Gene for regulating and controlling grain shape of millet and application thereof

Abstract

The invention provides a gene for regulating and controlling the grain shape of millet and application of the gene, and belongs to the technical field of molecular breeding. The nucleotide sequence of the gene gl1 provided by the invention is shown as SEQ ID NO.1 or SEQ ID NO.2; the amino acid sequence of the protein is as shown in SEQ ID NO. 4 or SEQ ID NO. 5. When the gene gl1 is expressed in a millet plant, the length of the millet grains can be increased, and the width of the millet grains can be reduced. The gene gl1 provided by the invention is obtained by performing point mutation on the basis of the gene GL1, after the gene GL1 is mutated into the gene gl1, the grain length of millet is longer than that of a wild type, and a new thought and a new genetic material are provided for breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular breeding, and in particular relates to a gene for regulating millet grain shape and application thereof. Background Art

[0002] Millet (Setaria italica) is one of the important grain crops of the Poaceae family, belonging to the subfamily Panicaceae and the genus Setaria. Millet is produced in China and has a long history of cultivation. The main cultivation areas are the temperate and subtropical regions of Eurasia and the middle and upper reaches of the Yellow River in China. As a traditional food and feed crop, millet has the characteristics of rich nutrition, drought resistance, short growth cycle and high photosynthetic efficiency. Millet is rich in nutrition and high in protein, with an average content of 12.71%. The average fat content in millet is 4.28%, which is higher than that of rice and wheat. Unsaturated fatty acids account for about 85%, which is beneficial for softening blood vessels and preventing cardiovascular diseases such as atherosclerosis.

[0003] Grain shape is not only an important factor affecting yield, but also determines commercial traits. Therefore, high-quality grain shape is the basic guarantee for the development of millet yield. However, people in different regions have different preferences for the appearance of rice grains, so obtaining millets with different grain shapes has become one of the current breeding goals. Grain shape is mainly determined by the plant's own genetic mechanism, but the molecular mechanism that regulates millet is still unclear. Due to the small size and light weight of millet grains, it is difficult to discover genes that regulate millet grain shape. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a gene for regulating millet grain shape and its application; compared with wild genes, the gene can make millet grains longer, can be used in millet breeding, and provide new genetic material for breeding.

[0005] The present invention provides a gene gl1 for regulating millet grain shape, wherein the nucleotide sequence of the gene gl1 is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0006] The present invention provides a protein encoded by the gene gl1, and the amino acid sequence of the protein is shown in SEQ ID NO.4 or SEQ ID NO.5.

[0007] The present invention provides biological materials for expressing the gene gl1, and the biological materials include recombinant DNA, expression cassette, recombinant plasmid, recombinant bacteria or recombinant cells.

[0008] The present invention provides the use of the gene gl1, the protein or the biological material in regulating the shape of millet grains. Expressing the gene gl1, the protein or introducing the biological material into millet plants can increase the length and reduce the width of millet grains.

[0009] The present invention provides the application of the gene gl1, the protein or the biological material in millet breeding. Expressing the gene gl1, the protein or introducing the biological material into millet plants can increase the length of millet grains.

[0010] The invention provides a method for increasing millet grain length, which comprises expressing the gene gl1 in millet plants.

[0011] Preferably, the gene gl1 is expressed in millet plants by Agrobacterium-mediated genetic transformation.

[0012] Preferably, the variety of the millet plant is Jingu 21.

[0013] Compared with the prior art, the present invention has the following beneficial effects: the gene gl1 for regulating millet grain shape provided by the present invention is obtained by point mutation on the basis of gene GL1. After gene GL1 mutates to gene gl1, the millet grain traits become longer than the wild type. The present invention provides new ideas and genetic materials for breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Comparison of the grain length of millet with gl1 mutation and wild type millet Figure 1 , scale bar = 2000 μm;

[0015] Figure 2 Comparison of the grain length of millet with gl1 mutation and wild type millet Figure 2 , scale bar = 2000 μm. DETAILED DESCRIPTION

[0016] The present invention provides a gene gl1 for regulating millet grain shape, wherein the nucleotide sequence of the gene gl1 is shown in SEQ ID NO.1 or SEQ ID NO.2. In the present invention, the nucleotide sequence of one chain of the gene gl1 is shown in SEQ ID NO.1, and the nucleotide sequence of the other chain is shown in SEQ ID NO.2, which is specifically as follows:

[0017] SEQ ID NO.1:

[0018] C CCGAGGCCGGTACTAGTCATGCTGGGTCGGATGTTACAGCAGAGGCCATTGCTGCCAATTCAGCCGCCTCAGTTGGGTCTAGCATCGCTTCTGGCAGTATTCCTGATAGCTTCGAGGAACAGATGATGCTGGCCATGGCTCTTTCGTTGGTCGATGCCCGTACGAGGGCAAATTCTCCGGGGTTAGCTTGGCGGTAG。

