CN102234329B - Plant-type associated and/or yield associated protein, coding gene thereof, and application thereof - Google Patents
Plant-type associated and/or yield associated protein, coding gene thereof, and application thereofDownload PDFInfo
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Abstract
Translated fromChineseDescription
Translated fromChinese技术领域technical field
本发明涉及一种与株型相关和/或与产量相关蛋白及其编码基因与应用。The invention relates to a protein related to plant type and/or related to yield, its coding gene and application.
背景技术Background technique
株型(plant type)，是指与作物品种产量能力有关的一组特征或植物体在空间的排列方式，即长势长相。理想株型(Ideal plant type)亦称为理想型(Ideotype)，指由有利植株光合作用、生长发育和籽粒产量的性状所组成的理想化株型，它能最大限度地提高群体光能利用率，增加生物学产量和提高经济系数等。有关水稻株型的研究，20世纪50年代末期，日本科学家角田重三郎从他对水稻、大豆、甘蔗等的研究实践中总结出适于多肥集约栽培的品种应具有厚、小、直立且深绿色的叶片，短而坚韧的茎秆以及中等分蘖力的理想株型理论(角田重三郎.农业及园艺.1987，62(1)：25～29.)。不断改良株型也是我国水稻超高产育种的基本经验。从农家品种的高秆披叶型逐步改良为矮秆直叶型，从注重形态改良逐渐发展到形态和机能改良并重，不但改善了光能利用效率，增加了抗倒伏能力，还增强了水稻的生理功能。Plant type refers to a group of characteristics related to the yield capacity of crop varieties or the arrangement of plants in space, that is, the growth and appearance. Ideal plant type (Ideal plant type), also known as ideal type (Ideotype), refers to the ideal plant type composed of traits that are beneficial to plant photosynthesis, growth and development, and grain yield, which can maximize the use of light energy in the group , increase biological yield and improve economic coefficient, etc. Regarding the research on rice plant type, in the late 1950s, Japanese scientist Shigesaburo Kakuda concluded from his research practice on rice, soybeans, sugarcane, etc. that the varieties suitable for intensive cultivation with multiple fertilizers should have thick, small, upright and dark green The ideal plant type theory of leaves, short and tough stalks and medium tillering ability (Kakuda Shigesaburo. Agriculture and Horticulture. 1987, 62(1): 25～29.). Continuous improvement of plant type is also the basic experience of super-high-yielding rice breeding in my country. Gradually improve from the high-stalked leaf type of farm varieties to the short-stalked straight-leaf type, and gradually develop from focusing on shape improvement to both shape and function improvement, which not only improves the efficiency of light energy use, but also increases the lodging resistance of rice, and also enhances the rice's Physiological function.
水稻叶片性状是株型构成的重要因素，直接关系到叶片的光合面积和光能利用率，进而对产量产生重要影响，因此对叶型的研究是育种家、遗传学家和分子生物学家共同关注的焦点之一，而叶片的宽度和卷曲情况是其中的重要组成部分。研究表明，水稻窄叶和卷叶性状主要受质量性状基因控制。Rice leaf traits are an important factor in the composition of the plant type, which is directly related to the photosynthetic area and light energy utilization rate of the leaf, and then has an important impact on the yield. Therefore, the study of leaf type is the common concern of breeders, geneticists and molecular biologists. One of the focal points, and the width and curl of the blade is an important part of it. Studies have shown that the narrow and rolling leaf traits of rice are mainly controlled by quality trait genes.
发明内容Contents of the invention
本发明的一个目的是提供一种蛋白及其编码基因。One object of the present invention is to provide a protein and its coding gene.
本发明所提供的蛋白，是如下a)或b)的蛋白质：The protein provided by the present invention is the protein of a) or b) as follows:
a)由SEQ ID NO：2所示的氨基酸序列组成的蛋白质；a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
b)将SEQ ID NO：2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物株型相关和/或与产量相关的由a)衍生的蛋白质。b) The amino acid sequence shown in SEQ ID NO: 2 undergoes substitution and/or deletion and/or addition of one or several amino acid residues and is related to plant type and/or related to yield by a) derived protein .
所述蛋白的编码基因为如下1)、2)、3)或4)的基因：The coding gene of the protein is the following 1), 2), 3) or 4) gene:
1)其核苷酸序列是SEQ ID NO：1中自5′末端第140-1201位核苷酸所示DNA分子；1) Its nucleotide sequence is the DNA molecule shown in the 140th-1201th nucleotide from the 5' end in SEQ ID NO: 1;
2)其核苷酸序列是SEQ ID NO：1所示DNA分子；2) Its nucleotide sequence is a DNA molecule shown in SEQ ID NO: 1;
3)在严格条件下与1)或2)限定的DNA序列杂交且编码所述蛋白的DNA分子；3) a DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) and encodes the protein under stringent conditions;
4)与1)或2)限定的DNA序列具有90％以上的同源性且编码所述蛋白的DNA分子。4) A DNA molecule that has more than 90% homology with the DNA sequence defined in 1) or 2) and encodes the protein.
所述高严谨条件为在0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。The high stringency condition is to hybridize and wash the membrane at 65° C. in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.
为了使(a)中的蛋白便于纯化，可在由序列表中SEQ ID NO：2所示的氨基酸序列组成的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。In order to make the protein in (a) easy to purify, the amino-terminal or carboxy-terminal of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing can be linked with the tags shown in Table 1.
