用75％的乙醇灭菌5min；使用25％次氯酸钠，加1滴吐温，于摇床200r/min，震荡40min；以无菌水清洗3-5次，用灭菌滤纸吸干表面水分。The seeds ofrice cultivar 0667 are sterilized with 75% ethanol for 5 minutes; use 25% sodium hypochlorite, add 1 drop of Tween, shake on a shaker at 200r/min for 40 minutes; wash 3-5 times with sterile water, and use sterilized filter paper Blot surface moisture.

愈伤诱导：将灭菌后的种子，放到Os1培养基上，于培养室中31℃光照培养5-6天。待水稻愈伤长至大小约与种子大小一致时，即可用于侵染。Callus induction: Put the sterilized seeds on the Os1 medium, and culture them in a culture room at 31° C. under light for 5-6 days. When the rice callus grows to about the same size as the seed, it can be used for infection.

侵染：a.活化好的EHA105-pYLCRISPR/Cas9-MH-sgRNA1-sgRNA2用灭菌牙签挑至50mL Os-inf染液中，28℃，220r/min振荡培养至OD_600nm为1左右；b.将a所得菌液加至上述诱导得到的水稻愈伤中，放置摇床100r/min侵染30min；c.取出用无菌滤纸吸干。Infection: a. Pick the activated EHA105-pYLCRISPR/Cas9-MH-sgRNA1-sgRNA2 into 50mL Os-inf staining solution with a sterilized toothpick, culture at 28°C and 220r/min shaking until the OD_600nm is about 1; b. Add the bacterial solution obtained in a to the rice callus induced above, place on a shaker at 100r/min to infect for 30min; c. take it out and blot it dry with sterile filter paper.

共培养：将吸干的愈伤，接至Os-cocu培养基。Co-cultivation: The blotted callus was connected to Os-cocu medium.

脱菌：200mL无菌水加头孢曲松钠(cef，终浓度500mg/L)；分5次清洗愈伤：第1-2次，轻摇清洗后弃去废液；第3次摇床100r/min，震荡清洗90min；第4-5次，轻摇清洗后弃去废液。最后，用无菌滤纸吸干愈伤。Sterilization: add ceftriaxone sodium (cef, final concentration 500mg/L) to 200mL sterile water; wash the callus in 5 times: the 1st-2nd time, shake gently and discard the waste liquid after washing; the 3rd time shaker 100r /min, shake and wash for 90 minutes; for the 4th or 5th time, discard the waste liquid after gently shaking and washing. Finally, the callus was blotted dry with sterile filter paper.

筛选：将吸干的愈伤，接至OsHn50培养基。每隔10d继代1次，培养基为OsHn50，继代至第3-4次时，外植体周围出现新生长的愈伤，为筛选成功的阳性愈伤。Screening: The blotted callus was connected to OsHn50 medium. Subculture once every 10 days, the medium is OsHn50, when subcultured to the 3rd or 4th time, a new growth callus appeared around the explant, which was a positive callus that was successfully screened.

分化：将筛选出的阳性愈伤接至Os2培养基，分化。每隔10-12d进行一次继代，培养基为Os2培养基，约继代3-4次。期间：分化开始7d，愈伤开始出现绿色；再约10d绿色愈伤分化成约2mm长的小芽；再约10-20d，小芽生长至3-5cm长的阳性苗。Differentiation: Inoculate the screened positive calli to Os2 medium for differentiation. Subculture was carried out every 10-12 days, and the medium was Os2 medium, about 3-4 times. Period: 7 days after the differentiation started, the callus began to appear green; about 10 days later, the green callus differentiated into small buds about 2 mm long; about 10-20 days later, the small buds grew to 3-5 cm long positive seedlings.

生根：待分化的阳性幼苗长至3-5cm，清理好基部愈伤，接种至Os3培养基，生根后炼苗移栽，得到T0代潮霉素抗性阳性水稻植株。Rooting: Positive seedlings to be differentiated grow to 3-5cm, clean the base callus, inoculate into Os3 medium, harden and transplant the seedlings after rooting, and obtain T0 hygromycin resistance positive rice plants.

4、突变体筛选和鉴定4. Screening and identification of mutants

CTAB法提取T0代潮霉素抗性阳性水稻植株的DNA作为模板进行PCR分子检测，以野生型水稻0667为对照。设计引物包含靶点Hd2和Hd4在内的PCR引物，进行PCR扩增，并将扩增产物进行测序。其中扩增hd2的引物为：hd2F(作为正向引物)和hd2R(作为反向引物)；扩增hd4的引物为：hd4F(作为正向引物)和hd4R(作为反向引物)。The DNA of hygromycin resistance-positive rice plants of the T0 generation was extracted by CTAB method as a template for PCR molecular detection, and wild-type rice 0667 was used as a control. PCR primers including target Hd2 and Hd4 were designed for PCR amplification, and the amplified products were sequenced. The primers for amplifying hd2 are: hd2F (as a forward primer) and hd2R (as a reverse primer); the primers for amplifying hd4 are: hd4F (as a forward primer) and hd4R (as a reverse primer).

hd2F：5’-CTCTTCGAGTCAGGCTGATG-3’；hd2F: 5'-CTCTTCGAGTCAGGCTGATG-3';

hd2R：5’-GACAGAAGTGAGAAGGTAG-3’。hd2R: 5'-GACAGAAGTGAGAAGGTAG-3'.

hd4F：5’-AGTGACCTCACCTGCTATAG-3’；hd4F: 5'-AGTGACCTCACCTGCTATAG-3';

hd4R：5’-GTAGTCATCGAACAGCCAG-3’。hd4R: 5'-GTAGTCATCGAACAGCCAG-3'.

PCR产物经1％琼脂糖凝胶电泳检测后，表明共获得阳性转基因植株22株。After the PCR products were detected by 1% agarose gel electrophoresis, it was shown that 22 positive transgenic plants were obtained.

将22株阳性转基因植株的PCR产物与pGEM-T载体(Promega公司)4℃过夜连接，通过热激转化导入大肠杆菌感受态细胞中，蓝白斑筛选后，对若干白斑进行菌液PCR鉴定，每株最少5个为重组子菌液送检测序，所得序列与受体野生型0667(WT)序列进行比对分析，确定编辑植株及编辑结果，详见表3、表4。The PCR products of 22 positive transgenic plants were connected to the pGEM-T vector (Promega Company) overnight at 4°C, and then introduced into E. coli competent cells through heat shock transformation. At least 5 of the strains were recombinant bacterial liquids and sent for detection sequences, and the obtained sequences were compared and analyzed with the recipient wild-type 0667 (WT) sequence to determine the edited plants and the edited results, see Table 3 and Table 4 for details.

表3 T0代转基因植株中hd2基因突变类型Table 3 Mutation types of hd2 gene in transgenic plants of T0 generation

编辑类型edit type靶点序列target sequence插入/缺失insertion/deletionWTWT<![CDATA[TTGATAGCGATGACTCCACC<u>AGG</u>]]><![CDATA[TTGATAGCGATGACTCCACC<u>AGG</u>]]>E1E1<![CDATA[TTGATAGCGATGACTCC<u>G</u>ACC<u>AGG</u>]]><![CDATA[TTGATAGCGATGACTCC<u>G</u>ACC<u>AGG</u>]]>+1+1E2E2<![CDATA[TTGATAGCGATGACTCC<u>A</u>ACC<u>AGG</u>]]><![CDATA[TTGATAGCGATGACTCC<u>A</u>ACC<u>AGG</u>]]>+1+1E3E3<![CDATA[TTGATAGCGAT----------<u>GG</u>]]><![CDATA[TTGATAGCGAT----------<u>GG</u>]]>-10-10E4E4<![CDATA[TTGATAGCGATGACTCC<u>C</u>ACC<u>AGG</u>]]><![CDATA[TTGATAGCGATGACTCC<u>C</u>ACC<u>AGG</u>]]>+1+1

表4 T0代转基因植株中hd4基因突变类型Table 4 Types of hd4 gene mutation in transgenic plants of T0 generation

编辑类型edit type靶点序列target sequence插入/缺失insertion/deletionWTWT<![CDATA[CGGAGAAGGATGTGGCCTGTG<u>CGG</u>]]><![CDATA[CGGAGAAGGATGTGGCCTGTG<u>CGG</u>]]>E1E1<![CDATA[CGGAGAAGGATGTGGCC-GTG<u>CGG</u>]]><![CDATA[CGGAGAAGGATGTGGCC-GTG<u>CGG</u>]]>-1-1E2E2<![CDATA[CGGAGAAGGATGTGGCCT<u>T</u>GTG<u>CGG</u>]]><![CDATA[CGGAGAAGGATGTGGCCT<u>T</u>GTG<u>CGG</u>]]>+1+1

注：表3、表4中，“-”表示缺失1个核苷酸，下划线指示的核苷酸为插入的核苷酸。Note: In Table 3 and Table 4, "-" indicates the deletion of 1 nucleotide, and the nucleotide indicated by the underline is the inserted nucleotide.

以T0代的基因编辑水稻自交后收获的T1代种子种植得到T1代植株，获得了5株在hd2和/或hd4基因的sgRNA1和sgRNA2位置发生编辑的水稻植株，即为T1代基因编辑植株，编号Mu-1、Mu-2、Mu-3、Mu-4和Mu-5。T1 generation plants were planted with T1 generation seeds harvested after selfing of T0 gene-edited rice, and 5 rice plants edited at the sgRNA1 and sgRNA2 positions of hd2 and/or hd4 genes were obtained, which were T1 generation gene-edited plants , numbered Mu-1, Mu-2, Mu-3, Mu-4 and Mu-5.

分别提取Mu-1、Mu-2、Mu-3、Mu-4和Mu-5的DNA采用PCR体系扩增，其中扩增hd2的引物为：hd2F(作为正向引物)和hd2R(作为反向引物)；扩增hd4的引物为：hd4F(作为正向引物)和hd4R(作为反向引物)。PRC产物4℃过夜连接，通过热激转化导入大肠杆菌感受态细胞中，蓝白斑筛选后，对若干白斑进行菌液PCR鉴定，所得序列与受体野生型0667(WT)序列进行比对分析，详见表4。The DNAs of Mu-1, Mu-2, Mu-3, Mu-4 and Mu-5 were respectively extracted and amplified by PCR system, wherein the primers for amplifying hd2 were: hd2F (as forward primer) and hd2R (as reverse primer) primers); the primers for amplifying hd4 are: hd4F (as a forward primer) and hd4R (as a reverse primer). The PRC product was ligated overnight at 4°C, and introduced into Escherichia coli competent cells through heat shock transformation. After the blue and white spots were screened, several white spots were identified by bacterial liquid PCR, and the obtained sequence was compared with the wild-type 0667 (WT) sequence of the receptor. See Table 4 for details.

表4 T1代基因编辑植株靶点序列突变情况Table 4 Mutations of target sequence in T1 gene-edited plants

其中，hd2基因和hd2基因的靶点序列均纯合的为Mu-2和Mu-5，具体如下：Among them, the hd2 gene and the target sequence of the hd2 gene are both homozygous for Mu-2 and Mu-5, as follows:

与野生型水稻栽培品种0667相比，Mu-2的基因组中，hd2基因未发生基因突变，hd4基因发生了突变两条同源染色体的hd4基因靶点Hd4上，均在序列表中序列4的第403位和第404位之间插入核苷酸T，共插入1bp，导致hd4基因CDS第二个外显子移码突变，得到hd4-1基因，推导hd4-1基因编码的氨基酸序列为“MGMANEESPNYQVKKGGRIPPPRSSLIYPFMSMGPAAGEGCGLVRRRRWRLLLPPSPR-”，与序列6差异较大，最终致使hd4功能丧失，从而将hd4基因敲除。Compared with the wild-type rice cultivar 0667, in the genome of Mu-2, the hd2 gene has no gene mutation, and the hd4 gene has a mutation. The hd4 gene target Hd4 of the two homologous chromosomes is listed in sequence 4 in the sequence table. Nucleotide T was inserted between the 403rd and 404th positions, and a total of 1 bp was inserted, resulting in a frameshift mutation in the second exon of the CDS of the hd4 gene, and the hd4-1 gene was obtained, and the amino acid sequence encoded by the hd4-1 gene was deduced as " MGMANEESPNYQVKKGGRIPPPRSSLIYPFMSMGPAAGEGCGLVRRRRWRLLLPPSPR-", which is quite different from sequence 6, which eventually leads to the loss of hd4 function, thereby knocking out the hd4 gene.