[0019] SEQ ID NO.2:

[0020]

[0021] In the present invention, the gene gl1 regulating the shape of millet grains is obtained by point mutation based on the gene GL1. For the convenience of distinction, in the present invention, the wild-type gene GL1 is represented by uppercase, and the mutated gene gl1 is represented by lowercase. The bold and underlined site in SEQ ID NO.1 is the mutation site, which is missing in SEQ ID NO.2; the nucleotide sequence of the wild-type gene GL1 is shown in SEQ ID NO3, which is as follows:

[0022]

[0023] Among them, "TCAGTGGTCAGAGATCGCCG" is a gene editing target. In the present invention, the design of the gene editing target is preferably used targetDesign-scau.edu.cn The target design was performed by referring to the website (http: / / skl.scau.edu.cn / targetdesign / ) and the steps in the literature “A Convenient Software Toolkit for CRISPR-Based GenomeEditing.”

[0024] In the present invention, preferably, the mutant material is obtained by using Crispr / Cas9 technology and millet genetic transformation technology. The transgenic material is obtained by referring to the genetic transformation steps in the article "Amini foxtail millet with an Arabidopsis-like life cycle as a C4 model system".

[0025] The present invention provides a protein encoded by the gene gl1, the amino acid sequence of the protein is shown in SEQ ID NO.4 or SEQ ID NO.5, specifically as follows:

[0026] SEQ ID NO.4:

[0027] MGNRVGGRRRRPAVEERYTRPQGLYPHPDIDLRKLRRLILEAKLAPCHPGADDPRADLDECPICFLFYPSLNRSKCCAKGICTECFLQMKSPTSCRPTQCPYC KTLNYAVEYRGVKTKEEKGIEQLEEQRVIEAQIRMRQQELQEDAERMKNKQTAASTDSVATAQVESCDADGTSTTVASSEQGNDAVSSQVQHSELLLRNSEAFK QMRGNNFDMDLEEVMLMEAIWLSIQDQEALGNPGCVATTPPSIQRPYDGSMTTTAEAASSGGFACAVAALAEQQHIHGESSSTPTCQTTRFDTLSRSDRSYTEDLSIVGSSSSDSRVEEPSSSGTHRTVEGAESSNDQWSEIPEAGTSHAGSDVTAEAIAANSAASVGSSIASGSIPDSFEEQMMLAMALSLVDARTRANSPGLAWR.

[0028] SEQ ID NO.5:

[0029] MGNRVGGRRRRPAVEERYTRPQGLYPHPDIDLRKLRRLILEAKLAPCHPGADDPRADLDECPICFLFYPSLNRSKCCAKGICTECFLQMKSPTSCRPTQCPYCKTLNYAVEYRGVKTKEEKGIEQLEEQRVIEAQIRMRQQELQEDAERMKNKQTAASTDSVATAQVESCDADGTSTTVASSEQGNDAVSSQVQH SELLLRNSEAFKQMRGNNFDMDLEEVMLMEAIWLSIQDQEALGNPGCVATTPPSIQRPYDGSMTTTAEAASSGGFACAVAALAEQQHIHGESSSTPTCQTTRFDTLSRSDRSYTEDLSIVGSSSSDSRVEEPSSSGTHRTVEGAESSNDQWSEIPRPVLVMLGRMLQQRPLLPIQPPQLGLASLLAVFLIASRNR.

[0030] The wild-type amino acid sequence is shown in SEQ ID NO.6, and is as follows:

[0031] MGNRVGGRRRRPAVEERYTRPQGLYPHPDIDLRKLRRLILEAKLAPCHPGADDPRADLDECPICFLFYPSLNRSKCCAKGICTECFLQMKSPTSCRPTQCPYC KTLNYAVEYRGVKTKEEKGIEQLEEQRVIEAQIRMRQQELQEDAERMKNKQTAASTDSVATAQVESCDADGTSTTVASSEQGNDAVSSQVQHSELLLRNSEAFK QMRGNNFDMDLEEVMLMEAIWLSIQDQEALGNPGCVATTPPSIQRPYDGSMTTTAEAASSGGFACAVAALAEQQHIHGESSSTPTCQTTRFDTLSRSDRSYTEDLSIVGSSSSDSRVEEPSSSGTHRTVEGAESSNDQWSEIAEAGTSHAGSDVTAEAIAANSAASVGSSIASIPDSFEEQMMLAMALSLVDARTRANSPGLAWR.

[0032] The present invention also provides biological materials expressing the gene gl1, wherein the biological materials include but are not limited to recombinant DNA, expression cassette, recombinant plasmid, recombinant bacteria or recombinant cells.

[0033] The present invention also provides the use of the gene gl1, the protein or the biological material in regulating the shape of millet grains. Expressing the gene gl1, the protein or introducing the biological material into millet plants can increase the length and reduce the width of millet grains.