表1 标签的序列Table 1 Sequence of labels
上述(a)中的蛋白可人工合成，也可先合成其编码基因，再进行生物表达得到。上述(a)中的蛋白的编码基因可通过将SEQ ID NO：1所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。The protein in (a) above can be synthesized artificially, or its coding gene can be first synthesized and then biologically expressed. The coding gene of the protein in (a) above can be deleted by the codon of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 1, and/or carry out the missense of one or several base pairs mutation, and/or link the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.
扩增上述任一所述编码基因全长或其任意片段的引物对也属于本发明的保护范围。A pair of primers for amplifying the full length of any of the above-mentioned coding genes or any fragment thereof also falls within the protection scope of the present invention.
所述引物对中的一条引物序列如SEQ ID NO：3所示，所述引物对中的另一条引物序列如SEQ ID NO：4所示。A primer sequence in the primer pair is shown in SEQ ID NO: 3, and another primer sequence in the primer pair is shown in SEQ ID NO: 4.
含有上述任一所述编码基因的重组载体、重组菌、转基因细胞系或表达盒也属于本发明的保护范围。Recombinant vectors, recombinant bacteria, transgenic cell lines or expression cassettes containing any of the above-mentioned coding genes also belong to the protection scope of the present invention.
所述重组载体是将上述任一所述编码基因插入载体pCAMBIA2300-Actin的多克隆位点得到的。The recombinant vector is obtained by inserting any of the above-mentioned coding genes into the multiple cloning site of the vector pCAMBIA2300-Actin.
为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所用植物表达载体进行加工，如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因(GUS基因、萤光素酶基因等)、具有抗性的抗生素标记物(庆大霉素标记物、卡那霉素标记物等)或是抗化学试剂标记基因(如抗除莠剂基因)等。从转基因植物的安全性考虑，可不加任何选择性标记基因，直接以逆境筛选转化植株。In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) Genes, etc.), antibiotic resistance markers (gentamycin markers, kanamycin markers, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.
携带有本发明基因的植物表达载体可通过使用Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、微注射、电导、农杆菌介导等常规生物学方法转化植物细胞或组织，并将转化的植物细胞或组织培育成植株。The plant expression vector carrying the gene of the present invention can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conduction, Agrobacterium mediation, and transform the Plant cells or tissues grown into plants.
上述任一所述编码基因在改良植物株型和/或改良产量表型和/或提高产量中的应用也属于本发明的保护范围。The application of any of the above-mentioned coding genes in improving plant architecture and/or improving yield phenotype and/or increasing yield also belongs to the protection scope of the present invention.
上述任一所述蛋白在改良植物株型和/或改良产量表型和/或提高产量中的应用也属于本发明的保护范围。The application of any of the above-mentioned proteins in improving plant architecture and/or improving yield phenotype and/or increasing yield also falls within the protection scope of the present invention.
本发明的最后一个目的是提供一种改良植物株型和/或改良产量表型和/或提高产量的方法。A final object of the present invention is to provide a method for improving plant architecture and/or improving yield phenotype and/or increasing yield.
本发明所提供的改良植物株型和/或改良产量表型和/或提高产量的方法，为如下1)或2)或3)所述：The method for improving plant type and/or improving yield phenotype and/or increasing yield provided by the present invention is as described in 1) or 2) or 3) as follows:
1)向出发植物中导入权利要求2或3所述编码基因，得到与出发植物相比株型改良的目的植物；1) introducing the coding gene described in claim 2 or 3 into the starting plant to obtain a target plant with improved plant type compared with the starting plant;
2)向出发植物中导入权利要求2或3所述编码基因，得到与出发植物相比产量表型改良的目的植物；2) introducing the coding gene of claim 2 or 3 into the starting plant to obtain the target plant with improved yield phenotype compared with the starting plant;
3)向出发植物中导入权利要求2或3所述编码基因，得到与出发植物相比产量提高的目的植物。3) Introducing the coding gene of claim 2 or 3 into the starting plant to obtain the target plant with increased yield compared with the starting plant.
上述任一所述编码基因是通过上述任一所述重组载体导入的。Any of the above-mentioned coding genes is introduced by any of the above-mentioned recombinant vectors.
上述任一所述应用和上述任一改良植物株型方法中，所述株型改良为目的植物的植株主茎高大于出发植物、目的植物的植株叶片宽大于出发植物，和/或目的植物的植株叶片长大于出发植物；In any of the above-mentioned applications and any of the above-mentioned methods for improving plant type, the plant type improvement is that the height of the main stem of the target plant is greater than that of the starting plant, the width of the plant leaves of the target plant is greater than that of the starting plant, and/or the height of the target plant is greater than that of the starting plant. The leaves of the plant are longer than the starting plant;
所述产量表型改良为目的植物的植株主穗长大于出发植物和/或目的植物的植株主穗粒数多于出发植物。The improvement of the yield phenotype is that the length of the main panicle of the target plant is greater than that of the starting plant and/or the number of grains in the main panicle of the target plant is greater than that of the starting plant.