与野生型水稻栽培品种0667相比，Mu-5基因组中，hd2基因和hd4基因均发生了突变：两条同源染色体的hd2基因靶点Hd2上，均在序列表中序列1的第216位和217位之间插入核苷酸T，共插入1bp，导致hd2基因CDS第一个外显子移码突变，得到hd2-1基因，推导hd2-1基因编码的氨基酸序列为“MMGTAHHNQTAGSALGVGVGDANDAVPGAGGGGYSDPDGGPISGVQRPPQVCWERFIQKKTIKVLLVDSDDSHQAGGQCPASSLHV-”，与序列3差异较大，最终致使hd2功能丧失，从而将hd2基因敲除；两条同源染色体的hd4基因靶点Hd4上，均在序列表中序列4的第403位和第404位之间插入核苷酸T，共插入1bp，导致hd4基因CDS第二个外显子移码突变，得到hd4-1基因，推导hd4-1基因编码氨基酸序列“MGMANEESPNYQVKKGGRIPPPRSSLIYPFMSMGPAAGEGCGLVRRRRWRLLLPPSPR-”，与序列6差异较大，最终致使hd4功能丧失，从而将hd4基因敲除。Compared with the wild-type rice cultivar 0667, in the Mu-5 genome, both the hd2 gene and the hd4 gene are mutated: the hd2 gene target Hd2 of the two homologous chromosomes is both at the 216th position of sequence 1 in the sequence table Nucleotide T was inserted between position 217 and 1bp in total, resulting in frameshift mutation of the first exon of hd2 gene CDS, and hd2-1 gene was obtained. The amino acid sequence encoded by hd2-1 gene was deduced as "MMGTAHHNQTAGSALGVGVGDANDAVPGAGGGGYSDPDGGPISGVQRPPQVCWERFIQKKTIKVLLVDSDDSHQAGGQCPASSLHV-", The difference from sequence 3 is quite large, which eventually leads to the loss of hd2 function, thereby knocking out the hd2 gene; the hd4 gene target Hd4 of the two homologous chromosomes is between the 403rd and 404th positions of sequence 4 in the sequence table Nucleotide T was inserted, and a total of 1 bp was inserted, resulting in a frameshift mutation in the second exon of the CDS of the hd4 gene, and the hd4-1 gene was obtained. The amino acid sequence encoded by the hd4-1 gene was deduced "MGMANEESPNYQVKKGGRIPPPPRSSLIYPFMSMGPAAGEGCGLVRRRRWRLLLPPSPR-", which was quite different from Sequence 6. Ultimately resulting in the loss of hd4 function, thereby knocking out the hd4 gene.

三、基因编辑植株抽穗性状测定3. Determination of heading traits in gene-edited plants

选择基因编辑植株Mu-5，将T1代Mu-5自交至收获T2代种子。将T2代Mu-5的水稻种子和野生型水稻0667(WT)，于2020年4月下旬催芽育苗，5月下旬插秧于试验田，株距12cm，行距50cm，行长1.0m，设三个生物学重复，每个生物学重复3行。8月上旬于田间调查抽穗状况，可见Mu-5(图1的左侧)均已抽穗，对照组野生型水稻0667(WT)(图1的右侧)尚未抽穗。The gene-edited plant Mu-5 was selected, and the T1 generation Mu-5 was selfed to harvest the T2 generation seeds. The rice seeds of T2 generation Mu-5 and wild-type rice 0667 (WT) were germinated in late April 2020, and transplanted in the experimental field in late May, with a plant spacing of 12 cm, a row spacing of 50 cm, and a row length of 1.0 m. Three biological Repeat with 3 rows per biological replicate. In the first ten days of August, the heading status was investigated in the field. It can be seen that Mu-5 (the left side of Fig. 1) has already headed, while the control wild-type rice 0667 (WT) (the right side of Fig. 1 ) has not yet headed.

到2020年10月8日收割田间材料并调查成熟情况，具体见图2和图3，Mu-5稻穗已经黄熟(图2的右侧)，米粒为白色(图3的左侧)，说明此时Mu-5已经成熟。而同期对照0667(WT)的稻穗还带有青色，未彻底黄熟(图2的左侧)，米粒仍带有明显的青色(图3的右侧)，还未成熟。By October 8, 2020, the field materials will be harvested and the maturity status will be investigated. See Figure 2 and Figure 3 for details. Mu-5 rice ears are already yellow and ripe (right side of Figure 2), and rice grains are white (left side of Figure 3), indicating that At this point Mu-5 has matured. And the rice ear of contrast 0667 (WT) in the same period also has cyan, not fully yellow ripe (the left side of Fig. 2), and the rice grain still has obvious cyan (right side of Fig. 3), is immature.

以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

本发明所涉序列如下：The sequences involved in the present invention are as follows:

序列1sequence 1

ATGATGGGAACCGCTCATCACAACCAAACCGCCGGCTCTGCCCTCGGAGTCGGAGTCGGAGATGCCAACGACGCCGTGCCTGGGGCTGGGGGTGGGGGCTACAGCGACCCGGATGGCGGACCAATCTCCGGTGTGCAGCGGCCACCGCAGGTCTGCTGGGAGCGCTTCATCCAGAAGAAGACTATCAAAGTCTTGCTAGTTGATAGCGATGACTCCACCAGGCAGGTGGTCAGTGCCCTGCTTCGTCACTGCATGTATGAAGGTCTGTCTCTTCTTTCCTCCTTCTACCTTCTCACTTCTGTCTATTTATTTATATGTTTGCCTTTTTCACAACATATTTTCCTATTATTACTTACTATCTGACTCTCAAACTGTTAATTTTTCTGCTCCTCATCTCTGTTTAATCACACGTACCGCCTCTGCTTTACAATTTGCTGTAGTGATTTTATCCGGAAACTATCTTCTTAAGGATTGTACCAAACACATTTTTCACCACAAATTAGACGATTGTCAAGTTCAGAACAAGATTTTGAACACCTACATCACAAGGACGATAAGCAAGCAACAAGATCATTTGGTACATAAATCTGTTGAATACTTTGTTTTGAAACATAAAGTATGCTGTTCCAAAGTCTTGTTTGAGAGACTGACTTGAACCCTGCAAAATCCATGCACACTTTGTCATGCTAGCCGCTAAAGCCAAATTTAAACTAGGCCTGATTAGGTCAACTGGAAACTCCTTCTGATTTTTTTTTGTTGAAAAAAGGGGTGCAGATTGTGGTACTGATAATCATCTCAAGGTCCTCCTTTTTTTTTCTACTGAAATCATGTCAAGGTCCTTGAACGGACACGTTACCAAATCAATTTGTTGAAGAAGGGAGAGATAACTTTTTAATCCAAGATTAATCTCCACGTTGTATGAGAGGCCACCTTGAATTGATGGTTTCTAATGATTTTGGGTTTTCTTTTTTATTTCAATACTTGTATGAATTTCTAGTGCCAATATTTTGGACCAATGCTCTTGTATTCCTTATCTTTAATTTGGATGCAACTTTTCCATTGGATACATCAAACGAGAATGAAGTCATAGGGATAGTTTGTTGTTGTTGTGTTGTGTTGGATACCTCCTTTTGGCACCCAGTTCACCTTCTTCTTGTAAGCCAATGGGTACCACCTAGAATCTCCAGCCATCTGTGGACAGCACTATATATTGCAGGCTTGTTTTCTTCAAAGTATTTTTATGCCTTCCCTGTCTTCGTATCATATTGATGTATGATTTTGGACTGGAAAATGATTTGATTCTATTTCGATCGCTAGCCTACTGTTAGGTTTGTCCTATTCTGCTGCTTTGGTGTGGTTGCTTCAGCTCATGAAGGGGGTTGCTGCGGATGGGCACATCAAAACCATTTCTTTTTCATTCCATTGGTAGCATCACTTCTGGGGAGCGACCATCAAATGCTACGTAGACAACCATACCTTGTCCTTCTCCCTTGGTTGTTGGCCATGGGAGAATTGTTTTCATTGCCTAAGCCAGAGGGTCCTTTTCATCGCATGTTGGAAAGTCCAGCTATGCAGTAGCCCGAAACTCCTCTTCTGCCACCCAACATTGCCTCCCTACAGGTGACATGTGCCAAATGGCGACGGCAGCAAGACTAGACCACCCAGCTTGGCGATAGGAATTGGAGGTTGTCGGCCTCTTCTTGTATGTGGGGTGGCAGATTTACTGCCACCACCATTAGAGTCTGTTTAAAAGAGTATATATACTTGGACATCAAAAAGCACATGCTAGATTACATGAAAGTAGGAAAAGTCTAGGTAGCTCAATGAAGGTTTTGTTATAACATGGTCAAGTAAGGGTGTAGGGCCTGATTACAGGGCACTCCCAATTTGATTGATGAGTACTTCTACAGTGCTCTCTGAATACTACAAGTATTGTTACCAATAGAAGCGATAGAAGTCAGTGATTTATGAGTTCTACTACCACTAGAAAAAAAATTTCTGCTACCAGTAGTTGAGGTGGTAGCATAAATAGATATGGCCCATTGGATGAGAAAAGATGACAGGACTAGATTGATTTTACTACCAGTAAAAACCACCGAATAAAGTTTCTAACTATTCTTTCCTCACACTATTGAGAAGAAGGCACTTCCATTAAAAAGGAAAACATTTTCGATATCAAGGCATCAATTTGTTTTTCATTAAAAAGGAAACTGAATGAGGGGATAAAAACCTTCCGTTGGGCTTTGCATGCGAATAGTCTAATAAATACTCCCTCTGTTTTCTTTTATTTGACACCTTTGACTCCACACACAAAACTAGGCAATCATTGTACTTTCTAGAGATGACTATTTTACTCTTAGAGAAATTTAGTGTTGTTGGTAGGGTATGCTCACACAAGAACTTTCTAGATTAGTGTGGATGAATACATAGTTGTTTCTTATAAGGGCAAAATGGGAAAATAAAGATTGAGTTTCGTGCCGTGCCAATTATTTGCAAACAACTTTGGTCAAAGTTGTCTAATTAAAAGAGAACTGAGGGAGTAAAGAATTCTAGCTCAGCCTAGGAAAGTAGAGCCAACAACATAAGTCAATGAGTCCATGTTGCTTTTGAGCCAACTAATTGAGCAACAAGTTTGGCATCTACTACAAAAGAGAGAATAACCATGTGAGAAGGGTATATCATTTAGTAGGAAGGGTATATCCTGGAATACCTCGCTTGACCTCTTTCAAATGATTGTCTTGGCTTGGCTTCGGTTTAGCTGGCACATTTATTAGGAGGAAGGGTTTCTTGGAGCTTTTCTTCATTGCTTGCTGGCACATTTGGAAACAGCGTAATGAAAAGATTTTTCAGCATATTGATCCTTCTTTGATGCCTGGTGGTCTGCCTTTCAGACATGAGATTCTCCTATATAACTATAGGATGAATGGCCCCTACGACAAATAGTTGTTGATTGGTTACTACTTTTGTAGTTCTTTGGTTTTTGTTTTTGCCTTTTATGTACATATTCCTATAAAACTTTTTTGTTCTCTGACATGGGGTAGTTCTTCCCCTAAAACAAACACTCAATTCACCGGTCTGACCTGTGGACCGGCTGGTTGAATCATGGTTCAATAGCGTATATATACATTGTATATTCTCTGTAGATCACAAGGGAAGAATGACAGGAATGGTAGGCTAAAGCTCAGAGTTCTCAGTGCACGGACCTGAGTTCAAGTCCCCATGCCACAACCTTTTCTAGGATTTTTATTAAAAAAACAGAAGGCATAAATTGGGCCTTGATGTGGCCCATTTATCTCTGCACGGTTCAACCGGGGCAGTTTAATGTCCAATTTAGCTTAAACTAGGCCGGTCCGCCCAGTCCAATCCAGCTCAAATGCATGTTCGGTCTTTGAGTGCAACCGTGGCGACCTTTTGTTCTGTTTTTTTAGGTCGACTTGTTGACCCGGTTCAGTTTTTTTACACTATGACGCCTGGAGATACAACTGCTGGGCTAAAATGGGTTGAGCAACTGTCGAGTTGGCCTGAGCAAAGGAAGGGCCCACACAACTCTGCCCATGTTTCTGGAAAGAGAATCATCGCTCCGATTCTTCTCTCTTCTAGGCTAGTTTGAGCAATTCCCAGGTCAGCTCATAACAATGTGATATCAGAGCTCGTCAATCCTAGAGAAGTCGTGATTCCGAACACGTATGCAGCAACAGGTCGAGGATATGGATTTTAGGAAAGTATTCCAAACACATAAGTGTGGGCCTTTCCTTGCTCGGCCCAACCCATCGGTTGCTTGACCCATTTCATCAGTTGACCCAAGAATTGTATTTCTAGGTGTATGTGGGTCCGCTATACGTGAGAGTGAAATCTAGATTTTGGAGGCACCGAGGCAGGGTGTGATATCAAGATTGTAATCCTACTCATGAGTAATACAATGAACATATCTTGATTGTTTTAAAAAAACCGACAAGGCTGTGATGTTCTTTTCACAAAAAGAAAGAAAGAAAAGAAGACGCCAATGTTGTGCTGGTCTTTTGGCCTGTAATGGCTCTTTATGGACGAGAACATTGTTATTAAGCTATTGTTGCTGGTGACTTGTCTAGAACACACTACACATACACTCTCCTCGGAAATTTCATTAGCCACAAGTGTTAGCACCTCCAACCTAATCCCATCCTGAAGGACCTAGAACTTGATTTTATCCATTATCTAAAAAGAAAGGACCTAGAACTTGCCTTCCCACAGAAAGGAATTACGAGGGTCTTGTCATGTTGCTAGCCGATGATAATATCTGCCAAATATTCTTGAAGTGCAGGAAGTTACCCTTGTAAGCATCGAAGAAACAGGATCCTAAGTGCCTCGCAAGCTTGTGCACAACATGACCCAATGGTTGGAATAGTGGCAAACACCGGACTAAAATTTAGTATAGACTAGTCCACTGGTTTGACATATGCTTGTTCTCAGTCTGACTATATTGGGGAGTAAAGGTTTTTTTTTTCATATTTCAACATTAGAAACTTTAAATTGTTGGTCTAGTACTCATACTTGGCTGTGTTCGCATATGTGGGTTGGGAACTCATTCCCTCCGCATGGAAAACGGAGTGGTCCATTAGCACGTGATTAATTAAGTATTAGCTATTTTTTTCAAAAATGGATTAATTTGATTTTTTTAAGCAACTTTCGTATAGAAACTTTTTACAAAAACATACCGTTTAGCAATTTGAAAAGCGTGCATGTGGAAAACGAGGGAGAAGGGTTGGGAAAAGGGGATGCCGAACACAGCCTTAAGTTTGTTGGTCGCACTGTACCCAAGCATGTGGAGCTTTGATTTCCTTCAGTCTGTACAATTTTGGGTGAGATATGCACAACAGCACAAGATAAATATAGCGTGTTCTAATATTTTAACTCACCCGCTAATTTACCCAGCTGCCAATATTTTTCATGATTGACGTAGCCTGTACAAATTCGATCCTTATTTTTTGTAATTTGTTATGGACTAGCTAGGGTAAGGGTTTGAATACAAGGTGCTCCGTGGGAACCATTAATGGCATCAGATGGACCTTTTTATACTGGAGTTTGAATGTTTGGGGAATAGACAAGAGACTTATGCATCATTTTATTGAGAAGGAAAGGGTCTGGTATAAATCCAAAAAATGAAAAACAAAACAACAAAGAAACTAGAAATGTCAAATGTGGTGGGTGGTGGTGAATAATGCTGCTTGCTAGCGGGAGATGCCTGCCCATTGGCTATCTCTCGCAAAATGATAGTGTGACCAAGAAAAGATAAAGCACTCTATGCCTTGAGCTGTGGGATCATCACTCCAGTCCAGCAGCACTTGATCCTTTTTTTCTCATTGCAGGCATTATATTCAATTTGCTAACGACTTTAACTGAGATCACTGATTTATGGTCCCCATTCTTTGACTAAAACTCATTTCTGCATTGTGCACTTGCTTGCTGTTTCAAGTATTATTCATATTTCTTGTGGATCTTCCATTCGATTATACTCTTGTTTATTCGGCTTCTTTGTTAAGGACAGGGAGCCAAGCACACTAGAGTAATATTCTGAATGCAGACTGGCTTTTGTGACCTTTGTCGGCCCCGGTTTTGGGTGCTTCTTCTTAGTTGCAATACCTCGGGGAAGTTCTTTCCCCTTGGGGCCAAGGTTTTTTTTTAATGCACAAGATATTTTATCTTTGTTGGTCTTTGACTGAGTGTTATTAATTTATTTCTGTGAAGTCATCCCTGCTGAAAATGGCCAGCAAGCATGGACATATCTAGAAGATATGCAAAACAGCATTGATCTTGTTTTGACAGAGGTTGTTATGCCTGGTGTATCTGGAATTTCTCTATTGAGTAGGATCATGAACCACAATATTTGCAAGAATATTCCAGTGATTAGTAAGTAGCTTTCCTCAACTCTACAGTACAATTCTTGTACAAAATGTTGCCCTTGTCATTTATTTACTTTCTATGCTTTTAGCTGCTACACTGTTGTGGTTTATGTGCAGTGATGTCTTCAAATGATGCTATGGGTACAGTTTTTAAGTGTTTGTCAAAGGGCGCTGTTGACTTCTTAGTCAAGCCCATACGTAAGAATGAACTTAAGAACCTATGGCAGCATGTGTGGAGACGGTGCCACAGCGTAAGTTGTTGTTGGTCCAGTTTTTCTTATATATTAGATGTTCAGGCAAATCCATCAAGTACTGACTGCTGCTTCACCATTTTATGGTTACAATATGTGATGACTCTAAACTAAAGTTGTTATTTTCAAATTCAGTCCAGTGGCAGTGGAAGTGAAAGTGGCATTCAGACACAAAAGTGTGCCAAATCAAAAAGTGGGGATGAATCCAATAATAACAATGGCAGCAATGACGATGATGACGACGATGGTGTAATCATGGGACTTAATGCAAGAGATGGCAGTGATAACGGCAGTGGCACTCAAGTATGAAACTTGATCTTTTTATTCCAACATAGCTTTACTACTACCTGTTAACAAAGCTGTAATTAGAATGAGAAGAAAAAACTGAAGTTAAAAACTGAATAAACCTGTCAGTAACAATGATTTCTGAAGGCATAAATGACATTTTTTTGCATAGCTGATAAATTTATTTTAGAATAGTGGGAAATAGGAAGAGTTCACCATGTCACTGTAAAGTTTTTGAATTAACCAAACCAGTAAAATACCATGGATCATCTGCATATAACAATCTTAATATTGTACAAACACAACAGATGAAACACATACTGAAGAAAATATAGTTATCGCCTCTCGGATAGTTTACATGTGTGTTCATGTGGCACTATCGTTTTGCTTAACTAATACCAAGGTGCCAAATAAACCAGCGGTTGGTTCATTGCATTTTGGAGCACATATAGTATTTATAAATCTTGATAGCGTGTGTGTAGAATGTTCCACCAGAAGTTGATAGCACTGTGCTTTTTGCATGTTCAAGGAATCAAATGGATTGTACAATACATGCAACCAAACTTTGATGTAGGGAATTTCTTTGTTTTGGCTTGGATGTCCAACTTATAGTTTCTCACCAGTTGGATTAACTTCTTTTCCTTGTTCTCCCATTGTAAACGTGAATTGAACAATCCTTGCCTTTTAAGATAAATGTGGGGTCTATGCTTTATGCTGCCCTTTAAATTTTTGAAATGCTAATAAGTAGTTTGCATGCATCGTGCTTCTCTAAATGTCATCAAGTTGACTAATATACAGTTCACTATCAATCAGCTAATACCACTTTATTTTATGTGTTTCCTATTATAAATTGAACATTTTGTTTCCTTACCAACTAGAAATCATAGTACAGATACATATTGTATAGTTATTTTCGGTTTATTCATGAATTTGTGTTAGAATTAGACTATATAGACATTGCTGGTTAAAAAGTACTCATCCCCTAACACTGTGGGAAGAGTACAAAACTACTAGTGTGTTAACACCATTGGCCTCTTGACATTCAAAACTGCTACCCAATGAACACATTCCCCATCAATGGTCCAATAATCCCGAAGTCTAAAGTCCTTAATCAACTCGTGCATAATTATTCACTTGGTAAGGAAATAGGAAAAGGATGGATGCACACAAATGTTTTGTCCATTTTTGCTAAGCCTCCAGTATTTTTACTTCTGTGTTTTGAGCTTCATAATGGTTTCCATGTCTTTTCTTCAATTAACGCTTTTGTCCTGTATTTTGATTTCTATTTGCACCATTTAATGAATCTTCCAAATTTGGGTTTTTCCTCTTGATGATTTTTAACTTCACATCTCAATTGATCTTTAAAATATGCCTAATTATTTAATACTACCTCCGTCCCAAAATATAGCAACCTAAGACTGGATAGGACATATCCTAGTCCAATGAATCTGGACAACCCTTGTCCAGATTCCTTGGACTAGGATATGTTCCATCCAATCCTAAATTGCTATATTTTGGGATGGAGAGAGTATCATCTAATTAATTACCTGCAAATCTGCAATGCTCTGCTTAAACATATTGTATATTTTCTATACTAATTTACTATACTTATTATCAAAATGAAACGTATATTAAAAATTAAATATCTGCTGCAATATGTGCCCTCAAAACTAGTATAAACATATTGTATATTTTCTTTTCAAAGTTACCTTTAATTTGGCGACATGTTTGGATTGATAAAATCAAGTCTAAGCTTGCAAATTTGGTTCTCCTGATGCTGTATGTTTGATTCCACTAAATAGATTCTCTTCAATGACCTAGGCAAGCTGTCGTAGTTTATCCCAAATGTGTTGAAAAGGTTGTTCTGGGTATCACCGTACAGATAGATTGATCCTGAAATTCGTATGCAGAGTAAACAAAATAGTCTTATCAACATAGATAATGCTAAACATCATATCATCAGCGCACTAGTGCAGGACTGCCTTGACATATAATGGTTGCCTGAAATGGCAATGAAATAAAAAATCTTTTATTTAAAAAGCAAGATTCAATTGTGAATTAGCAATAGTTGGAGAAATTTAGAGTTACAGGATATGGCACACGAGGTGTACTTGAGAACTGTCGGTTTGTAGCTAGAGATGGCTACGAATGCCGCCGGGGGGTACCTAGCTTATCGGCGGTGGATGAAAACTAGTGTAAACTTGAAATTAGTCCACGTTTCATGTGGATGAAATCTAGTGTAAACTTGAAATTAGTCCACGTTTATTATGAAACGTTGACTACAGACCCTACCGAGATAAACTGTATCTTATGTGTTGCTGTCAAGTTCTCTTTTATTACAGTGTTGTGGAGTGGGGTAAAATTTTTTTCCTGTCAGTTAGTTATGAATCACATAAGATTTTGCCACTGAAATGTTCAGTGACATGGCCGATATGTCAATGTAAGAAACAACTTAGAATAAATTCCACATTACAATATTCAGAAATAATTACTGTAGATCGGATCACTTTGTGAAGTGCATTTAAACTGGTTGCTCATCATTTTTTTAAGGCGCAGAGCTCATGGACAAAGCGCGCTGTTGAGATTGACAGTCCACAGGCTATGTCTCCAGATCAATTAGCTGATCCACCTGATAGCACTTGTGCACAAGTGATCCACCTGAAGTCAGATATATGCAGCAATAGATGGTTACCATGTACAAGCAACAAAAATTCCAAGAAACAAAAAGAAACTAATGGTATTGTATGCTCAACTGATTCATTGTGCAACTCGATAAAACAAAAGCTCCATAACACTGTATATTAATAAATTTCAACATGTTTTTTTTTCAGATGACTTCAAGGGGAAGGACTTGGAAATAGGTTCTCCTAGAAATTTAAACACAGCTTATCAATCCTCTCCGAATGAGAGATCCATCAAACCAACAGATAGACGGAATGAATATCCACTGCAAAACAATTCAAAGGAGGCAGCGATGGAAAATCTGGAGGAGTCAAGTGTTCGAGCTGCTGACTTAATTGGTTCGATGGCCAAAAACATGGATGCACAACAGGCAGCAAGAGCCGCAAATGCCCCTAATTGCTCCTCCAAAGTGCCAGAAGGGAAAGATAAGAACCGTGATAATATTATGCCATCACTTGAATTAAGTTTGAAAAGGTCAAGATCGACTGGGGATGGTGCAAACGCAATCCAAGAGGAACAACGGAATGTTTTGAGACGATCAGATCTCTCGGCATTTACGAGGTGCAAAACATAATATCAGTGTCGCTAGTGAGTTAGGAAACCATTGTTAAGTTGCATACTAACTGTTACTTTTGTTGCAAGGTACCATACACCTGTGGCTTCCAATCAAGGTGGGACAGGATTCATGGGAAGCTGTTCGCTGCATGATAATAGCTCAGAGGCTATGAAAACGGATTCTGCTTACAACATGAAGTCAAACTCAGATGCTGCACCAATAAAACAAGGTTCTAATGGTAGTAGCAATAACAATGACATGGGTTCCACTACAAAGAACGTTGTGACAAAGCCTAGTACAAATAAGGAGAGAGTAATGTCACCCTCAGCTGTTAAGGCTAATGGACACACATCAGCATTTCATCCTGCACAGCACTGGACGTCTCCAGCTAATACAACAGGAAAAGAAAAGACTGATGAAGTGGCTAACAATGCAGCAAAGAGGGCTCAGCCTGGTGAAGTACAGAGCAACCTCGTACAACACCCTCGCCCAATACTTCATTATGTTCATTTCGATGTGTCACGTGAGAATGGTGGATCCGGGGCCCCTCAATGTGGTTCATCCAATGTATTTGATCCTCCTGTCGAAGGTCATGCTGCCAACTATGGTGTCAATGGAAGCAACTCAGGCAGTAACAATGGAAGCAATGGGCAGAATGGGAGTACGACTGCTGTAAATGCTGAACGGCCAAATATGGAGATCGCTAATGGCACCATCAACAAAAGTGGACCTGGAGGTGGCAATGGAAGTGGAAGCGGCAGTGGCAATGACATGTATCTGAAACGCTTCACTCAACGAGAGCATAGAGTGGCTGCAGTGATCAAGTTTAGACAGAAAAGGAAAGAGCGCAACTTCGGAAAAAAGGTAGCCTGTTTTCAATTGCATGTTTTCTGTTCCTTTGGTTTTAGCATTCCTGTTTAACTCGTCTAAATTAGCTAAAGAACATGTTACTGGAAGTAGTTGTCAAAAGCATATTACTGGAAGTTTCTCCCAAACGACTAGCTAAATGGGATCGGGCATGAATATAATTTGTTTATATACTAGTATTATCTGATTTCTAAAAGGAATCTCACAAGATAATCTTCCAAAAAGTTGCACGTTTTTGGTCCCATAGCCGTGTTGCTGAAATTTCTTGCCAATGACATTTCTTGGATTTTTCTCATAATTTAATGGTAGCTACTTAGAGCGGCAATTCAATTTTACTCTTGAAACACTGTCCTTTACTTTTCGGCGCGGGTGAGTACAATTTGGATAGGAGGCTCTTTTTATATATAGAGAAACATACATACATACATACATATATATATATATATATATATATATATATATGATGTGCTTGATTAAGCTTTGGATAGGATCGGTTTCTTTCTGATGGCTGTCTGCCATTTTCAGGTGCGGTACCAGAGCAGAAAGAGGCTGGCCGAGCAGCGGCCAAGGGTCCGCGGACAGTTCGTGCGGCAAGCTGTGCAAGACCAACAACAGCAGGGTGGTGGGCGCGAAGCGGCAGCGGACAGATGAATGATGGGAACCGCTCATCACAACCAAACCGCCGGCTCTGCCCTCGGAGTCGGAGTCGGAGATGCCAACGACGCCGTGCCTGGGGCTGGGGGTGGGGGCTACAGCGACCCGGATGGCGGACCAATCTCCGGTGTGCAGCGGCCACCGCAGGTCTGCTGGGAGCGCTTCATCCAGAAGAAGACTATCAAAGTCTTGCTAGTT GATAGCGATGACTCCACCAGGCAGGTGGTCAGTGCCCTGCTTCGTCACTGCATGTATGAAGGTCTGTCCTTCTTTCCTCCTTCTACCTTCTCACTTCTGTCTATTTATTTATATGTTTTGCCTTTTTCACAACATATTTCCTATTTACTTACTATCTGACTCTCAAACTGTTAATTTTTCTGCTCCTCATCTCTGTTTAATCACACGTACCGCCTCTGCTTTACAATTTGCTG TAGTGATTTTATCCGGAAACTATTCTTCTTAAGGATTGTACCAAAACACATTTTTCACCACAAATTAGACGATTGTCAAAGTTCAGAACAAGATTTTGAACACCTACATCACAAGGACGATAAGCAAGCAACAAGATCATTTGGTACATAAATCTGTTGAATACTTTGTTTTGAAACATAAAGTATGCTGTTCCAAAGTCTTGTTTGAGAGACTGACTTGAACCCCTGCAAAATCCAT GCACACTTTGTCATGCTAGCCGCTAAAGCCAAATTTAAACTAGGCCTGATTAGGTCCAACTGGAAACTCCTTCTGATTTTTTTTTGTTGAAAAAAGGGGTGCAGATTGTGGTACTGATAATCATCTCCAAGGTCTCCTTTTTTTTTTCTACTGAAATCATGTCAAGGTCCTTGAACGGACACGTTACCAAAATCAATTTGTTGAAGAAGGGAGAGATAACTTTTAATC CAAGATTAATCTCCACGTTGTATGAGAGGCCACCTTGAATTGATGGTTTCTAATGATTTTGGGTTTTTCTTTTTTATTTCAATACTTGTGAATTTTCTAGTGCCAATATTTTGGACCAATGCTCTTGTATTCCTTTATCTTTAATTTGGATGCAACTTTTCCATTGGATACATCAAACGAGAATGAAGTCATAGGGATAGTTTGTTGTTGTTGTGTTGTGTTGG ATACCTCCTTTTGGCACCCAGTTCACCTTCTTCTTGTAAGCCAATGGGTACCACCTAGAATCTCCAGCCATCTGTGGACAGCACTATATTGCAGGCTTGTTTTTCTTCAAAGTATTTTTATGCCTTCCCTGTCTTCGTATCATATTGATGTATGATTTTGGACTGGAAAATGATTTGATTCTATTTCGATCGCTAGCCTACTGTTAGGTTTGTCCTATTCTGCTGCTTTG GTGTGGTTGCTTCAGCTCATGAAGGGGGTTGCTGCGGATGGGCACATCAAAACCATTTCTTTTTCATTCCATTGGTAGCATCACTTCTGGGGAGCGACCATCAAATGCTACGTAGACAACCATACCTTGTCCTTCTCCCTTGGTTGTTGGCCATGGGAGAATTGTTTTCATTGCCTAAGCCAGAGGGTCCTTTTCATCGCATGTTGGAAAGTCC AGCTATGCAGTAGCCCGAAACTCCTCTTCTGCCACCCAACATTGCCTCCCTACAGGTGACATGTGCCAAATGGCGACGGCAGCAAGACTAGACCACCCAGCTTGGCGATAGGAATTGGAGGTTGTCGGCCTCTTCTTGTATGTGGGGTGCAGATTTACTGCCACCACCATTAGAGTCTGTTTAAAGAGTATATACTTGGACATCAAAAGCACATGCT GATTACATGAAAGTAGGAAAAGTCTAGGTAGCTCAATGAAGGTTTTGTTATAACATGGTCAAGTAAGGGTGTAGGGCCTGATTACAGGGCACTCCCAATTTGATTGATGAGTACTTCTACAGTGCTCTCTGAATACTACAAGTATTGTTACCAATAGAAGCGATAGAAGTCAGTGATTTATGAGTTCTACTACCACTAGAAAAAAATTTCTGCTACCAGTAGTTGAGGT GGTAGCATAAATAGATATGGCCCATTGGATGAGAAAAGATGACAGGACTAGATTGATTTTACTACCAGTAAAAAACCACCGAATAAAGTTTCTAACTATTCTTTCTCACACTATTGAGAAGAAGGCACTTCCATTAAAAAGGAAAACATTTTCGATATCAAGGCATCAATTTGTTTTTCATTAAAAAAGGAAACTGAATGAGGGGATAAAAACCTTCCGTTGGGCTTT GCATGCGAATAGTCTAATAAATACTCCCTCTGTTTTTCTTTTATTTGACACCTTTGACTCCACACACAAACTAGGCAATCATTGTACTTTACTCTTAGAGAAATTTAGTGTTGTTGGTAGGGTATGCTCACACAAGAACTTTTCTAGATTAGTGTGGATGAATACATAGTTGTTTCTTATAAGGGCAAAATGGGAAAATAAAGATTGAGTTTC GTGCCGTGCCAATTATTTGCAAACAACTTTGGTCAAAGTTGTCTAATTAAAAGAGAACTGAGGGAGTAAAGAATTCTAGCTAGCCTAGGAAAGTAGAGCCAACAACATAAGTCAATGAGTCCATGTTGCTTTTGAGCCAACTAATTGAGCAACAAGTTTGGCATCTACTACAAAAGAGAGAATAACCATGTGAGAAGGGTATATCATTTAGTAGGAAGGGTATATCCTG GAATACCTCGCTTGACCTCTTTCAAATGATTGTCTTGGCTTGGCTTCGGTTTAGCTGGCACATTTATTAGGAGGAAGGGTTTCTTGGAGCTTTTCTTCATTGCTTGCTGGCACATTTGGAAACAGCGTAATGAAAAGATTTTTCAGCATATTGATCCTTCTTTGATGCCTGGTGGTCTGCCTTTCAGACATGAGATTCTCCTATAACTATAGGATGAAT GGCCCCTACGACAAATAGTTGTTGATTGGTTACTACTTTTGTAGTTCTTTGGTTTTTTGTTTTTGCCTTTTATGTACATATTTCCTATAAAACTTTTTTTGTTCTCTGACATGGGGTAGTTTCTTCCCCTAAAACAAACACTCAATTCACCGGTCTGACCTGTGGACCGGCTGGTTGAATCATGGTTCAATAGCGTATATATCCTGTATATTCTCTGTAGATCA CAAGGGAAGAATGACAGGAATGGTAGGCTAAAGCTCAGAGTTCTCAGTGCACGGACCTGAGTTCAAGTCCCATGCCACAACCTTTTCTAGGATTTTTATTAAAAAAACAGAAGGCATAAATTGGGCCTTGATGTGGCCCATTTATCTCTGCACGGTTCAACCGGGGCAGTTTAATGTCCAATTTAGCTTAAACTAGGCCGGTCCGCCCAGTCCAATCCAGCTCA AATGCATGTTCGGTCTTTGAGTGCAACCGTGGCGACCTTTTGTTCTGTTTTTTTAGGTCGACTTGTTGACCCGGTTCAGTTTTTTACACTATGACGCCTGGAGATACAACTGCTGGGCTAAAATGGGTTGAGCAACTGTCGAGTTGGCCTGAGCAAAGGAAGGGCCCACACAACTCTGCCCATGTTTCTGGAAAGAGAATCATCGCTCCGATTCTTCTCTC TTCTAGGCTAGTTTGAGCAATTCCCAGGTCAGCTCATAACAATGTGATATCAGAGCTCGTCAATCCTAGAGAAGTCGTGATTCCGAACACGTATGCAGCAACAGGTCGAGGATATGGATTTTAGGAAAGTATTCCAAACACATAAGTGTGGGCCTTTCCTTGCTCGGCCCAACCCATCGGTTGCTTGACCCATTTCATCAGTTGACCCAAGAATTGTATTTCTAGGTGTGTGT