CN106701818B - Method for cultivating common genic male sterile line of rice - Google Patents

Abstract

Translated fromChinese

本发明公开了一种培育水稻普通核不育系的方法，包括以下步骤：利用CRISPR/Cas9系统，根据水稻普通核不育基因设计靶位点序列；构建含靶位点序列片段的pCRISPR/Cas9重组载体；将所获得的pCRISPR/Cas9重组载体导入保持系的胚性愈伤组织中得到转基因苗；筛选转基因苗中的转基因阳性植株；筛选转基因阳性植株中的突变植株；将突变植株进行繁种，于后代植株中分离不含转基因成分的普通核不育系。本发明通过编辑普通核不育基因，实现快速培育普通核不育系的目标，育种周期短、成本低且实用性强。

The invention discloses a method for cultivating a rice common nuclear sterile line, comprising the following steps: using a CRISPR/Cas9 system to design a target site sequence according to a rice common nuclear sterile gene; constructing a pCRISPR/Cas9 containing a fragment of the target site sequence Recombinant vector; introduce the obtained pCRISPR/Cas9 recombinant vector into the embryogenic callus of the maintainer line to obtain transgenic seedlings; screen the transgenic positive plants in the transgenic seedlings; screen the mutant plants in the transgenic positive plants; propagate the mutant plants , and isolate the common sterile line without transgenic components in the progeny plants. The invention achieves the goal of rapidly cultivating the common sterility line by editing the common sterility gene, and has the advantages of short breeding cycle, low cost and strong practicability.

Description

Method for cultivating common genic male sterile line of rice

Technical Field

The invention relates to the field of rice biotechnology breeding, in particular to a method for cultivating a common genic male sterile line of rice.

Background

The common genic male sterility is controlled by a pair of recessive nuclear genes, is generally not influenced by external environmental factors, and always shows sterility regardless of the change of environmental conditions. In production application, the common genic male sterility has the following advantages: 1) the varieties with normal fertility are the restorer lines of the varieties, the restoration spectrum is extremely wide, and the probability of breeding excellent combinations is greatly increased due to the free matching of the varieties; 2) can follow the breeding steps of conventional rice, open up a new field of heterosis between subspecies of indica rice and japonica rice, and realize a higher yield target of rice yield on the basis of the existing hybrid rice. Therefore, the technique of applying the common genic male sterile line to hybrid rice seed production by Yuanyongpingheishi is called third generation hybrid rice. The propagation problem of the common genic male sterile line is overcome at present, Chinese patent documents No. ZL201210426678.7 and No. ZL201210426939.5 applied by the research center of Hunan hybrid rice describe a method for constructing an engineering maintainer line by using a genetic engineering means to propagate the common genic male sterile line on a large scale, and a foundation is laid for the application of the common genic male sterile line to actual production. However, the preparation of super rice combinations with high yield, high resistance and high quality by using the common genic male sterile line has a difficult problem, and the main problem is that the breeding of the excellent common genic male sterile line can only be obtained by inducing fertile materials such as an excellent maintainer line and a restorer line by a traditional physical and chemical method or obtained by backcrossing the fertile materials and the sterile line for multiple generations. The physical and chemical mutagenesis method has randomness, low efficiency and high labor intensity; backcross breeding requires background screening through multi-generation backcross, the time span is large, even through multi-generation backcross, the original genetic background is difficult to recover, and some original excellent characters are lost and the plant types of the ordinary genic male sterile lines bred by utilizing the engineering maintainer lines are inconsistent. Therefore, the two traditional methods have great limitations, and the application of the third generation hybrid rice technology in the production and seed production is delayed. To break through the limitations of the conventional methods, genetic engineering means must be used. In recent years, genome editing techniques have been developed in a breakthrough manner, and in particular, two efficient gene editing techniques, i.e., TALEN (Transcription activator-like effector genes) and CRISPR/Cas9(clustered regularly amplified polymorphic short palindromic repeats and CRISPR associated genes), have been developed and perfected, and have been widely applied to site-directed mutagenesis of genomes of various organisms. For example, patent nos. CN201410496037.8 and CN201510009526.0 report methods for realizing the goal of rapidly breeding two-line sterile lines by using TMS5 gene and P-TMS12-1 gene which control the photo-thermo-sensitive sterile character of rice through the site-specific mutagenesis by using CRISPR/Cas9 gene editing system. At present, no method for rapidly cultivating the common genic male sterile line by adopting a CRISPR/Cas9 genetic engineering means exists.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for obtaining a common genic-sterile line by site-specific mutation of a common genic-sterile gene by using a genome editing technology, the aim of rapidly cultivating the common genic-sterile line is fulfilled by completely inactivating the common genic-sterile gene, the breeding period is short, the cost is low, the practicability is high, and the method has important significance for promoting the wide application of third-generation hybrid rice.