上述任一所述应用和上述任一改良植物株型方法中，所述出发植物为单子叶植物；所述单子叶植物为水稻；所述水稻为水稻突变体nal1。In any of the above-mentioned applications and any of the above-mentioned methods for improving plant architecture, the starting plant is a monocot; the monocot is rice; and the rice is a rice mutant nal1.
实验证明，向水稻突变体nal1中导入本发明基因后，得到的转基因植株的株型和产量表型得到明显改良，具体表现在：植株主茎高大于突变体nal1、植株叶片宽大于突变体nal1、植株叶片长大于突变体nal1、植株主穗长大于突变体nal1、植株主穗粒数多于突变体nal1。株型改良后的植株的水稻产量会提高。本发明基因在植物的遗传育种领域、提高作物产量领域将有广阔的应用前景。Experiments have proved that after the gene of the present invention is introduced into the rice mutant nal1, the plant type and yield phenotype of the obtained transgenic plants are significantly improved, specifically manifested in: the height of the main stem of the plant is greater than that of the mutant nal1, and the width of the leaves of the plant is greater than that of the mutant nal1 , the leaf length of the plant is longer than that of the mutant nal1, the length of the main panicle of the plant is larger than that of the mutant nal1, and the number of grains in the main panicle of the plant is larger than that of the mutant nal1. The rice yield of the plants with the improved plant type will increase. The gene of the invention will have broad application prospects in the fields of plant genetic breeding and crop yield improvement.
附图说明Description of drawings
图1为SUN1mu基因图位克隆的遗传图谱。Figure 1 is the genetic map of SUN1mu gene map-site clone.
图2为转基因水稻与对照植株的表型。Figure 2 shows the phenotypes of transgenic rice and control plants.
图3为转基因水稻与对照植株的叶宽和叶长。Figure 3 shows the leaf width and leaf length of transgenic rice and control plants.
图4为转基因水稻与对照植株的穗及节间的表型。Figure 4 shows the phenotypes of panicles and internodes of transgenic rice and control plants.
图5为转基因水稻与对照植株的中间部位透明及横切比较图。Fig. 5 is a comparison diagram of the transparency and cross-section of the middle part of the transgenic rice and the control plant.
具体实施方式Detailed ways
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.
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 materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
实施例1、基因的发现
在nal1(narrow leaf 1)突变体背景下，用EMS诱变的方法筛选其恢复突变体，经过3代稳定遗传，得到一个表型明显回复的突变体，命名为sun1(suppressor of nal1)。In the background of the nal1 (narrow leaf 1) mutant, EMS mutagenesis was used to screen the recovery mutant, and after three generations of stable inheritance, a mutant with an obvious phenotype recovery was obtained, which was named sun1 (suppressor of nal1).
采用图位克隆法从sun1中分离与株型相关基因SUN1mu，具体方法如下：The gene SUN1mu related to plant type was isolated from sun1 by map-based cloning method, and the specific method was as follows:
1)以sun1突变体(籼稻背景)与nal1(粳稻背景)杂交所获得的F2代分离群体为材料，从中鉴定到了1600个具有sun1突变体表型的个体进行遗传学分析，把SUN1mu基因定位到水稻第3号染色体的一个BAC克隆(AC133340)上。1) Using the F2 segregation population obtained by crossing the sun1 mutant (indica rice background) and nal1 (japonica rice background) as materials, 1600 individuals with the phenotype of the sun1 mutant were identified for genetic analysis, and the SUN1mu gene was mapped to On a BAC clone (AC133340) of rice chromosome 3.
2)利用籼稻(9311)、粳稻(日本晴)之间在该区域DNA序列的差别设计CAPS标记，进一步把目标基因定位到一个29KB的范围内(见图1，数字代表分子标记与目的基因间重组体的个数，箭头表示在cds开始第214和215个碱基间1个a的插入)，对该DNA片段进行序列分析，发现只有一个编码基因ORF1，其cDNA具有序列表中SEQID NO：1的核苷酸序列，由1353个碱基组成，其编码框为自5’端第140-第1201位碱基，编码具有序列表中SEQ ID NO：2的氨基酸残基序列的蛋白质。2) Using the difference in the DNA sequence of indica rice (9311) and japonica rice (Nipponbare) to design CAPS markers in this region, and further locate the target gene within a range of 29KB (see Figure 1, the number represents the recombination between the molecular marker and the target gene The number of individuals, the arrow indicates the insertion of 1 a between the 214th and 215th bases at the beginning of cds), the sequence analysis of this DNA fragment found that there is only one coding gene ORF1, and its cDNA has SEQID NO: 1 in the sequence table The nucleotide sequence consists of 1353 bases, and its coding frame is from the 140th to the 1201st base at the 5' end, encoding a protein with the amino acid residue sequence of SEQ ID NO: 2 in the sequence listing.