GGGTCCGCTATACGTGAGAGTGAAATCTAGATTTTGGAGGCACCGAGGCAGGGTGTGATATCAAGATTGTAATCCTACTCATGAGTAATACAATGAACATATCTTGATTGTTTTAAAAAACCGACAAGGCTGTGATGTTCTTTTCCAAAAAGAAAGAAAGAAAAAGAAGACGCCAATGTTGTGCTGGTCTTTTGGCCTGTAATGGCTCTTTATGGACGAGAAC ATTGTTATTAAGCTATTGTTGCTGGTGACTTGTCTAGAACACACTACACATACACTCTCCTCGGAAATTTCATTAGCCACAAGTGTTAGCACCTCAACCTAATCCCATCCTGAAGGACCTAGAACTTGATTTTATCCATTATCTAAAAAGAAAGGACCTAGAACTTGCCTTCCCACAGAAAGGAATTACGAGGGTCTTGTCATGTTGCTAGCCGATGATAATATCTG CCAAATATTCTTGAAGTGCAGGAAGTTACCCTTGTAAGCATCGAAGAAACAGGATCCTAAGTGCCTCGCAAGCTTGTGCACAACATGACCCAATGGTTGGAATAGTGGCAAACACCGGACTAAAATTTAGTATAGACTAGTCCACTGGTTTGACATATGCTTGTTCTCAGTCTGACTATATTGGGGAGTAAAGGTTTTTTTTTTTCATATTTCAACATTAGAAACTT TAAATTGTTGGTCTAGTACTCATACTTGGCTGTGTTCGCATATGTGGGTTGGGAACTCATTCCCTCCGCATGGAAAACGGAGTGGTCCATTAGCACGTGATTAATTAAGTATTAGCTATTTTTTCAAAAATGGATTAATTTGATTTTTTTAAGCAACTTTTCGTATAGAAACTTTTACAAAAACATACCGTTTAGCAATTTGAAAAGCGTGCATGTGGAAAACGA GGGAGAAGGGTTGGGAAAAGGGGATGCCGAACACAGCCTTAAGTTTGTTGGTCGCACTGTACCCAAGCATGTGGAGCTTTGATTTCCTTCAGTCTGTACAATTTTGGGTGAGATATGCACAACAGCACAAGATAAATAGCGTGTTCTAATATTTTTAACTCACCCGCTAATTTACCCAGCTGCCAATATTTTTCATGATTGACGTAGCCTGTACAAATTCGA TCCTTATTTTTTGTAATTTGTTATGGACTAGCTAGGGTAAGGGTTTGAATACAAGGTGCTCCGTGGGAACCATTAATGGCATCAGATGGACCTTTTATACTGGAGTTTGAATGTTTGGGGAATAGACAAGAGACTTATGCATCATTTTATTGAGAAGGAAAGGGTCTGGTATAAATCCAAAAAATGAAAACAAAAACAACAAAGAAACTAGAAATGTCAA ATGTGGTGGGTGGTGGTGAATAATGCTGCTTGCTAGCGGGAGATGCCTGCCCATTGGCTATCTCTCGCAAAATGATAGTGTGACCAAGAAAAGATAAAGCACTCTATGCCTTGAGCTGTGGGATCATCACTCCAGTCCAGCAGCACTTGATCCTTTTTTTCTCATTGCAGGCATTATATTCAATTTGCTAACGACTTTAACTGAGATCACTGATTTATGGTCCCC ATTCTTTGACTAAAACTCATTTCTGCATTGTGCACTTGCTTGCTGTTTCAAGTATTATTCATATTTCTTGTGGATCTTCCATTCGATTATACTCTTGTTTATTCGGCTTCTTTGTTAAGGACAGGGAGCCAAGCACACTAGAGTAATATTCTGAATGCAGACTGGCTTTTGTGACCTTGTCGGCCCCGGTTTTGGGTGCTTCTTCTTAGTTGCAATACCTCGG GGAAGTTCTTTCCCCTTGGGGCCAAGGTTTTTTTTTAATGCACAAGATATTTTATCTTTGTTGGTCTTTGACTGAGTGTTATTAATTTATTTCTGTGAAGTCATCCCTGCTGAAAATGGCCAGCAAGCATGGACATATCTAGAAGATATGCAAAACAGCATTGATCTTGTTTTGACAGAGGTTGTTATGCCTGGTGTATCTGGAATTTCTCTATTGAGTAGGATCAT GAACCACAATATTTGCAAGAATATTCCAGTGATTAGTAAGTAGCTTTCCTCAACTCTACAGTACAATTCTTGTACAAAATGTTGCCCTTGTCATTTATTTACTTTCTATGCTTTTAGCTGCTACACTGTTGTGGTTTTATGTGCAGTGATGTCTTCAAATGATGCTATGGGTACAGTTTTTAAGTGTTTGTCAAAGGGCGCTGTTGACTTCTTAGTCAAGCCCATACGTAA GAATGAACTTAAGAACCTATGGCAGCATGTGTGGAGACGGTGCCACAGCGTAAGTTGTTGTTGGTCCAGTTTTTCTTATATTAGATGTTCAGGCAAATCCATCAAGTACTGACTGCTGCTTCACCATTTTATGGTTACAATATGTGATGACTCTAAACTAAAGTTGTTATTTTCAAATTCAGTCCAGTGGCAGTGGAAGTGAAAGTGGCATTCAGACACAAAA GTGTGCCAAAATCAAAAGTGGGGATGAATCCAATAATAACAATGGCAGCAATGACGATGATGACGACGATGGTGTAATCATGGGACTTAATGCAAGAGATGGCAGTGATAACGGCAGTGGCACTCAAGTGAAACTTGATCTTTTTATTCCAACATAGCTTTACTACTACCTGTTAACAAAGCTGTAATTAGAATGAGAAGAAAAAACTGAAGTTAAAA CTGAATAAACCTGTCAGTAACAATGATTTCTGAAGGCATAAATGACATTTTTTTGCATAGCTGATAAATTTATTTAGAATAGTGGGAAATAGGAAGAGTTCACCATGTCACTGTAAAGTTTTTGAATTAACCAAACCAGTAAAATACCATGGATCATCTGCATAACAATCTTAATATTGTACAAACACAACAGATGAAACACATACTGAAGAAAATATAGTTATCGCCTCTC GGATAGTTTACATGTGTGTTCATGTGGCACTATCGTTTTGCTTAACTAATACCAAGGTGCCAAATAAACCAGCGGTTGGTTCATTGCATTTTGGAGCACATAGTATTTAAATCTTGATAGCGTGTGTGTAGAATGTTCCACCAGAAGTTGATAGCACTGTGCTTTTTGCATGTTCAAGGAATCAAATGGATTGTACAATACATGCAACCAAACTTTGATGT AGGGAATTTCTTTGTTTTGGCTTGGATGTCCAACTTATAGTTTCTCACCAGTTGGATTAACTTCTTTTCCTTGTTCCTCCATTGTAAACGTGAATTGAACAATCCTTGCCTTTTAAGATAAATGTGGGGTCTATGCTTTATGCTGCCCTTTAAATTTTTGAAATGCTAATAAGTAGTTTGCATGCATCGTGCTTCTCTAAATGTCATCAAGTTGACTAATATACAGT TCACTATCAATCAGCTAATACCACTTTATTTTATGTGTTTCCTATTTATAAATTGAACATTTTGTTTCCTTACCAACTAGAAATCATAGTACAGATACATATTGTATAGTTATTTTCGGTTTATTCATGAATTTGTGTTAGAATTAGACTATATAGACATTGCTGGTTAAAAAGTACTCATCCCCTAACACTGTGGGAAGAGTACAAAACTACTAGTGTGTTAACACCATTGGCC TCTTGACATTCAAAACTGCTACCCAATGAACACATTCCCCCATCAATGGTCCAATAATCCCGAAGTCTAAAGTCCTTAATCAACTCGTGCATAATTATTCACTTGGTAAGGAAATAGGAAAAGGATGGATGCACACAAATGTTTTGTCCATTTTTGCTAAGCCTCCAGTTTTTACTTCTGTGTTTTGAGCTTCTAATGGTTTCCATGTCTTTTTCTTCAATTAACGCTTTT GTCCTGTATTTTGATTTCTATTTGCACCATTTAATGAATCTTCCAAATTTGGGTTTTTCCCTCTTGATGATTTTTAACTTCACATTCTCAATTGATCTTTAAAATATGCCTAATTATTACTACCTCCGTCCCAAATATAGCAACCTAAGACTGGATAGGACATATCCTAGTCCAATGAATCTGGACAACCCTTGTCCAGATTCCTTGGACTAGGATATGTTCCATCCAAT CCTAAATTGCTATATTTTGGGATGGAGAGAGTATCATCTAATTAATTACCTGCAAATCTGCAATGCTCTGCTTAAACATATTGTATATTTTCTATACTAATTTACTATACTTATTATCAAAATGAAACGTATATTAAAAATTAAATCTGCAATATGTGCCTCAAAAACTAGTATAAACATATTGTATTTTCTTTTCAAAGTTACCTTTTAATTTGGCGACATGTTT GGATTGATAAAATCAAGTCTAAGCTTGCAAATTTGGTTCTCCTGATGCTGTATGTTTGATTCCACTAAATAGATTCTCTTCAATGACCTAGGCAAGCTGTCGTAGTTTATCCCAAATGTGTTGAAAAGGTTGTTCTGGGTATCACCGTACAGATAGATTGATCCTGAAATTCGTATGCAGAGTAAACAAAATAGTCTTATCAACATAGATAATGCTAAACATCATATC ATCAGCGCACTAGTGCAGGACTGCCTTGACATATAATGGTTGCCTGAAATGGCAATGAAATAAAAAATCTTTTAAAAAAGCAAGATTCAATTGTGAATTAGCAATAGTTGGAGAAATTTAGAGTTACAGGATATGGCACACGAGGTGTACTTGAGAACTGTCGGTTTGTAGCTAGAGATGGCTACGAATGCCGCCGGGGGGTACCTAGCTTATCGGC GGTGGATGAAAACTAGTGTAAACTTGAAATTAGTCTCACGTTTCATGTGGATGAAATCTAGTGTAAACTTGAAATTAGTCCACGTTTATTATGAAACGTTGACTACAGACCCTACCGAGATAAACTGTATCTTATGTGTTGCTGTCAAAGTTTCTCTTTTATTATCAGTGTTGTGGAGTGGGGTAAATTTTTTTCCTGTCAGTTAGTTATGAATCACATAAGATTTT GCCACTGAAATGTTCAGTGACATGGCCGATATGTCAATGTAAGAAACAACTTAGAATAAATTCCACATTACAATATTCAGAAATAATTACTGTAGATCGGATCACTTTGTGAAGTGCATTTAAACTGGTTGCTCATCATTTTTTTAAGGCGCAGAGCTCATGGACAAAGCGCGCTGTTGAGATTGACAGTCCACAGGCTATGTCTCCAGATCAATTAGCTGATCC ACCTGATAGCACTTGTGCACAAGTGATCCACCTGAAGTCAGATATGCAGCAATAGATGGTTACCATGTACAAGCAACAAAAATTCCAAGAAACAAAAAGAAACTAATGGTATTGTATGCTCAACTGATTCATTGTGCAACTCGATAAAACAAAGCTCCATAACACTGTATATTAATAAATTTCAACATGTTTTTTTCAGATGACTTCAAGGGGAAGGACTTGGAAAT AGGTTTCCTAGAAATTTAAACACAGCTTATCAATCCTCTCCGAATGAGAGATCCATCAAACCAACAGATAGACGGAATGAATATCCACTGCAAAACAATTCAAAGGAGGCAGCGATGGAAAATCTGGAGGAGTCAAGTGTTCGAGCTGCTGACTTAATTGGTTCGATGGCCAAAAACATGGATGCACAACAGGCAGCAAGAGCCGCAAATGCCCCTAATTGCTCC TCCAAAGTGCCAGAAGGGAAAGATAAGAACCGTGATAATATTATGCCATCACTTGAATTAAGTTTGAAAAGGTCAAGATCGACTGGGGATGGTGCAAACGCAATCCAAGAGGAACAACGGAATGTTTTGAGACGATCAGATCTCTCGGCATTTACGAGGTGCAAAACATAATATCAGTGTCGCTAGTGAGTTAGGAAACCATTGTTAAGTTGCATACTAACTGTTA CTTTTGTTGCAAGGTACCATACACCTGTGGCTTCCAATCAAGGTGGGACAGGATTCATGGGAAGCTGTTCGCTGCATGATAATAGCTCAGAGGCTATGAAAACGGATTCTGCTTACAACATGAAGTCAAACTCAGATGCTGCACCAATAAACAAGGTTCTAATGGTAGTAGCAATAACAATGACATGGGTTCCACTACAAAGAACGTTGTGACAAAGCCT AGTACAAATAAGGAGAGAGTAATGTCACCCTCAGCTGTTAAGGCTAATGGACACACATCAGCATTTCATCCTGCACAGCACTGGACGTCTCCAGCTAATACAACAGGAAAAGAAAAGACTGATGAAGTGGCTAACAATGCAGCAAAGAGGCTCAGCCTGGTGAAGTACAGAGCAACCTCGTACAACACCCTCGCCCAATACTTCATTATGTTCATTTCGATGTGTC ACGTGAGAATGGTGGATCCGGGGCCCCTCAATGTGGTTCATCCAATGTATTTGATCCTCCTGTCGAAGGTCATGCTGCCAACTATGGTGTCAATGGAAGCAACTCAGGCAGTAACAATGGAAGCAATGGGCAGAATGGGAGTACGACTGCTGTAAATGCTGAACGGCCAAATATGGAGATCGCTAATGGCACCATCAACAAAGTGGACCTGGA GGTGGCAATGGAAGTGGAAGCGGCAGTGGCAATGACATGTATCTGAAACGCTTCACTCAACGAGAGCATAGAGTGGCTGCAGTGATCAAGTTTAGACAGAAAAGGAAAGAGCGCAACTTCGGAAAGCGCAACTTCGGAAAAAAAAGGTAGCCTGTTTTCTGTTCCTTTGGTTTTGCATTCCTGTTTAACTCGTCTAAATTAGCTAAAGAACATGTTACTGGAAG TAGTTGTCAAAAAGCATATTACTGGAAGTTTCTCCCAAACGACTAGCTAAATGGGATCGGGCATGAATATAATTTGTTTATATACTAGTATTATCTGATTTCTAAAAGGAATCTCACAAGATAAATCTTCCAAAAAGTTGCACGTTTTTTGGTCCCATAGCCGTGTTGCTGAAATTTCTTGCCAATGACATTTCTTGGATTTTTCTCATAATTTAATGGTAGCTACTTAGAGC GGCAATTCAATTTTACTCTTGAAACACTGTCCTTTACTTTTCGGCGCGGGTGAGTACAATTTGGATAGGAGGCTCTTTTATATAGAGAAACATACATACATACATACATATATATATATATATATATATATATATATGATGTGCTTGATTAAGCTTTGGATAGGATCGGTTCTTTCTGATGGCTGTCTGCCATTTTCAGGTGCGGTACCAGAGCAGAGAA AGGCTGGCCGAGCAGCGGCCAAGGGTCCGCGGACAGTTCGTGCGGCAAGCTGTGCAAGACCAACAACAGCAGGGTGGTGGGCGCGAAGCGGCAGCGGACAGATGA