Therefore, the invention provides a method for cultivating a common genic male sterile line of rice, which comprises the following steps:

s1, designing a target site sequence according to the rice common genic male sterile gene by using a CRISPR/Cas9 system;

s2, constructing a pCRISPR/Cas9 recombinant vector containing the target site sequence fragment;

s3, introducing the obtained pCRISPR/Cas9 recombinant vector into embryonic callus of a maintainer line to obtain a transgenic seedling;

s4, screening transgenic positive plants in the transgenic seedlings;

s5, screening mutant plants in the transgenic positive plants;

s6, breeding the mutant plant, and separating the common genic male sterile line without transgenic components from the progeny plant.

In the above method, preferably, one strand of the target site sequence designed in step S1 has one of the following structures: 5' - (N)_n-NGG-3’、5’-(N)_n-NAG-3’、5’-(N)_n-NGA-3', said N representing any one of A, T, C and G, 14. ltoreq. n.ltoreq.30.

The method preferably further comprises the step of selecting the general genic male sterile gene as the genemsp1、pair1、pair2、zep1、mel1、pss1、tdr、udt1、gamyb4、ptc1、api5、wda1、cyp704B2、dpw、mads3、osc6、rip1、csaAndaid1one kind of (1).

The method preferably further comprises the step of selecting the general genic male sterile gene as the geneptc1The Target site sequence comprises PTC1-Target1 and PTC1-Target2, the DNA sequence of the PTC1-Target1 is a sequence shown in SEQ ID NO.1, and the DNA sequence of the PTC1-Target1 is a sequence shown in SEQ ID NO. 2.

In the above method, preferably, the step S2 specifically includes the following steps:

s2-1, designing a joint primer with a sticky end according to the target site sequence and the information of the enzyme cutting site;

s2-2, enzyme cutting of the original vector;

s2-3, annealing the adaptor primer with the sticky end and then connecting the adaptor primer to an original vector subjected to enzyme digestion to obtain a recombinant gRNA expression cassette;

s2-4, carrying out PCR amplification on the recombinant gRNA expression cassette to obtain an amplification product;

s2-5, enzyme cutting the amplification product, connecting the amplification product after enzyme cutting to the pCRISPR/Cas9 carrier after enzyme cutting to obtain the recombinant carrier.

In the above method, preferably, the linker primer in step S2-1 includes PTC1-Target1-F, PTC1-Target1-R, PTC1-Target2-F and PTC1-Target 2-R; the DNA sequence of the PTC1-Target1-F is a sequence shown by SEQ ID NO.3, the DNA sequence of the PTC1-Target1-R is a sequence shown by SEQ ID NO.4, and the DNA sequence of the PTC1-Target2-F is a sequence shown by SEQ ID NO. 5; the DNA sequence of the PTC1-Target2-R is a sequence shown in SEQ ID NO. 6.

In the method, the original vector is preferably pU3-gRNA or pU6 a-gRNA.

In the above method, preferably, in step S2-2, BsaI is used to enzyme-cut the original vector; in the step S2-3, the adapter primer with sticky end is located between two Bsa I restriction sites of the original vector to form a recombinant gRNA expression cassette; in the step S2-5, BsaI is used to enzyme-cut the amplification product.

Preferably, in the above method, the step S5 specifically includes: extracting DNA of the transgenic positive plant, and carrying out PCR amplification to obtain an amplification product; sequencing the amplification product, and selecting T with the loss-of-function mutation at any target site₀The generation homozygous mutant is used as a mutant plant.