3)利用测序的方法找到基因突变。如果该基因是SUN1mu基因的候选基因，就应该在sun1突变体(籼稻背景)与nal1(籼稻背景)之间表现序列差异。具体方法为：对上述ORF进行PCR扩增并测序(检测引物序列为：5’-GATGGACTCCCCGTCGCCTAT和5’-GTGGTGGGAGCTAGAGGCTCA)，结果在sun1突变体(籼稻背景)与nal1(籼稻背景)之间表现序列差异；同时对其基因组的序列分析结果表明ORF1在sun1突变体内产生1个碱基的插入，该突变导致SUN1mu所编码的氨基酸残基序列中从第73位氨基酸残基以后发生移码突变。因此，推断该ORF1是SUN1mu的侯选基因。3) Use the method of sequencing to find the gene mutation. If this gene is a candidate gene for the SUN1mu gene, it should show sequence differences between the sun1 mutant (indica background) and nal1 (indica background). The specific method is: perform PCR amplification and sequencing on the above ORF (detection primer sequence: 5'-GATGGACTCCCCGTCGCCTAT and 5'-GTGGTGGGAGCTAGAGGCTCA), the result shows sequence differences between the sun1 mutant (indica rice background) and nal1 (indica rice background) ; At the same time, the sequence analysis of its genome showed that ORF1 produced a 1-base insertion in the sun1 mutant, which caused a frameshift mutation in the sequence of amino acid residues encoded by SUN1mu from the 73rd amino acid residue onwards. Therefore, it was inferred that this ORF1 is a candidate gene of SUN1mu.
由此，得到的基因SUN1mu的cDNA核苷酸序列如SEQ ID NO：1所示，由1353个碱基组成，自其5’端第140-1201位碱基编码蛋白，编码的蛋白的氨基酸序列为SEQ IDNO：2。Thus, the cDNA nucleotide sequence of the obtained gene SUN1mu is shown in SEQ ID NO: 1, consisting of 1353 bases, from the 140th to 1201st bases at the 5' end of the encoded protein, the amino acid sequence of the encoded protein is SEQ ID NO:2.
实施例2、基因的制备与功能验证Embodiment 2, preparation and function verification of gene
野生型水稻浙辐802(zf802)在文献(郑雷英，朱旭东，钱前，赵忠，张建军，胡筱荷，林鸿宣，罗达.2003.水稻穗部突变体C1的形态和定位分析.科学通报第48卷第3期)中公开过，由中国科学院遗传与发育生物学研究所提供。Wild type rice Zhefu 802 (zf802) in the literature (Zheng Leiying, Zhu Xudong, Qian Qian, Zhao Zhong, Zhang Jianjun, Hu Xiaohe, Lin Hongxuan, Luo Da. 2003. Morphological and localization analysis of rice panicle mutant C1. Science Bulletin Vol.48 Issue 3), provided by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.
pMD19-T载体购自takara，产品目录号为D102A。The pMD19-T vector was purchased from takara, the product catalog number is D102A.
载体pCAMBIA2300-Actin在文献(Kejian Wang，Ding Tang，Lilan Hong，WenyingXu，Jian Huang，Ming Li，Minghong Gu，Yongbiao Xue，Zhukuan Cheng.2010.DEPandAFORegulate Reproductive Habit in Rice.January 2010.Volume 6.)中公开过，由中国科学院遗传与发育生物学研究所提供。The vector pCAMBIA2300-Actin is disclosed in the literature (Kejian Wang, Ding Tang, Lilan Hong, WenyingXu, Jian Huang, Ming Li, Minghong Gu, Yongbiao Xue, Zhukuan Cheng.2010.DEPandAFORegulate Reproductive Habit in Rice.January 2010.Volume 6.) However, provided by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.
根癌农杆菌EH105在文献(赵志强，傅亚萍，杨鹍，张玉满，颜永胜，方荣祥，孙宗修，陈晓英.2008.水稻小G蛋白OsPra2基因的克隆及功能分析.Chin J Biotech2008，December 25；24(12)：2027-2033中公开过，由中国科学院遗传与发育生物学研究所提供。Agrobacterium tumefaciens EH105 in literature (Zhao Zhiqiang, Fu Yaping, Yang Kun, Zhang Yuman, Yan Yongsheng, Fang Rongxiang, Sun Zongxiu, Chen Xiaoying. 2008. Cloning and functional analysis of rice small G protein OsPra2 gene. Chin J Biotech2008, December 25; 24(12) : Published in 2027-2033, provided by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.
水稻突变体nal1(Dong Fenggao，Xiong Zhenmin，Qian Qian，Zu Xudong，Chen Shihua(1994).Breeding Near-isogenic lines of morphological markers in indica rice.Chinese J.Rice sci.8，135-139。(由中国科学院遗传与发育生物学研究所提供)。Rice mutant nal1 (Dong Fenggao, Xiong Zhenmin, Qian Qian, Zu Xudong, Chen Shihua (1994). Breeding Near-isogenic lines of morphological markers in indica rice. Chinese J.Rice sci.8, 135-139. (by China Institute of Genetics and Developmental Biology, Academy of Sciences).
一、基因制备1. Gene preparation
人工合成SEQ ID NO：1中自5’端起第140-1201位碱基所示的DNA，也可按照如下方法制备SEQ ID NO：1中自5’端起第140-1201位碱基所示的DNA。SEQ ID NO：1中自5’端起第140-1201位碱基所示的DNA编码的蛋白的氨基酸序列如SEQ ID NO：2所示。Artificially synthesize the DNA shown in base 140-1201 from the 5' end in SEQ ID NO: 1, and the DNA shown in base 140-1201 from the 5' end in SEQ ID NO: 1 can also be prepared according to the following method DNA shown. The amino acid sequence of the protein encoded by the DNA shown in base 140-1201 from the 5' end in SEQ ID NO: 1 is shown in SEQ ID NO: 2.