序列2sequence 2

ATGATGGGAACCGCTCATCACAACCAAACCGCCGGCTCTGCCCTCGGAGTCGGAGTCGGAGATGCCAACGACGCCGTGCCTGGGGCTGGGGGTGGGGGCTACAGCGACCCGGATGGCGGACCAATCTCCGGTGTGCAGCGGCCACCGCAGGTCTGCTGGGAGCGCTTCATCCAGAAGAAGACTATCAAAGTCTTGCTAGTTGATAGCGATGACTCCACCAGGCAGGTGGTCAGTGCCCTGCTTCGTCACTGCATGTATGAAGTCATCCCTGCTGAAAATGGCCAGCAAGCATGGACATATCTAGAAGATATGCAAAACAGCATTGATCTTGTTTTGACAGAGGTTGTTATGCCTGGTGTATCTGGAATTTCTCTATTGAGTAGGATCATGAACCACAATATTTGCAAGAATATTCCAGTGATTATGATGTCTTCAAATGATGCTATGGGTACAGTTTTTAAGTGTTTGTCAAAGGGCGCTGTTGACTTCTTAGTCAAGCCCATACGTAAGAATGAACTTAAGAACCTATGGCAGCATGTGTGGAGACGGTGCCACAGCTCCAGTGGCAGTGGAAGTGAAAGTGGCATTCAGACACAAAAGTGTGCCAAATCAAAAAGTGGGGATGAATCCAATAATAACAATGGCAGCAATGACGATGATGACGACGATGGTGTAATCATGGGACTTAATGCAAGAGATGGCAGTGATAACGGCAGTGGCACTCAAGCGCAGAGCTCATGGACAAAGCGCGCTGTTGAGATTGACAGTCCACAGGCTATGTCTCCAGATCAATTAGCTGATCCACCTGATAGCACTTGTGCACAAGTGATCCACCTGAAGTCAGATATATGCAGCAATAGATGGTTACCATGTACAAGCAACAAAAATTCCAAGAAACAAAAAGAAACTAATGATGACTTCAAGGGGAAGGACTTGGAAATAGGTTCTCCTAGAAATTTAAACACAGCTTATCAATCCTCTCCGAATGAGAGATCCATCAAACCAACAGATAGACGGAATGAATATCCACTGCAAAACAATTCAAAGGAGGCAGCGATGGAAAATCTGGAGGAGTCAAGTGTTCGAGCTGCTGACTTAATTGGTTCGATGGCCAAAAACATGGATGCACAACAGGCAGCAAGAGCCGCAAATGCCCCTAATTGCTCCTCCAAAGTGCCAGAAGGGAAAGATAAGAACCGTGATAATATTATGCCATCACTTGAATTAAGTTTGAAAAGGTCAAGATCGACTGGGGATGGTGCAAACGCAATCCAAGAGGAACAACGGAATGTTTTGAGACGATCAGATCTCTCGGCATTTACGAGGTACCATACACCTGTGGCTTCCAATCAAGGTGGGACAGGATTCATGGGAAGCTGTTCGCTGCATGATAATAGCTCAGAGGCTATGAAAACGGATTCTGCTTACAACATGAAGTCAAACTCAGATGCTGCACCAATAAAACAAGGTTCTAATGGTAGTAGCAATAACAATGACATGGGTTCCACTACAAAGAACGTTGTGACAAAGCCTAGTACAAATAAGGAGAGAGTAATGTCACCCTCAGCTGTTAAGGCTAATGGACACACATCAGCATTTCATCCTGCACAGCACTGGACGTCTCCAGCTAATACAACAGGAAAAGAAAAGACTGATGAAGTGGCTAACAATGCAGCAAAGAGGGCTCAGCCTGGTGAAGTACAGAGCAACCTCGTACAACACCCTCGCCCAATACTTCATTATGTTCATTTCGATGTGTCACGTGAGAATGGTGGATCCGGGGCCCCTCAATGTGGTTCATCCAATGTATTTGATCCTCCTGTCGAAGGTCATGCTGCCAACTATGGTGTCAATGGAAGCAACTCAGGCAGTAACAATGGAAGCAATGGGCAGAATGGGAGTACGACTGCTGTAAATGCTGAACGGCCAAATATGGAGATCGCTAATGGCACCATCAACAAAAGTGGACCTGGAGGTGGCAATGGAAGTGGAAGCGGCAGTGGCAATGACATGTATCTGAAACGCTTCACTCAACGAGAGCATAGAGTGGCTGCAGTGATCAAGTTTAGACAGAAAAGGAAAGAGCGCAACTTCGGAAAAAAGGTGCGGTACCAGAGCAGAAAGAGGCTGGCCGAGCAGCGGCCAAGGGTCCGCGGACAGTTCGTGCGGCAAGCTGTGCAAGACCAACAACAGCAGGGTGGTGGGCGCGAAGCGGCAGCGGACAGATGAATGATGGGAACCGCTCATCACAACCAAACCGCCGGCTCTGCCCTCGGAGTCGGAGTCGGAGATGCCAACGACGCCGTGCCTGGGGCTGGGGGTGGGGGCTACAGCGACCCGGATGGCGGACCAATCTCCGGTGTGCAGCGGCCACCGCAGGTCTGCTGGGAGCGCTTCATCCAGAAGAAGACTATCAAAGTCTTGCTAGTT GATAGCGATGACTCCACCAGGCAGGTGGTCAGTGCCCTGCTTCGTCACTGCATGTATGAAGTCATCCCTGCTGAAAATGGCCAGCAAGCATGGACATATCTAGAAGATATGCAAAACAGCATTGATCTTGTTTTGACAGAGGTTGTTATGCCTGGTGTTCTATTGAGTAGGATCATGAACCACAATATTTGCAAGAATATTCCAGTGATTATGATGT CTTCAAATGATGCTATGGGTACAGTTTTTAAGTGTTTGTCAAAGGGCGCTGTTGACTTCTTAGTCAAGCCCATACGTAAGAATGAACTTAAGAACCTATGGCAGCATGTGTGGAGACGGTGCCACAGTCCAGTGGCAGTGGAAGTGAAAGTGGCATTCAGACACAAAGTGTCAAATCAAAAAGTGGGGATGAATCCAATAATAACAATGGCAGCAAT GACGATGATGACGACGATGGTGTAATCATGGGACTTAATGCAAGAGATGGCAGTGATAACGGCAGTGGCACTCAAGCGCAGAGCTCATGGACAAAGCGCGCTGTTGAGATTGACAGTCCACAGGCTATGTCTCCAAGATCAATTAGCTGATCCACCTGATAGCACTTGTGCAAGTGATCCACCTGAAGTCAGATATGCAGCAATAGATGGTTACCATG TACAAGCAACAAAAATTCCAAGAAACAAAAAGAAACTAATGATGACTTCAAGGGGAAGGACTTGGAAATAGGTTCCTAGAAATTTAAACACAGCTTATCAATCCTCTCCGAATGAGAGATCCATCAAACCAACAGATAGACGGAATGAATATCACTGCAAAACAATTCAAAGGAGGCAGCGATGGAAAATCTGGAGGAGTCCAAGTGTTCGAGCTGCTGACTTAAT TGGTTCGATGGCCAAAAACATGGATGCACAACAGGCAGCAAGAGCCGCAAATGCCCCTAATTGCTCCTCAAAGTGCCAGAAGGGAAAGATAAGAACCGTGATAATATTATGCCATCACTTGAATTAAGTTTGAAAAGGTCAAGATCGACTGGGGATGGTGCAAACGCAATCCAAGAGGAACAACGGAATGTTTTGAGACGATCAGATCTCTCGGCATTTAC GAGGTACCATACCACCTGTGGCTTCCAATCAAGGTGGGACAGGATTCATGGGAAGCTGTTCGCTGCATGATAATAGCTCAGAGGCTATGAAAACGGATTCTGCTTACAACATGAAGTCAAACTCAGATGCTGCACCAATAAAACAAGGTTCTAATGGTAGTAGCAATAACAATGACATGGGTTCCACTACAAAGAACGTTGTGACAAAGCCTAGTACAAATAAG GAGAGAGTAATGTCACCCTCAGCTGTTAAGGCTAATGGACACACATCAGCATTTCATCCTGCACAGCACTGGACGTCTCCAGCTAATACAACAGGAAAAGAAAGACTGATGAAGTGGCTAACAATGCAGCAAAGAGGGCTCAGCTGGTGAAGTACAGAGCAACCTCGTACAACACCCTCGCCCAATACTTCATTATGTTCATTTCGATGTGTCACGTGAGAATG GTGGATCCGGGGCCCCTCAATGTGGTTCATCCAATGTATTTGATCCTCCTGTCGAAGGTCATGCTGCCAACTATGGTGTCAATGGAAGCAACTCAGGCAGTAACAATGGAAGCAATGGGCAGAATGGGAGTACGACTGCTGTAAATGCTGAACGGCCAAATATGGAGATCGCTAATGGCACCATCAAAAAGTGGACCTGGAGGTGGCAAT GGAAGTGGAAGCGGCAGTGGCAATGACATGTATCTGAAACGCTTCACTCAACGAGAGCATAGAGTGGCTGCAGTGATCAAGTTTAGACAGAAAAGGAAAGAGCGCAACTTCGGAAAAAAGGTGCGGTACCAGAGCAAGAGGCTGGCCGAGCAGCGGCCAAGGGTCCGCGGACAGTTCGTGCGGCAAGCTGTGCAAGACCAACAACAGCAGGGTG GTGGGCGCGAAGCGGCAGCGGACAGATGA