Preferably, in the above method, step S6 specifically includes: and backcrossing the mutant plant and the maintainer line, and separating the common genic male sterile line without transgenic ingredients from the progeny plant.

Compared with the prior art, the invention has the advantages that:

(1) the invention provides a method for obtaining a common genic-sterile line by target mutation of a common genic-sterile gene based on a CRISPR/Cas9 system, the cultured common genic-sterile line does not contain transgenic components, and the method is not essentially different from the common genic-sterile line obtained by a physical-chemical mutagenesis method and a backcross breeding method, so that potential risks brought by transgenosis are avoided.

(2) The invention provides a method for culturing a common genic male sterile line of rice, which realizes the rapid culture of the common genic male sterile line, has higher efficiency and less genome damage than a physicochemical mutagenesis method; compared with the traditional backcross breeding method, the time is saved (the traditional backcross breeding method generally needs 4-6 years, but the method for the common genic male sterile line of the rice provided by the invention only needs two years), and the defects that the traditional backcross breeding method causes loss of some original excellent characters and the plant types of the common genic male sterile line bred by using the engineering maintainer line are inconsistent can be overcome.

Drawings

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.

FIG. 1 is a flow chart of the present invention for breeding a common genic male sterile line.

FIG. 2 shows the mutant types of five positive plants, in which WT is wild type 832B; PTC-M1 to PTC-M5 are 5 positive plants.

FIG. 3 is a partial result of PTC1 genotyping of individuals in BC1F2 population with PT6-F/PT6-R primer.

FIG. 4 is the comparison of the glume flower morphology of the heterozygous single plant and the single plant of the common genic male sterile line. Wherein the heterozygous single-plant anther is light yellow, contains normal pollen and shows fertile traits; the anther of the common genic male sterile line is white, normal pollen does not exist in the anther, and the anther shows sterile character.

FIG. 5 shows the result of PCR detection of hygromycin gene, where the heterozygous strain contains hygromycin gene and thus contains transgenic components; the common genic male sterile line does not contain hygromycin gene, so that the common genic male sterile line does not contain transgenic components.

Detailed Description

The invention is further described below with reference to the drawings and specific preferred embodiments of the description, without thereby limiting the scope of protection of the invention.

Examples

The materials and equipment used in the following examples are commercially available.

Example 1:

the method for rapidly breeding the common genic-sterile line is specifically applied to obtain the common genic-sterile line from the PTC1 gene (the cDNA sequence of the PTC1 gene is shown in SEQ ID No.7, and the qDNA sequence of the PTC1 gene is shown in SEQ ID No. 8) in the CRISPR/Cas9 system site-specific mutagenesis maintainer line 832B, and specifically comprises the following steps (the process flow can be seen in figure 1):

(1) designing double target sites according to the exon sequence of the PTC1 gene:

PTC1-Target1（SEQ ID NO.1）：CATGGTGGTCACCAAGTACC；

PTC1-Target2（SEQ ID NO.2）：GGCCACAAGCTGCTCAGCCT。

the two sites are respectively positioned at 886bp-905bp and 917bp-936bp of the coding region of the PTC1 gene.

PTC1-Target1 has the structure of 5 '- (N) N-NGG-3'; PTC1-Target2 has a 5 '- (N) N-NGA-3' structure.

(2) Construction of CRISPR/Cas9 recombinant vector (pCRISPR/Cas 9-PTC 1) containing PTC1-Target1 and PTC1-Target2 double targets:

2.1 synthesizing two complementary paired nucleotide single strands of the target site according to the target site sequence:

PTC1-Target1-F（SEQ ID NO.3）: ggcaCATGGTGGTCACCAAGTACC；

PTC1-Target1-R（SEQ ID NO.4）: aaacGGTACTTGGTGACCACCATG；

PTC1-Target2-F（SEQ ID NO.5）: gccGGCCACAAGCTGCTCAGCCT；

PTC1-Target2-R（SEQ ID NO.6）: aaacAGGCTGAGCAGCTTGTGGC。

2.2 construction of CRISPR/Cas9-PTC1 recombinant vector:

2.2.1 the two complementary paired single nucleotide strands in step 2.1 are mixed in equal amount, and then denatured at 90 ℃ for 3min and annealed at 20 ℃ for 5min to form a double-stranded linker with sticky ends.