提取sun1突变体的总RNA，以其反转录产物为模板，用如下引物P1/P2进行PCR扩增，得到PCR扩增产物。Extract the total RNA of the sun1 mutant, use its reverse transcription product as a template, and perform PCR amplification with the following primers P1/P2 to obtain a PCR amplification product.
P1：5’-ATGGACTCCCCGTCGCCTAT(SEQ ID NO：3)；P1: 5'-ATGGACTCCCCGTCGCCTAT (SEQ ID NO: 3);
P2：5’-TCACACACGAGGAGGAATTGGA(SEQ ID NO：4)。P2: 5'-TCACACACGAGGAGGAATTGGA (SEQ ID NO: 4).
将PCR产物进行1％琼脂糖凝胶电泳检测，回收并纯化1062bp的DNA片段，与pMD-19T载体连接，将连接产物转化大肠杆菌，抗性筛选，挑取单克隆；将单克隆分别进行液体培养，提取质粒，将质粒进行测序。结果，pMD-19T载体中插入的基因序列如SEQ ID NO：1中自5’端起第140-1201位碱基所示，表明构建的重组载体正确，将其命名为pMD-19T-SUN1mu。Perform 1% agarose gel electrophoresis detection on the PCR product, recover and purify the 1062bp DNA fragment, connect it to the pMD-19T vector, transform the ligated product into Escherichia coli, screen for resistance, and pick a single clone; Cultivate, extract the plasmid, and sequence the plasmid. As a result, the gene sequence inserted in the pMD-19T vector is shown in the 140-1201 bases from the 5' end of SEQ ID NO: 1, indicating that the constructed recombinant vector is correct, and it was named pMD-19T-SUN1mu.
二、基因转化突变体nal12. Gene transformation mutant nal1
1、重组表达载体构建1. Construction of recombinant expression vector
用限制性内切酶Xba I和Sal I酶切pMD-19T- SUN1mu，回收目的基因片段；用限制性内切酶Xba I和Sal I酶切载体pCAMBIA2300-Actin，回收载体大片段；目的基因片段与载体大片段连接，得到重组载体。将重组载体转化大肠杆菌，抗性筛选，挑取单克隆；将单克隆分别进行液体培养，提取质粒，将质粒进行测序。结果，pCAMBIA2300-Actin载体的Xba I和SalI酶切位点间插入的基因序列如SEQ ID NO：1中自5’端起第140-1201位碱基所示，表明构建的重组载体正确，将其命名为pActin::SUN1-mu。Digest pMD-19T-SUN1mu with restriction endonuclease Xba I and Sal I, and recover the target gene fragment; use restriction endonuclease Xba I and Sal I to digest the vector pCAMBIA2300-Actin, and recover the large fragment of the vector; the target gene fragment Ligated with the large fragment of the vector to obtain the recombinant vector. The recombinant vector is transformed into Escherichia coli, the resistance is screened, and single clones are picked; the single clones are cultured in liquid respectively, the plasmids are extracted, and the plasmids are sequenced. As a result, the gene sequence inserted between the Xba I and SalI restriction sites of the pCAMBIA2300-Actin vector is shown in the 140th-1201th base from the 5' end in SEQ ID NO: 1, indicating that the recombinant vector constructed is correct. It was named pActin::SUN1-mu.
2、重组农杆菌构建2. Construction of recombinant Agrobacterium
用热击法将重组表达载体pActin::SUN1-mu转化根癌农杆菌EH105，筛选阳性重组子，得到含有重组表达载体pActin::SUN1-mu的根癌农杆菌EH105，记作EH105-pActin::SUN1-mu。The recombinant expression vector pActin::SUN1-mu was transformed into Agrobacterium tumefaciens EH105 by heat shock method, and positive recombinants were screened to obtain Agrobacterium tumefaciens EH105 containing the recombinant expression vector pActin::SUN1-mu, denoted as EH105-pActin: :SUN1-mu.
3、转基因植株构建3. Construction of transgenic plants
用农杆菌介导的水稻愈伤组织转化方法将pActin::SUN1-mu转化水稻突变体nal1，对转化植株进行PCR鉴定，PCR鉴定所用引物为上述P1/P2，结果得到23株阳性植株，均为T0代植株，此阳性植株即为转入基因SUN1mu的植株，记作转基因植株。Using Agrobacterium-mediated transformation of rice callus, pActin::SUN1-mu was transformed into rice mutant nal1, and the transformed plants were identified by PCR. The primers used for PCR identification were the above-mentioned P1/P2. As a result, 23 positive plants were obtained. It is the T0 generation plant, and the positive plant is the plant with the gene SUN1mu transferred, which is recorded as the transgenic plant.
同时以转入空载体pCAMBIA2300-Actin的植株作为对照，以未经任何处理的水稻突变体nal1作为对照。At the same time, the plants transformed with the empty vector pCAMBIA2300-Actin were used as the control, and the rice mutant nal1 without any treatment was used as the control.
4、转基因植株的表型分析4. Phenotype analysis of transgenic plants
观察统计转基因植株、对照植株、野生型浙辐802(简称zf802)的表型。再对植株的主茎高度、主穗长度、主穗粒数进行统计分析。实验设3次重复，结果取平均值±标准差。Observe and count the phenotypes of transgenic plants, control plants, and wild-type Zhefu 802 (zf802 for short). The height of the main stem, the length of the main panicle and the number of grains in the main panicle of the plants were statistically analyzed. The experiment was repeated three times, and the results were average ± standard deviation.