序列3sequence 3

MMGTAHHNQTAGSALGVGVGDANDAVPGAGGGGYSDPDGGPISGVQRPPQVCWERFIQKKTIKVLLVDSDDSTRQVVSALLRHCMYEVIPAENGQQAWTYLEDMQNSIDLVLTEVVMPGVSGISLLSRIMNHNICKNIPVIMMSSNDAMGTVFKCLSKGAVDFLVKPIRKNELKNLWQHVWRRCHSSSGSGSESGIQTQKCAKSKSGDESNNNNGSNDDDDDDGVIMGLNARDGSDNGSGTQAQSSWTKRAVEIDSPQAMSPDQLADPPDSTCAQVIHLKSDICSNRWLPCTSNKNSKKQKETNDDFKGKDLEIGSPRNLNTAYQSSPNERSIKPTDRRNEYPLQNNSKEAAMENLEESSVRAADLIGSMAKNMDAQQAARAANAPNCSSKVPEGKDKNRDNIMPSLELSLKRSRSTGDGANAIQEEQRNVLRRSDLSAFTRYHTPVASNQGGTGFMGSCSLHDNSSEAMKTDSAYNMKSNSDAAPIKQGSNGSSNNNDMGSTTKNVVTKPSTNKERVMSPSAVKANGHTSAFHPAQHWTSPANTTGKEKTDEVANNAAKRAQPGEVQSNLVQHPRPILHYVHFDVSRENGGSGAPQCGSSNVFDPPVEGHAANYGVNGSNSGSNNGSNGQNGSTTAVNAERPNMEIANGTINKSGPGGGNGSGSGSGNDMYLKRFTQREHRVAAVIKFRQKRKERNFGKKVRYQSRKRLAEQRPRVRGQFVRQAVQDQQQQGGGREAAADRMMGTAHHNQTAGSALGVGVGDANDAVPGAGGGGYSDPDGGPISGVQRPPQVCWERFIQKKTIKVLLVDSDDSTRQVVSALLRHCMYEVIPAENGQQAWTYLEDMQNSIDLVLTEVVMPGVSGISLLSRIMNHNICKNIPVIMMSSNDAMGTVFKCLSKGAVDFLVKPIRKNELKNLWQHV WRRCHSSSGSGSESGIQTQKCAKSKSGDESNNNNGSNDDDDDGVIMGLNARDGSDNGSGTQAQSSWTKRAVEIDSPQAMSPDQLADPPDSTCAQVIHLKSDICSNRWLPCTSNKNSKKQKETNDDFKGKDLEIGSPRNLNTAYQSSPNERSIKPTDRRNEYPLQNNSKEAAMENLEESSVRADLIGSMAK NMDAQQAARAANAPNCSSKVPEGKDKNRDNIMPSLELSLKRSRSTGDGANAIQEEQRNVLRRSDLSAFTRYHTPVASNQGGTGFMGSCSLHDNSSEAMKTDSAYNMKSNSDAAPIKQGSNGSSNNNDDMGSTTKNVVTKPSTNKERVMSPSAVKANGHTSAFHPAQHWTSPANTTGKEKTDEVANNA AKRAQPGEVQSNLVQHPRPILHYVHFDVSRENGGSGAPQCGSSNVFDPPVEGHAANYGVNGSNSGSNNGSNGQNGSTTAVNAERPNMEIANGTINKSGPGGGNGSGSGSGNDMYLKRFTQREHRVAAVIKFRQKRKERNFGKKVRYQSRKRLAEQRPRVRGQFVRQAVQDQQQQGGGREAAAD R

序列4sequence 4

ATGGGGATGGCCAATGAGGAGTCGCCAAATTATCAGGTGAAAAAAGGCGGCCGGATTCCTCCACGTAAGGACCAAATCCATCCACAGATCGCCCCGCTCTCCTCGATCGATCATAATATGATCTCGCAATGGCCCCCCTACCTTTCCCTCATCCCCAACTTGCCCTGTCTTCTTCTTCTTCTTCTTCTTGTACCTATATTATTACAAGTCATCGATCTCGCTGATCGATCAGTGATCACAAGCATTTCACAACCCTAGCTAGCTGAGCTGATCGAGCTCAAGTGACCTCACCTGCTATAGCTAACTTACTAGCTAGCTCTAGCTAGTTGTTGTTTGTAGCTCGATCGAGTTTGATTTATCCGTTCATGTCGATGGGACCAGCAGCCGGAGAAGGATGTGGCCTGTGCGGCGCCGACGGTGGCGGCTGTTGCTCCCGCCATCGCCACGATGATGATGGATTCCCCTTCGTCTTCCCGCCGAGTGCGTGCCAGGGGATCGGCGCCCCGGCGCCACCGGTGCACGAGTTCCAGTTCTTCGGCAACGACGGCGGCGGCGACGACGGCGAGAGCGTGGCCTGGCTGTTCGATGACTACCCGCCGCCGTCGCCCGTTGCTGCCGCCGCCGGGATGCATCATCGGCAGCCGCCGTACGACGGCGTCGTGGCGCCGCCGTCGCTGTTCAGGAGGAACACCGGCGCCGGCGGGCTCACGTTCGACGTCTCCCTCGGCGAACGGCCCGACCTGGACGCCGGGCTCGGCCTCGGCGGCGGCGGCGGCCGGCACGCCGAGGCCGCGGCCAGCGCCACCATCGTGAGTATCAATCCAATAATCCTGATCCGGCCGGCATGATCGGCTCGATTGAGCCGTGTCGATTATTAATTTCCATCTTATATATTATTAATTGATGAATTCTTGATTGATTCATCGATCCTCCTCGTCTTTTCTTGGCTTCTTTGTTTTTGTTATTTAGTCAAAAACAACTCTTCATTTCTGCTGCCTATATGCCGTACAACTTCAAACTATCAAAGGTCAAATAATCGATCAATATATACCAAGTTTGAATTAATTTGGAGCTTAATTAATTAATTACTGGCTTGCAGCAGCTGGTTTATAGTATTGTTTCTAGCTATATATGTGAGGGCCGTGTGTGGGATGTGATTTGCATCTTTCGATGGCGACTTAATTAATTCGATGATATATTTCATTGCATATGCATACGGATCCAGCCTCTGTCTATACTGTACGATTCCACATACGTATATGTACGGTTAAGTCAGTATATATACTTTTAGATAGTCGCGTGTGCTTTTCGAGTTCGGTAGCTATATTTTAGATTGTAAAAACAAGTCAGAGGCTAATTTTATAATCTAGAAATACTTATTTCCCCATATATAAGCGTATGTTAAATATTGATGGTGTAATCTACTTATATGTCAGGAAACATCATTGCTTGCTTTCTGGCGCTTTCTTCTACATATCAGTAGAGGAAAATGGAAAAAAAAAGATGAATTTTGATGTTGTAGTTTGCTATATTCAGCATATATACCATCAGTTATACATATGCAGATCTTGCTAAAACCAAAATAAAAATAGAACTGTAAGGAGATATTGTGCTTCTCGGTCTATTTACTTACAGTTTGTTGAGAAGTAATACGAGCAAGCAAATGTATATATATATTTCTTTAGAACTGCAAGGAGATGCATATACATGTGTGATTCAAACACACGTACTGCACATTCAAACTATAAAAACAACTTGATTGCCGTAGAAGTTAAAAGGGAGACATATCCATGGGTTTCGGATTCTAAATCAATCTATGTGTAAATGAAACTTTAGTATAGTAGGAAATAGGTTTTCAAAAAAAAAAGTATAGTAGGAAATAGTATGTGTATATGCCTTTTTAACCCTTAATTACAAGTTGTTATAATTCAGTGTTAACAAAGTCACGGACTCACAGAGTGTGCCCTTACACAATTTCAGACTAATTTGTAAATGCATCGATCGTCACATTTTATGTGGTTCAATTATCTGACACAGTTAATTAATGGTGGCCGATCGATGTATGCTCTTCTAGCTTTCCAGCTATATGCGTATGTAATAAATGAATAAAACGTGTAGGATGAAATGTGAATACGCATCATTGTAATTAATTTGATTAATGCTAGTAAAAAATCTGCAAATTTGTCTTTTTGAAATTAAAATATGCCTTATAAAATTAATGGACCCAGGCCCCTAGGGCAAAATATATTGGGGCACAAAATCATGTCCATATATACATTCTTATTTGAAAGTAGACTCTGAAACAAAATATGCCCATATAAATCAAGGGAGGTTACAACTAACTGCATTTGCTTATGCGTACATCTGGATTGTAACTTCTATGTTTTGTACATACGATGATTAATTGTATTCGAGCTTCTTAATTGTACATCTATTAACTAACTAGTTTTGCAGATGTCATATTGTGGGAGCACGTTCACTGACGCAGCGAGCTCGATGCCCAAGGAGATGGTGGCCGCCATGGCCGATGATGGGGAGAGCTTGAACCCAAACACGGTGGTTGGCGCAATGGTGGAGAGGGAGGCCAAGCTGATGAGGTACAAGGAGAAGAGGAAGAAGAGGTGCTACGAGAAGCAAATCCGGTACGCGTCCAGAAAAGCCTATGCCGAGATGAGGCCCCGAGTGAGAGGTCGCTTCGCCAAAGAACCTGATCAGGAAGCTGTCGCACCGCCATCCACCTATGTCGATCCTAGTAGGCTTGAGCTTGGACAATGGTTCAGATAGATGGGGATGGCCAATGAGGAGTCGCCAAATTATCAGGTGAAAAAAGGCGGCCGGATTCCTCCACGTAAGGACCAAATCCATCCACAGATCGCCCCGCTCTCCTCGATCGATCATAATATGATCTCGCAATGGCCCCCCTACCTTTCCCTCATCCCCAACTTGCCCTGTCTTCTTCTTCTTCTTCTTGTACCTATATTATTACAAGTCATCGATCTCG CTGATCGATCAGTGATCACAAGCATTTCACAACCCTAGCTAGCTGAGCTGATCGAGCTCAAGTGACCTCACCTGCTATAGCTAACTTACTAGCTAGCTCTAGCTAGTTGTTGTTTGTAGCTCGATCGAGTTTGATTTATCCGTTCATGTCGATGGGACCAGCAGCCGGAGAAGGATGTGGCCTGTGCGGCGCCGACGGTGGCGGCTGTTGCTCCCGCCATCG CCACGATGATGATGGATTCCCCTTCGTCTTCCCGCCGAGTGCGTGCCAGGGGATCGGCGCCCCGGCGCCACCGGTGCACGAGTTCCAGTTCTTCGGCAACGACGGCGGCGGCGACGACGGCGAGAGCGTGGCCTGGCTGTTCGATGACTACCCGCCGCCGTCGCCCGTTGCTGCCGCCGCCGGGATGCATCATCGGCAGCCGCCGT ACGACGGCGTCGTGGCGCCGCCGTCGCTGTTCAGGAGGAACACCGGCGCCGGCGGGCTCACGTTCGACGTCTCCCTCGGCGAACGGCCCGACCTGGACGCCGGGCTCGGCCTCGGCGGCGGCGGCGGCCGCCACGCCGAGGCCGCGGCCAGCGCCACCATCGTGAGTATCAATCCAATAATCCTGATCCGGCCGGCATGATC GGCTCGATTGAGCCGTGTCGATTATTAATTTCCATCTTATATATTATTAATTGATGAATTCTTGATTGATTCATCGATCCTCCTCGTCTTTTCTTGGCTTCTTTGTTTTTGTTATTTTAGTCAAAAAAACTCTTCATTTCTGCTGCCTATATGCCGTACAACTTCAAACTATCAAAAGGTCAAATAATCGATCAATATATACCAAGTTTGAATTAATTTGGAGCTTAATTAATTA ATTACTGGCTTGCAGCAGCTGGTTTATAGTATTGTTTCTAGCTATATGTGAGGGCCGTGTGTGGGATGTGATTTGCATCTTTCGATGGCGACTTAATTAATTCGATGATATATTTCATTGCATATGCATACGGATCCAGCCTCTGTCTATACTGTACGATTCCACATACGTATATGTACGGTTAAGTCAGTATATATACTTTTAGATAGTCGCGTGTGCTTTTC GAGTTCGGTAGCTATATTTTTAGATTGTAAAAACAAGTCAGAGGCTAATTTATAATCTAGAAATACTTATTTCCCCCATATAAGCGTATGTTAAATATTGATGGTGTAATCTACTTATATGTCAGGAAACATCATTGCTTGCTTTCTGGCGCTTTCTTCTACATATCAGTAGAGGAAAATGGAAAAAAAAAAGATGAATTTTGATGTTGTAGTTTGCTATATTCAGCATAT ATACCATCAGTTATACATATGCAGATCTTGCTAAAACCAAAATAAAAATAGAACTGTAAGGAGATATTGTGCTTCTCGGTCTATTACTTACAGTTTGTTGAGAAGTAATACGAGCAAGCAAATGTATATATATTTCTTTAGAACTGCAAGGAGATGCATATACATGTGTGATTCAAACACACGTACTGCACATTCAAACTATAAAAACAACTTGATTGCCGTAGAAGTTAAA AGGGAGACATATCCATGGGTTTCGGATTCTAAATCAATCTATGTGTAAATGAAACTTTAGTATAGTAGGAAATAGGTTTTCAAAAAAAAAAGTATAGTAGGAAATAGTATGTGTATATGCCTTTTTAACCCTTAATTACAAGTTGTTATAATTCAGTGTTAACAAAGTCACGGACTCACAGGTGTGCCCTTACACAATTTCAGACTAATTTGTAAATGCATCGATCGTCACATT TTATGTGGTTCAATTATCTGACACAGTTAATTAATGGTGGCCGATCGATGTATGCTCTTCTAGCTTTCCAGCTATATGCGTATGTAATAAATGAATAAAACGTGTAGGATGAAATGTGAATACGCATCATTGTAATTAATTTGATTAATGCTAGTAAAAAATCTGCAAATTTGTCTTTTTGAAATTAAAATATGCCTTATAAAATTAATGGACCCAGGCCCCTAGGGCAA AATATATTGGGGCACAAAATCATGTCCATATATACATTCTTATTTGAAAGTAGACTCTGAAACAAAATATGCCCATAAATCAAGGGAGGTTACAACTAACTGCATTTGCTTATGCGTACATCTGGATTGTAACTTCTATGTTTTGTAACATACGATGATTAATTGTATTCGAGCTTCTTAATTGTACATCTAACTAACTAGTTTTGCAGATGTCATATTGTGGGAGC ACGTTCACTGACGCAGCGAGCTCGATGCCCAAGGAGATGGTGGCCGCCATGGCCGATGATGGGGAGAGCTTGAACCCAAACACGGTGGTTGGCGCAATGGTGGAGAGGGAGGCCAAGCTGATGAGGTACAAGGAGAAGAGGAAGAAGAGGTGCTACGAGAAGCAAATCCGGTACGCGTCCAGAAAAGCCTATGCCGAGATGAGGCCCC GAGTGAGAGGTCGCTTCGCCAAAGAACCTGATCAGGAAGCTGTCGCACCGCCATCCACCTATGTCGATCCTAGTAGGCTTGAGCTTGGACAATGGTTCAGATAG