2.2.2 enzyme digestion of circular pU3-gRNA and pU6a-gRNA vectors to obtain linearized pU3-gRNA and pU6a-gRNA vectors: the pU3-gRNA and pU6a-gRNA vectors were digested with 10U BsaI in a 20. mu.L reaction system for 20min, and the vector was inactivated by leaving the vector at 70 ℃ for 5min to obtain linearized pU3-gRNA and pU6agRNA vectors.

2.2.3 ligation of the double-stranded linker with sticky ends obtained in step 2.2.1 to linearized pU3-gRNA, pU6a-gRNA vectors. The connection steps are as follows: taking 1. mu.l of 10x T4 DNA ligase buffer, 0.5. mu.l of pU3-gRNA/pU6a-gRNA vector (12 ng), 1. mu.l of PTC1-Target 1/PTC 1-Target1, 1. mu. l T4 DNA ligase, and finally adding ddH₂And O to the total volume of 10 mu l, connecting for 30min at the temperature of 20-25 ℃ to obtain a recombinant guide-RNA expression cassette intermediate vector: pU3-PTC1-Target1-gRNA, pU6 a-PTC 1-Target 2-gRNA.

2.2.4 PCR amplification of expression cassettes: amplifying the pU3-PTC1-Target1-gRNA obtained in the step 2.2.3 by using primers U3-F and U3-R; the pU6 a-PTC 1-Target2-gRNA obtained in step 2.2.3 was amplified with primers U6a-F and U6 a-R. Wherein the sequences of the primers are respectively as follows:

U3-F：TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG；

U3-R：AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC；

U6a-F：TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG；

U6a-R：AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC；

the reaction system is as follows: 1 ul of pU3-PTC1-Target 1-gRNA/pU6 a-PTC 1-Target2-gRNA vector, 1.5 ul of each primer, 10 ul of dNTP, 25 ul of 2xbuffer, 1 ul of KOD enzyme, and ddH₂O to a total volume of 50. mu.L.

The reaction procedure is as follows: pre-denaturation at 95 ℃ for 3min, 35 cycles: denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 15s, and extension at 68 ℃ for 20 s.

The amplified PTC1-Target1-gRNA and PTC1-Target2-gRNA were purified using a PCR purification kit.

2.2.5 PTC1-Target1-gRNA and PTC1-Target2-gRNA are connected to pCRISPR/Cas9 vector to construct pCRISPR/Cas9-PTC1 recombinant vector. The specific construction method comprises the following steps: 1. mu.l of PTC1-Target1-gRNA and 1. mu.l of PTC1-Target2-gRNA, 1. mu.L of pCRISPR/Cas9 vector, 1. mu.L of BsaI, 1.5. mu.L of 10xSmart Buffer, 1.5. mu.L of ATP (1 mM), 1. mu. L T4 DNA ligand, and finally ddH₂O to a total volume of 15. mu.L.

The reaction system is reacted on PCR, and the reaction program is 5min at 37 ℃, 5min at 10 ℃ and 5min at 20 ℃.

(3) Adopting agrobacterium-mediated technology to integrate pCRISPR/Cas9-PTC1 recombinant vector into embryogenic callus of maintainer line 832B and obtaining 16 strains of T through callus differentiation₀Transgenic plants are generated. The method comprises the following specific steps:

3.1 the constructed pCRISPR/Cas9-PTC1 recombinant vector is introduced into Agrobacterium EHA105 to obtain EHA105 containing the pCRISPR/Cas9-PTC1 recombinant vector; and (3) inducing the callus on an induction culture medium by using the mature seeds of 832B to obtain the rice callus.

3.2 the EHA105 containing the recombinant vector pCRISPR/Cas9-PTC1 was inoculated on YM agar medium and cultured at 28 ℃ for 2 days to obtain a culture solution.

3.3 adding the collected culture solution into NB liquid medium containing 100mol/L acetosyringone to obtain bacterial solution with OD600 of 0.5.