转基因植株与对照植株的表型如图2、3、4所示。图4中，每组图从左至右依次为zf802、对照植株、转基因植株。统计结果如表1所示。表明转基因植株株高变高、叶片明显变宽、颜色变浅。The phenotypes of transgenic plants and control plants are shown in Figures 2, 3 and 4. In Fig. 4, each group of pictures is zf802, control plants and transgenic plants from left to right. The statistical results are shown in Table 1. It indicated that the height of the transgenic plants became higher, the leaves became wider and the color became lighter.
结果：株型变化：转基因植株的主茎高度(82.5±3.4cm)比对照植株(72.8±5.1cm)明显变高；产量表型变化：转基因植株的主穗长度(26.3±1.9cm)比对照植株(20.7±2.3cm)明显变长，转基因植株的主穗粒数(208.1±30.1)比对照植株(114.5±30.1)明显增多。Result: Plant type change: the main stem height (82.5±3.4cm) of the transgenic plant was significantly higher than that of the control plant (72.8±5.1cm); Yield phenotype change: the main panicle length (26.3±1.9cm) of the transgenic plant was higher than that of the control The plants (20.7±2.3cm) became significantly longer, and the number of grains in the main panicle of the transgenic plants (208.1±30.1) was significantly higher than that of the control plants (114.5±30.1).
图2-4中和表1中对照植株指未经任何处理的水稻突变体nal1；转入空载体pCAMBIA2300-Actin的植株与未经任何处理的水稻突变体nal1的结果相同。The control plants in Figures 2-4 and in Table 1 refer to the rice mutant nal1 without any treatment; the results of the plants transformed with the empty vector pCAMBIA2300-Actin are the same as those of the rice mutant nal1 without any treatment.
表1、转基因植株表型统计分析Table 1. Statistical analysis of transgenic plant phenotypes
实施例3、基因作用机理Embodiment 3, gene mechanism of action
将实施例2中的转基因植株、对照植株和野生型浙辐802的叶片和明显缩短的第四节间进行石蜡切片分析。The leaves of the transgenic plants, control plants and wild type Zhefu 802 in Example 2 and the significantly shortened fourth internode were analyzed by paraffin section.
结果如表2和图5所示。转基因植株叶片(blade)中小维管束(sv)总数与对照植株相比内有明显增加，甚至超过了野生型。同时转基因植株叶片(blade)中两相邻大维管束(lv)之间的小维管束(sv)数与对照植株相比也明显增加，从而导致转基因植株明显变宽。表明SUN1mu基因可能通过调控叶片(blade)中小维管束(sv)的形成影响叶片的宽窄和大小，同时SUN1mu基因对维管束的形成方式也起到重要的调控作用。表2中对照植株指未经任何处理的水稻突变体nal1；转入空载体pCAMBIA2300-Actin的植株与未经任何处理的水稻突变体nal1的结果相同。The results are shown in Table 2 and Figure 5. Compared with the control plants, the total number of small vascular bundles (sv) in the leaves (blade) of the transgenic plants increased significantly, and even exceeded that of the wild type. At the same time, the number of small vascular bundles (sv) between two adjacent large vascular bundles (lv) in the leaves (blade) of the transgenic plants was also significantly increased compared with that of the control plants, resulting in the obvious widening of the transgenic plants. It indicated that SUN1mu gene may affect the width and size of leaves by regulating the formation of small vascular bundles (sv) in leaves (blade), and SUN1mu gene also plays an important role in regulating the formation of vascular bundles. The control plants in Table 2 refer to the rice mutant nal1 without any treatment; the plants transformed with the empty vector pCAMBIA2300-Actin had the same results as the rice mutant nal1 without any treatment.