序列5sequence 5

ATGGGGATGGCCAATGAGGAGTCGCCAAATTATCAGGTGAAAAAAGGCGGCCGGATTCCTCCACCTCGATCGAGTTTGATTTATCCGTTCATGTCGATGGGACCAGCAGCCGGAGAAGGATGTGGCCTGTGCGGCGCCGACGGTGGCGGCTGTTGCTCCCGCCATCGCCACGATGATGATGGATTCCCCTTCGTCTTCCCGCCGAGTGCGTGCCAGGGGATCGGCGCCCCGGCGCCACCGGTGCACGAGTTCCAGTTCTTCGGCAACGACGGCGGCGGCGACGACGGCGAGAGCGTGGCCTGGCTGTTCGATGACTACCCGCCGCCGTCGCCCGTTGCTGCCGCCGCCGGGATGCATCATCGGCAGCCGCCGTACGACGGCGTCGTGGCGCCGCCGTCGCTGTTCAGGAGGAACACCGGCGCCGGCGGGCTCACGTTCGACGTCTCCCTCGGCGAACGGCCCGACCTGGACGCCGGGCTCGGCCTCGGCGGCGGCGGCGGCCGGCACGCCGAGGCCGCGGCCAGCGCCACCATCATGTCATATTGTGGGAGCACGTTCACTGACGCAGCGAGCTCGATGCCCAAGGAGATGGTGGCCGCCATGGCCGATGATGGGGAGAGCTTGAACCCAAACACGGTGGTTGGCGCAATGGTGGAGAGGGAGGCCAAGCTGATGAGGTACAAGGAGAAGAGGAAGAAGAGGTGCTACGAGAAGCAAATCCGGTACGCGTCCAGAAAAGCCTATGCCGAGATGAGGCCCCGAGTGAGAGGTCGCTTCGCCAAAGAACCTGATCAGGAAGCTGTCGCACCGCCATCCACCTATGTCGATCCTAGTAGGCTTGAGCTTGGACAATGGTTCAGATAGATGGGGATGGCCAATGAGGAGTCGCCAAATTATCAGGTGAAAAAAGGCGGCCGGATTCCTCCACCTCGATCGAGTTTGATTTATCCGTTCATGTCGATGGGACCAGCAGCCGGAGAAGGATGTGGCCTGTGCGGCGCCGACGGTGGCGCTGTTGCTCCCGCCATCGCCACGATGATGATGGATTCCCCTTCGTCTTCCCGCCGAGT GCGTGCCAGGGGATCGGCGCCCCGGCGCCACCGGTGCACGAGTTCCAGTTCTTCGGCAACGACGGCGGCGGCGACGACGGCGAGAGCGTGGCCTGGCTGTTCGATGACTACCCGCCGCCGTCGCCCGTTGCTGCCGCCGCCGGGATGCATCATCGGCAGCCGCCGTACGACGGCGTCGTGGCGCCGCCGTCGCTGTTCAGGAG GAACACCGGCGCCGGCGGGCTCACGTTCGACGTCTCCCTCGGCGAACGGCCCGACCTGGACGCCGGGCTCGGCCTCGGCGGCGGCGGCGGCCGGCACGCCGAGGCCGCGGCCAGCGCCACCATCATGTCATATTGTGGGAGCACGTTCACTGACGCAGCGAGCTCGATGCCCAAGGAGATGGTGGCCGCCATGGCCGATG ATGGGGAGAGCTTGAACCCAAACACGGTGGTTGGCGCAATGGTGGAGAGGGAGGCCAAGCTGATGAGGTACAAGGAGAAGAGGAAGAAGAGGTGCTACGAGAAGCAAATCCGGTACGCGTCCAGAAAAAGCCTATGCCGAGATGAGGCCCCGAGTGAGAGGTCGCTTCGCCAAAGAACCTGATCAGGAAGCTGTCGCACCGCCATCCACCTATGTC GATCCTAGTAGGCTTGAGCTTGGACAATGGTTCAGATAG

序列6sequence 6

MGMANEESPNYQVKKGGRIPPPRSSLIYPFMSMGPAAGEGCGLCGADGGGCCSRHRHDDDGFPFVFPPSACQGIGAPAPPVHEFQFFGNDGGGDDGESVAWLFDDYPPPSPVAAAAGMHHRQPPYDGVVAPPSLFRRNTGAGGLTFDVSLGERPDLDAGLGLGGGGGRHAEAAASATIMSYCGSTFTDAASSMPKEMVAAMADDGESLNPNTVVGAMVEREAKLMRYKEKRKKRCYEKQIRYASRKAYAEMRPRVRGRFAKEPDQEAVAPPSTYVDPSRLELGQWFR。MGMANEESPNYQVKKGGRIPPPPRSSLIYPFMSMGPAAGEGCGLCGADGGGCCSRHRHDDDGFPFVFPPSACQGIGAPAPPVHEFQFFGNDGGGDDGESVAWLFDDYPPPSPVAAAGMHHRQPPYDGVAPPSLFRRNTGAGGLTFDVSLGERPDLDAGLGLGGGGGRHAEAAASATIMSYCGSTTFDAASSMP KEMVAAMADDGESLNPNTVVGAMVEREAKLMRYKEKRKKRCYEKQIRYASRKAYAEMRPRVRGRFAKEPDQEAVAPPSTYVDPSRLEGQWFR.

Claims (10)

1. Use of a substance that modulates the activity or content of protein a and/or protein B, or a substance that modulates the expression of a gene encoding said protein a and/or a gene encoding said protein B, characterized in that: the application is any one of the following:

p1, application in regulating and controlling the maturity stage of rice or promoting the premature of rice;

p2, the application in regulating and controlling the heading date of rice or promoting the early heading of rice;

p3, application in regulating and controlling the plant height of rice or dwarf rice;

p4, application in rice breeding;

the protein A is the protein A1), A2) or A3) as follows:

a1 Amino acid sequence is protein of sequence 3 in the sequence table;

a2 A protein which is derived from A1) or has 80% or more identity with the protein shown in A1) and has the same function and is obtained by substituting and/or deleting and/or adding more than one amino acid residue into the amino acid sequence shown in A1);

a3 Fusion proteins obtained by attaching protein tags to the N-terminal or/and C-terminal of A1), A2) or A3);

the protein B is the protein B1), B2) or B3) as follows:

b1 Amino acid sequence is protein of sequence 6 in the sequence table;

b2 A protein which is derived from B1) or has 80% or more identity with the protein shown in B1) and has the same function and is obtained by substituting and/or deleting and/or adding more than one amino acid residue in the amino acid sequence shown in B1);

b3 Fusion proteins obtained by linking protein tags at the N-terminus or/and C-terminus of B1), B2) or B3).

2. The use according to claim 1, characterized in that: the protein A and the protein B are both derived from rice.

3. Use according to claim 1 or 2, characterized in that: the coding gene of the protein A is a DNA molecule shown in the following a 1) or a 2) or a 3):

a1 A coding sequence is a DNA molecule shown as a sequence 2 in a sequence table;

a2 A DNA molecule having 90% or more identity to the nucleotide sequence defined in a 1) and encoding said protein A;

a3 A DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined under a 1) or a 3) and which codes for said protein A;

the coding gene of the protein B is a DNA molecule shown in the following B1) or B2) or B3):

b1 A DNA molecule with a coding sequence shown as a sequence 5 in a sequence table;

b2 A DNA molecule which has 90% or more identity to the nucleotide sequence defined in B1) and which encodes said protein B;

b3 A DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined under B1) or B3) and which codes for said protein B.

4. A use according to any one of claims 1-3, characterized in that: the substance regulating the activity or content of the protein A and/or the protein B or the substance regulating the expression of the encoding gene of the protein A and/or the encoding gene of the protein B is any one of the following c 1) -c 4):

c1 A nucleic acid molecule that inhibits or reduces expression of the protein a encoding gene and/or the protein B encoding gene;

c2 An expression cassette comprising c 1) said nucleic acid molecule;

c3 A recombinant vector comprising c 1) said nucleic acid molecule, or a recombinant vector comprising c 2) said expression cassette;

c4 A recombinant microorganism comprising c 1) said nucleic acid molecule, or a recombinant microorganism comprising c 2) said expression cassette, or a recombinant microorganism comprising c 3) said recombinant vector.

5. The use according to any one of claims 1-4, characterized in that: c1 The nucleic acid molecule is a sgRNA or a DNA molecule expressing the sgRNA targeted to the gene encoding protein a according to any one of claims 1 to 3 and/or the gene encoding protein B according to any one of claims 1 to 3.

6. The use according to claim 5, characterized in that: the sgRNA is the sgRNA named as the sgRNA1 and the sgRNA named as the sgRNA2, the target sequence of the sgRNA1 is the 200 th-222 th site of the sequence 1, and the target sequence of the sgRNA2 is the 387 th-406 th site of the sequence 4 of the sequence table.

7. A method for the early stage of rice maturation and/or early stage of rice heading and/or shortening of rice growth comprising the early stage of rice maturation and/or early stage of rice heading and/or shortening of rice growth by inhibiting or reducing the expression level of a protein a-encoding gene according to any one of claims 1 to 3 and/or a protein B-encoding gene according to any one of claims 1 to 3 in the genome of rice.

8. The method according to claim 7, wherein: the method comprising introducing into the rice a substance that reduces the activity or amount of the protein a of claim 1 and/or the protein B of claim 1, or a substance that reduces the expression of the gene encoding the protein a of any one of claims 1 to 3 and/or the gene encoding the protein B of any one of claims 1 to 3; the substance which reduces the activity or content of protein a according to claim 1 and/or protein B according to claim 1, or which reduces the expression of the gene encoding protein a according to any one of claims 1 to 3 and/or the gene encoding protein B according to any one of claims 1 to 3 is any one of the following c 1) to c 4):

c1 A nucleic acid molecule that inhibits or reduces expression of the protein a encoding gene and/or the protein B encoding gene;

c2 An expression cassette comprising c 1) said nucleic acid molecule;

c3 A recombinant vector comprising c 1) said nucleic acid molecule, or a recombinant vector comprising c 2) said expression cassette;

c4 A recombinant microorganism comprising c 1) said nucleic acid molecule, or a recombinant microorganism comprising c 2) said expression cassette, or a recombinant microorganism comprising c 3) said recombinant vector.

9. The method according to claim 8, wherein: c1 The nucleic acid molecule is a sgRNA or a DNA molecule expressing the sgRNA targeted to the gene encoding protein a according to any one of claims 1 to 3 and/or the gene encoding protein B according to any one of claims 1 to 3.

10. The method according to claim 7, wherein: the inhibiting or reducing expression level of the protein A coding gene of any one of claims 1-3 and/or the protein B coding gene of any one of claims 1-3 in rice genome is to replace the protein A coding gene shown in sequence 1 in rice genome with hd2-1 gene and replace the protein B coding gene shown in sequence 4 with hd4-1 gene, wherein the hd2-1 gene is a DNA molecule obtained by inserting nucleotide T between 216 th and 217 th positions of sequence 1 in a sequence table, and keeping other nucleotides of the sequence 1 in the sequence table unchanged; the hd4-1 gene is a DNA molecule obtained by inserting a nucleotide T between the 403 th position and the 404 th position of a sequence 4 in a sequence table and keeping other nucleotides of the sequence 4 in the sequence table unchanged.

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