3.4 soaking the rice callus obtained in the step 3.1 in the bacterial liquid obtained in the step 3.3 for 30min, washing with sterile water for 3 times, drying, and transferring the rice callus to an NB culture medium for dark culture at 28 ℃ for 3 days. Then transferring the rice callus to a culture medium containing 50mg/L hygromycin, placing the culture medium at 28 ℃ for dark culture, and subculturing once every 15 days for 2 times. After resistance screening, transferring the transgenic seedlings into a regeneration culture medium, and differentiating 16 transgenic seedlings. 5 transgenic positive plants are obtained by hygromycin PCR detection.

(4) Identification of mutation sites:

4.1 extracting DNA of 5 positive plants.

4.2 design detection primers according to the positions of two target sites:

PT 6-F: CCTCCGACATGATGCCCCGGTAGTCCAT and

PT6-R：GAGCTCCCCATGGTGGTCACCAAGTACCAG。

4.3 PT6-F is upstream of Target site PTC1-Target1, PT6-R is downstream of Target site PTC1-Target 2.5 positive strains of DNA are taken as a template, PT6-F and PT6-R are taken as primers to carry out PCR amplification to obtain a PCR amplification product. The reaction system of PCR amplification is as follows: mu.l of each of PCR Mix, PT6-F and PT 6-R0.2. mu.l, 1. mu.l of template, ddH₂Make up to 10. mu.l of O. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 3min, 35 cycles: denaturation at 94 ℃ for 10sAnnealing at 60 ℃ for 30s and extension at 72 ℃ for 30 s.

4.4 the PCR amplification product was sent to sequencing company for sequencing. The sequencing results are shown in FIG. 2, which represents base deletions. From FIG. 2 it is shown that two of the 5 positive plants are homozygous mutant lines, T₀Representing the sterile character, are respectively named as PTC-M1 and PTC-M2. PTC-M1 lacks 26 bases between the two Target sites, PTC-M2 lacks 1 base A at the PTC1-Target1 Target site; the other three are heterozygous fertile mutant plants which are respectively named as PTC-M3, PTC-M4 and PTC-M5; PTC-M3 lacks a base C near the PTC1-Target2 Target site; the PTC-M4 lacks two base GC near the PTC1-Target2 Target site; PTC-M5 has a deletion of one base T near the Target site of PTC1-Target 2.

(5) Selecting PTC-M1 strain from two homozygous mutant strains, backcrossing the PTC-M1 strain with 832B for 1 generation to obtain BC1F1 progeny; the progeny of BC1F1 is selfed for 1 generation to obtain 378 strains of BC1F2 progeny. Because 26 bases are deleted from the PTC1 gene of the PTC-M1 strain, when the primers PT6-F and PT6-R are used for amplifying DNA of the 832B strain and the PTC-M1 strain, the sizes of PCR products of the two strains are different by 26 bases, and the PCR product of a heterozygous single strain is different by 26 bases. Therefore, the PTC1 gene of the backcross progeny can be genotyped according to the size and the number of the amplified bands of the PT6-F and PT6-R primers, and the sterile plants, hybrid plants and fertile plants in the backcross progeny can be identified through genotyping.

The method comprises the following specific steps:

5.1 taking 832B (electrophoresis band of PCR product is larger) and sterile mutant PTC-M1 (electrophoresis band of PCR product is smaller) as contrast, respectively amplifying DNA of 378 BC1F2 offspring by PT6-F and PT6-R primers, carrying out polyacrylamide gel electrophoresis on the amplified product, and identifying sterile single plants, heterozygous fertile single plants and homozygous fertile single plants according to the size and number of electrophoresis bands. See figure 3 for results: m is 50bp marker; p1 is 832B band type, and is homozygous fertile wild type; p2 is PTC-M1 banding pattern, is homozygous sterile mutation banding pattern, and double banding is fertile heterozygosis. Therefore, among individuals No. 1-24, individuals No.1, 2, 6, 7, 15, 16 are fertile individuals,individuals 8, 9, 10, 11, 14, 18, 21, 22, 23, 24 are mutated sterile individuals, and other double-banded plants are heterozygous fertile plants.

By the method, sterile single strains 89, heterozygous fertile strains 183 and homozygous fertile strains 106 are identified from 378 strains BC1F2 progeny.