表2、转基因植株维管束数目Table 2. Number of vascular bundles in transgenic plants
序列表sequence listing
<110>中国科学院遗传与发育生物学研究所<110> Institute of Genetics and Developmental Biology, Chinese Academy of Sciences
<120>一种与株型相关和/或与产量相关蛋白及其编码基因与应用<120> A protein related to plant type and/or related to yield, its coding gene and application
<160>4<160>4
<210>1<210>1
<211>1375<211>1375
<212>DNA<212>DNA
<213>稻属水稻(Oryza sativa)<213>Oryza sativa
<400>1<400>1
tgaccccaac cccaaaccca ctctactcta ctgtgcctca cctcttgcca ctactatttc 60tgaccccaac cccaaaccca ctctactcta ctgtgcctca cctcttgcca ctactatttc 60
tagtagtcgt gtatcatcat ttcagatatc atatcgccac ctctcgtttt tttaataata 120tagtagtcgt gtatcatcat ttcagatatc atatcgccac ctctcgtttt tttaataata 120
tcagcggcga gcgagcgaga tggactcccc gtcgcctatg gcggcgcagg cggccgacct 180tcagcggcga gcgagcgaga tggactcccc gtcgcctatg gcggcgcagg cggccgacct 180
gtcgctgacg ctggcgccgt cgggaggggg tggtggggga ggaggaggcg gcggcggtgg 240gtcgctgacg ctggcgccgt cgggagggggg tggtggggga ggaggaggcg gcggcggtgg 240
tgggtcgtcg tcggcgtgca tcgacggcaa ggacgtgcgg ctgttcccgt gcttgttctg 300tgggtcgtcg tcggcgtgca tcgacggcaa ggacgtgcgg ctgttcccgt gcttgttctg 300
caacaagaag ttcttgaagt cgcaggcgct aggcgggcac cagaacgcgc acaaagaagg 360caacaagaag ttcttgaagt cgcaggcgct aggcgggcac cagaacgcgc acaaagaagg 360
agcggagcat cgggtggaat ccctacttct acatgccgcc gacgccgcac cccgccggca 420agcggagcat cgggtggaat ccctacttct acatgccgcc gacgccgcac cccgccggca 420
atgccgccgc cgccgccgcg gcggcgacgc ccggtgggat gtcgtccgtc acgacgccgt 480atgccgccgc cgccgccgcg gcggcgacgc ccggtgggat gtcgtccgtc acgacgccgt 480
ccgggagcta cggcgtcgtc ggtggtgccg ccgccgcggc ggcggctgtc gtcggggcta 540ccgggagcta cggcgtcgtc ggtggtgccg ccgccgcggc ggcggctgtc gtcggggcta 540
ctgctggcgt tgggggcgga ggtggagtgg gaggggggct tctcccggcg cacgcgtacg 600ctgctggcgt tgggggcgga ggtggagtgg gaggggggct tctcccggcg cacgcgtacg 600
ccgggcacgg gtacgccgcg gtgccgacgt cgttccccat cgcgtcgcac agctcgagcg 660ccgggcacgg gtacgccgcg gtgccgacgt cgttccccat cgcgtcgcac agctcgagcg 660
tggttggctc cggtgggctg cagtactacg ctggtaccga ctgcggcgcg gcggcggcgg 720tggttggctc cggtgggctg cagtactacg ctggtaccga ctgcggcgcg gcggcggcgg 720
gtgcggcgaa gacgacgacg acgacggcgg cggcggcggc gacggccgtg gcggggagcg 780gtgcggcgaa gacgacgacg acgacggcgg cggcggcggc gacggccgtg gcggggagcg 780
agagcggcgt gcaggtgccc cggttcgcga cgcaccagca ccatctcctg gcggtggtga 840agagcggcgt gcaggtgccc cggttcgcga cgcaccagca ccatctcctg gcggtggtga 840
gcagcgggcg cgcgatgctg gcggcgcccg accagccggg cgccgggcgc gacgacatga 900gcagcgggcg cgcgatgctg gcggcgcccg accagccggg cgccgggcgc gacgacatga 900
tcgacatgct caactggagg cgaggctccc acggccccac cgcctccgcc gccgccacca 960tcgacatgct caactggagg cgaggctccc acggccccac cgcctccgcc gccgccacca 960
cgccctcccc ggcaagcacc accaccacgc tcaccacctt cgccagcgcc gacggcagca 1020cgccctcccc ggcaagcacc accacacgc tcaccacctt cgccagcgcc gacggcagca 1020
acaacggcga ggagaacgag gagctcgacc tcaacttgag cctctagctc ccaccaccac 1080acaacggcga ggagaacgag gagctcgacc tcaacttgag cctctagctc ccaccaccac 1080
cacctcctcc tccgccgccg ccgccgccgc cgcgcaatcc aagaaggcaa ggtcaatcaa 1140cacctcctcc tccgccgccg ccgccgccgc cgcgcaatcc aagaaggcaa ggtcaatcaa 1140
tcgccatgtt cttcttctcc aagctccacc tactcctctt ccaattcctc ctcgtgtgtg 1200tcgccatgtt cttcttctcc aagctccacc tactcctctt ccaattcctc ctcgtgtgtg 1200
attaatcccc ctcttcttgc tgcctgcgta cgtactcctt aattaattag ctcttaggga 1260attaatcccc ctcttcttgc tgcctgcgta cgtactcctt aattaattag ctcttaggga 1260
cgttaattaa tctcagttct tggctctctt ctcctctcct ctcctctcct ctcatctcac 1320cgttaattaa tctcagttct tggctctctt ctcctctcct ctcctctcct ctcatctcac 1320
ttgtatgtta atgttagtac tccttgtaat cgatcaatca gtcctctttt tttgc 1375ttgtatgtta atgttagtac tccttgtaat cgatcaatca gtcctctttt tttgc 1375
<210>2<210>2
<211>353<211>353
<212>PRT<212>PRT
<213>稻属水稻(Oryza sativa)<213>Oryza sativa