[0034] The present invention provides the application of the gene gl1, the protein or the biological material in millet breeding. Expressing the gene gl1, the protein or introducing the biological material into millet plants can increase the length of millet grains.

[0035] The present invention provides a method for increasing millet grain length, wherein the gene gl1 is expressed in millet plants. In the present invention, the gene gl1 is preferably expressed in millet plants by Agrobacterium-mediated genetic transformation. The present invention has no special limitation on the Agrobacterium-mediated genetic transformation operation, and conventional Agrobacterium-mediated genetic transformation operations in the art can be used. In the present invention, the variety of the millet plant is preferably Jingu 21.

[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] Example 1

[0038] Obtaining the sequence of mutant gene gl1

[0039] Design and utilization of gene editing targets targetDesign-scau.edu.cn The website (http: / / skl.scau.edu.cn / targetdesign / ) was used to refer to the steps in the literature "A Convenient Software Toolkit for CRISPR-Based Genome Editing." The wild-type gene sequence is shown in SEQ ID NO..3, and the gene editing target is TCAGTGGTCAGAGATCGCCG

[0040] The mutant material was obtained by using Crispr / Cas9 technology and millet genetic transformation technology. The transgenic material was obtained by referring to the genetic transformation steps in the article "Aminifoxtail millet with an Arabidopsis-like life cycle as a C4 model system".

[0041] After obtaining the transgenic gene editing material, the DNA of the sample was extracted as a template, primers were designed for the GL1 gene, and PCR amplification was performed. The PCR product was sequenced using Sanger to finally obtain the mutant gene sequence. The nucleotide sequence of one chain is shown in SEQ ID NO.1, and the nucleotide sequence of the other chain is shown in SEQ ID NO.2.

[0042] Example 2

[0043] Compared with wild-type materials, the mutant materials obtained by genetic transformation showed that the grains of the mutants were longer.

[0044] The process of obtaining mutant materials

[0045] 1) Genetically transform the wild type JG21 of millet using Agrobacterium tumefaciens-mediated genetic transformation to create millet gene-edited mutant materials. Millet genetic transformation: refer to the genetic transformation steps in the article "A mini foxtail millet with an Arabidopsis-like lifecycle as a C4 model system" to obtain transgenic materials.

[0046] The length and width of millet grains of mutant materials and wild-type materials were measured using Wanshen SG-G automatic seed copying instrument, which can automatically measure the shape parameters of each grain (including length and width). The results are shown in Table 1.

[0047] Table 1 The length and width measurement results of millet grains of mutant materials and wild-type materials (unit: mm)

[0048] Material Long mean Long Standard Deviation Wide mean Wide standard deviation gl1-1 2.581 0.495 1.491 0.219 gl1-2 2.493 0.381 1.434 0.18 Wild-type JG21-1 2.225 0.294 1.629 0.194 Wild-type JG21-2 2.204 0.279 1.601 0.188

[0049] Among them, gl1-1 and gl1-2 are two repeats, and the wild type JG21-1 and wild type JG21-2 are two repeats.

[0050] It can be seen from the above embodiments that the gene gl1 for regulating millet grain shape provided by the present invention is obtained by point mutation on the basis of gene GL1. After gene GL1 mutates to gene gl1, the millet grain traits become longer than the wild type. The present invention provides new ideas and genetic materials for breeding.

[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A gene gl1 for regulating millet grain shape, characterized in that: The nucleotide sequence of the gene gl1 is shown as SEQ ID NO.1 or SEQ ID NO.

2.

2. The protein encoded by the gene gl1 according to claim 1, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.4 or SEQ ID NO.

5.

3. A biological material expressing the gene gl1 according to claim 1, characterized in that: The biological material includes recombinant DNA, expression cassette, recombinant plasmid, recombinant bacteria or recombinant cells.

4. Use of the gene gl1 according to claim 1, the protein according to claim 2 or the biological material according to claim 3 in regulating the shape of millet grains, characterized in that: Expressing the gene gl1 described in claim 1, the protein described in claim 2, or transferring the biological material described in claim 3 into millet plants can increase the length and reduce the width of millet grains.

5. Use of the gene gl1 according to claim 1, the protein according to claim 2 or the biological material according to claim 3 in millet breeding, characterized in that: Expressing the gene gl1 described in claim 1, the protein described in claim 2, or transferring the biological material described in claim 3 into millet plants can increase the length of millet grains.

6. A method for increasing millet grain length, characterized in that: The gene gl1 of claim 1 is expressed in millet plants.

7. The method according to claim 6, characterized in that Expression of the gene gl1 in foxtail millet plants by Agrobacterium-mediated genetic transformation.

8. The method according to claim 6, characterized in that The variety of the millet plant is Jingu 21.

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