Performing field fertility character investigation at the heading and flowering stage, wherein the heterozygous single plant and the common genic male sterile line single plant have glume flower forms in thegraph 4, wherein the heterozygous single plant anther is light yellow, contains normal pollen and shows fertility characters; the anther of the common genic male sterile line is white, normal pollen does not exist in the anther, and the anther shows sterile character. The results of the fertility investigation were completely consistent with the above genotyping results.

Carrying out hygromycin gene PCR detection on 89 sterile single plants, wherein hygromycin gene PCR detection is carried out in the figure 5, and the heterozygous plants contain hygromycin gene and therefore contain transgenic components; the common genic male sterile line does not contain hygromycin gene, so that the common genic male sterile line does not contain transgenic components. As a result, 2 ordinary genic male sterile lines containing no transgenic component were selected from 89 sterile individuals.

The hygromycin gene PCR detection primer is HPT-F: GACGTCTGTCGAGAAGTTTC and HPT-R: GCTGTTATGCGGCCATTGTC, the reaction sequence is: pre-denaturation at 95 ℃ for 3min, 35 cycles: denaturation at 95 ℃ for 10s, annealing at 58 ℃ for 15s, and extension at 72 ℃ for 30 s.

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make many possible variations and modifications to the disclosed embodiments, or equivalent modifications, without departing from the spirit and scope of the invention, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.

SEQUENCE LISTING

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ctggtcgttc cagctcgagc tgcaccgcca cccccccacc gtcgtgaggc tcttcgtcgt 300