<400>2<400>2
Met Asp Ser Pro Ser Pro Met Ala Ala Gln Ala Ala Asp Leu Ser LeuMet Asp Ser Pro Ser Pro Met Ala Ala Gln Ala Ala Asp Leu Ser Leu
1 5 10 151 5 10 15
Thr Leu Ala Pro Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly GlyThr Leu Ala Pro Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
20 25 3020 25 30
Gly Gly Gly Ser Ser Ser Ala Cys Ile Asp Gly Lys Asp Val Arg LeuGly Gly Gly Ser Ser Ser Ser Ala Cys Ile Asp Gly Lys Asp Val Arg Leu
35 40 4535 40 45
Phe Pro Cys Leu Phe Cys Asn Lys Lys Phe Leu Lys Ser Gln Ala LeuPhe Pro Cys Leu Phe Cys Asn Lys Lys Phe Leu Lys Ser Gln Ala Leu
50 55 6050 55 60
Gly Gly His Gln Asn Ala His Lys Glu Gly Ala Glu His Arg Val GluGly Gly His Gln Asn Ala His Lys Glu Gly Ala Glu His Arg Val Glu
65 70 75 8065 70 75 80
Ser Leu Leu Leu His Ala Ala Asp Ala Ala Pro Arg Arg Gln Cys ArgSer Leu Leu Leu His Ala Ala Asp Ala Ala Pro Arg Arg Gln Cys Arg
85 90 9585 90 95
Arg Arg Arg Arg Gly Gly Asp Ala Arg Trp Asp Val Val Arg His AspArg Arg Arg Arg Gly Gly Asp Ala Arg Trp Asp Val Val Arg His Asp
100 105 110100 105 110
Ala Val Arg Glu Leu Arg Arg Arg Arg Trp Cys Arg Arg Arg Gly GlyAla Val Arg Glu Leu Arg Arg Arg Arg Arg Trp Cys Arg Arg Arg Gly Gly
115 120 125115 120 125
Gly Cys Arg Arg Gly Tyr Cys Trp Arg Trp Gly Arg Arg Trp Ser GlyGly Cys Arg Arg Gly Tyr Cys Trp Arg Trp Gly Arg Arg Trp Ser Gly
130 135 140130 135 140
Arg Gly Ala Ser Pro Gly Ala Arg Val Arg Arg Ala Arg Val Arg ArgArg Gly Ala Ser Pro Gly Ala Arg Val Arg Arg Ala Arg Val Arg Arg
145 150 155 160145 150 155 160
Gly Ala Asp Val Val Pro His Arg Val Ala Gln Leu Glu Arg Gly TrpGly Ala Asp Val Val Pro His Arg Val Ala Gln Leu Glu Arg Gly Trp
165 170 175165 170 175
Leu Arg Trp Ala Ala Val Leu Arg Trp Tyr Arg Leu Arg Arg Gly GlyLeu Arg Trp Ala Ala Val Leu Arg Trp Tyr Arg Leu Arg Arg Gly Gly
180 185 190180 185 190
Gly Gly Cys Gly Glu Asp Asp Asp Asp Asp Gly Gly Gly Gly Gly AspGly Gly Cys Gly Glu Asp Asp Asp Asp Asp Gly Gly Gly Gly Gly Asp
195 200 205195 200 205
Gly Arg Gly Gly Glu Arg Glu Arg Arg Ala Gly Ala Pro Val Arg AspGly Arg Gly Gly Glu Arg Glu Arg Arg Ala Gly Ala Pro Val Arg Asp
210 215 220210 215 220
Ala Pro Ala Pro Ser Pro Gly Gly Gly Glu Gln Arg Ala Arg Asp AlaAla Pro Ala Pro Ser Pro Gly Gly Gly Glu Gln Arg Ala Arg Asp Ala
225 230 235 240225 230 235 240
Gly Gly Ala Arg Pro Ala Gly Arg Arg Ala Arg Arg His Asp Arg HisGly Gly Ala Arg Pro Ala Gly Arg Arg Ala Arg Arg His Asp Arg His
245 250 255245 250 255
Ala Gln Leu Glu Ala Arg Leu Pro Arg Pro His Arg Leu Arg Arg ArgAla Gln Leu Glu Ala Arg Leu Pro Arg Pro His Arg Leu Arg Arg Arg
260 265 270260 265 270
His His Ala Leu Pro Gly Lys His His His His Ala His His Leu ArgHis His Ala Leu Pro Gly Lys His His His His Ala His His Leu Arg
275 280 285275 280 285
Gln Arg Arg Arg Gln Gln Gln Arg Arg Gly Glu Arg Gly Ala Arg ProGln Arg Arg Arg Gln Gln Gln Arg Arg Gly Glu Arg Gly Ala Arg Pro
290 295 300290 295 300
Gln Leu Glu Pro Leu Ala Pro Thr Thr Thr Thr Ser Ser Ser Ala AlaGln Leu Glu Pro Leu Ala Pro Thr Thr Thr Thr Thr Ser Ser Ser Ala Ala
305 310 315 320305 310 315 320
Ala Ala Ala Ala Ala Gln Ser Lys Lys Ala Arg Ser Ile Asn Arg HisAla Ala Ala Ala Ala Gln Ser Lys Lys Ala Arg Ser Ile Asn Arg His
325 330 335325 330 335
Val Leu Leu Leu Gln Ala Pro Pro Thr Pro Leu Pro Ile Pro Pro ArgVal Leu Leu Leu Gln Ala Pro Pro Thr Pro Leu Pro Ile Pro Pro Arg
340 345 350340 345 350
ValVal
<210>3<210>3
<211>20<211>20
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<220><220>
<223><223>
<400>3<400>3
atggactccc cgtcgcctat 20atggactccc cgtcgcctat 20
<210>4<210>4
<211>22<211>22
<212>DNA<212>DNA
<213>人工序列<213> Artificial sequence
<220><220>
<223><223>
<400>4<400>4
tcacacacga ggaggaattg ga 22tcacacacga ggaggaattg ga 22
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