cgaggaggag gtcgccgcct cgccgcaccg ccagtgccac ctctgccgcc atattggtcc 360

gtcgaacaaa ctacaattaa tcaatcaacc tttacatagg attgatccga tcgatgccat 420

ggtgttgtag ggtgggggag gcatctgata tgcagcaaga ggtatcactt cttgctgccg 480

aggagggaat cggcggcgga agccgacggc ctgtgcttcg cgatcaacca cggcggcggc 540

ggtggcgcgg agaaagcgtc gtcgaaaggg acgacgacga cggcctccag cagaggccac 600

ctgctacacg gcgtcgtgca cctcaacggc tacggccacc tcgtcgccct ccacggcctc 660

gagggcggct ccgacttcgt ctccggccac cagatcatgg acctctggga ccgcatttgc 720

tcagccttgc acgtaaggta gtagtagtat acatgtgcgt gtgcatgcat gcaagcaatg 780

caacgatgtc gggctgcgtg tgagaacatt tgcttgggca tggtgtggtg tatgcaagga 840

cggtgagcct ggtggacacg gcgaggaagg gccacatgga gctgaggctg ctgcacggcg 900

tcgcgtacgg cgagacgtgg ttcgggcggt gggggtacag gtacggccgg ccgagctacg 960

gcgtcgcgct gccgtcgtac cggcagtcgc tgcacgtgct cggctccatg ccgctctgcg 1020

tgctggtgcc gcacctgtcg tgcttcagcc aggagctccc catggtggtc accaagtacc 1080

aggccatcag cggccacaag ctgctcagcc tcggcgacct cctccgcttc atgctcgagc 1140

tgcgcgcccg cctgccggcc acctccgtca cggccatgga ctaccggggc atcatgtcgg 1200

aggcctcgtg ccggtggtcg gcgaagcgcg tcgacatggc ggcgcgcgcc gtcgtggacg 1260

cgctccgccg cgccgagccg gcggcgcggt gggtcacgcg gcaggaggtg cgcgacgcgg 1320

cgcgcgccta catcggcgac acgggcctcc tcgacttcgt gctcaagtcc ctcggcaacc 1380

acatcgtcgg caactacgtc gtgcgccgca ccatgaaccc ggtgaccaag gtgctcgagt 1440

actgcctcga ggacgtctcc agcgtgctcc cggcggtcgc cgccggcggc ggcgtgccgg 1500

cgcagggcaa gatgagggtg aggttccagc tcacgcgtgc gcagctcatg agggacctgg 1560

tgcacctgta ccggcacgtg ctcaaggagc ccagccaggc gctcaccggc ggcgcgttcg 1620

gcgcgatccc ggtggcggtg cggatggtcc tggacatcaa gcacttcgtc aaagattacc 1680

acgaaggaca agccgcggcg agcagcaatg gcggtggcgg attcgggcat ccccacatca 1740

acctgtgctg cacgctgctc gtgagcaacg ggagcccgga gctagctcca ccgtacgaga 1800

cggtgaccct gccggcgcac gcgacggtgg gcgagctgaa gtgggaggcg cagagggtgt 1860

tcagcgagat gtacctcggc ctgaggagct tcgcggcgga ctccgtcgtc ggggtcggcg 1920

ccgaccagga gggcctcccg gtgctcgggc tggtcgacgt cggaagcgcc gtcgtggtgc 1980

aagggagcgt gggcgagcag ataaacgggg aggaccacga gaggaaggag gaggcggcgg 2040

cggcggccgt gtgcgagggg agcggcggcg gcgagcgcgt cgtggactgc gcgtgcggcg 2100

cggtggacga cgacggcgag cgcatggcgt gctgcgacat ctgcgaggcg tggcagcaca 2160

cgcggtgcgc cgggatcgcg gacaccgagg acgcgccgca cgtcttcctc tgcagccggt 2220

gcgacaacga cgtcgtgtcg ttcccgtcct tcaactgtta gatgtgatgc tgctgctgct 2280

actgctacta ctactgcctc tgctgctata tatgatgcta cctagtacaa gtgatcgaga 2340

attcaatttg ttttctcggc aaaaccaaaa tgaaaacgaa ggtaaaacca agtgaacttc 2400

agatcaa 2407

Claims (1)

1. The method for cultivating the rice common genic male sterile line is characterized by comprising the following steps:

s1, designing a target site sequence according to the rice common genic male sterile gene by using a CRISPR/Cas9 system; the common genic male sterile geneptc1The Target site sequence comprises PTC1-Target1 and PTC1-Target2, the DNA sequence of the PTC1-Target1 is a sequence shown in SEQ ID NO.1, and the DNA sequence of the PTC1-Target1 is a sequence shown in SEQ ID NO. 2;

s2, constructing a pCRISPR/Cas9 recombinant vector containing the target site sequence fragment;

s3, introducing the obtained pCRISPR/Cas9 recombinant vector into embryonic callus of a maintainer line to obtain a transgenic seedling;

s4, screening transgenic positive plants in the transgenic seedlings;

s5, extracting DNA of the transgenic positive plant, and performing PCR amplification to obtain an amplification product; sequencing the amplification product, and selecting T with the loss-of-function mutation at any target site₀The generation homozygous mutant is used as a mutant plant;

s6, backcrossing the mutant plant and the maintainer line, and separating the common genic male sterile line without transgenic components from the progeny plant;

the step S2 specifically includes the following steps:

s2-1, designing a joint primer with a sticky end according to the target site sequence and the information of the enzyme cutting site; the joint primer comprises PTC1-Target1-F, PTC1-Target1-R, PTC1-Target2-F and PTC1-Target 2-R; the DNA sequence of the PTC1-Target1-F is a sequence shown in SEQ ID NO.3, the DNA sequence of the PTC1-Target1-R is a sequence shown in SEQ ID NO.4, and the DNA sequence of the PTC1-Target2-F is a sequence shown in SEQ ID NO. 5; the DNA sequence of the PTC1-Target2-R is a sequence shown in SEQ ID NO. 6;

s2-2, adopting BsaI to carry out enzyme digestion on an original vector, wherein the original vector is pU3-gRNA or pU6 a-gRNA;

s2-3, annealing the adaptor primer with the cohesive end, and then connecting the adaptor primer with the cohesive end to an original vector subjected to enzyme digestion, wherein the adaptor primer with the cohesive end is positioned between two Bsa I enzyme digestion sites of the original vector, so that a recombinant gRNA expression cassette is obtained;

s2-4, carrying out PCR amplification on the recombinant gRNA expression cassette to obtain an amplification product;

and S2-5, adopting BsaI to enzyme-cut the amplification product, and connecting the enzyme-cut amplification product to the enzyme-cut pCRISPR/Cas9 vector to obtain the recombinant vector.

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