A gene for regulating the grain shape of foxtail millet and its application

Abstract

The present invention provides a gene for regulating the grain shape of foxtail millet and its application, belonging to the technical field of molecular breeding. The nucleotide sequence of the gene gl1 provided by the present invention is shown in SEQ ID NO.1 or SEQ ID NO.2; the amino acid sequence of the protein is shown in SEQ ID NO.4 or SEQ ID NO.5. Expressing the gene gl1 in foxtail millet plants can increase the grain length and decrease the width of foxtail millet. The gene gl1 provided by the present invention is obtained by point mutation on the basis of the gene GL1. After the gene GL1 mutates into the gene gl1, the grain length of foxtail millet becomes longer than that of the wild type. The present invention provides new ideas and genetic materials 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 an application thereof. Background Art

[0002] Millet (Setaria italica) is a key grain crop in the Poaceae family, belonging to the subfamily Paniculoideae and the genus Setaria. Native to China, millet has a long history of cultivation, primarily cultivated in the temperate and subtropical zones of Eurasia and the middle and upper reaches of the Yellow River in China. As a traditional food and feed crop, millet boasts rich nutrition, drought and barrenness tolerance, a short growth cycle, and high photosynthetic efficiency. Millet is highly nutritious, with a high protein content (average 12.71%). The average fat content in millet is 4.28%, higher than that of rice and wheat. Unsaturated fatty acids account for approximately 85% of this content, contributing to blood vessel softening and the prevention of cardiovascular diseases such as atherosclerosis.

[0003] Grain shape is not only a key factor influencing yield but also determines commercial characteristics. Therefore, high-quality grain shape is fundamental to millet production. However, preferences for grain shape vary from region to region, making obtaining diverse grain shapes a current goal in millet breeding. Grain shape is primarily determined by the plant's own genetic mechanisms, but the molecular mechanisms regulating millet are still unclear. Due to millet's small size and light weight, the discovery of genes regulating grain shape has been difficult. 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. 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 a biological material for expressing the gene gl1, wherein the biological material comprises recombinant DNA, an expression cassette, a recombinant plasmid, a recombinant bacterium or a recombinant cell.

[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 present 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 foxtail millet plants by Agrobacterium-mediated genetic transformation.

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

[0013] Compared with the existing technology, the present invention has the following beneficial effects: the gene gl1 for regulating the shape of millet grains provided by the present invention is obtained by point mutation on the basis of gene GL1. After gene GL1 mutates into 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 millet grain length between gl1 mutant and wild-type millet Figure 1 , scale bar = 2000 μm;

[0015] Figure 2 Comparison of millet grain length between gl1 mutant 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. 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, 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, which regulates millet grain shape, was obtained by point mutation of the gene GL1. For ease of distinction, the wild-type gene GL1 is represented in uppercase, and the mutated gene gl1 is represented in lowercase. The bold and underlined sites in SEQ ID NO. 1 are mutation sites; these sites are missing in SEQ ID NO. 2. The nucleotide sequence of the wild-type gene GL1 is shown in SEQ ID NO. 3 and 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 which 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, which is as follows:

[0031] MGNRVGGRRRRPAVEERYTRPQGLYPHPDIDLRKLRRLILEAKLAPCHPGADDPRADLDECPICFLFYPSLNRSKCCAKGICTECFLQMKSPTSCRPTQCPYC KTLNYAVEYRGVKTKEEKGIEQLEEQRVIEAQIRMRQQELQEDAERMKNKQTAASTDSVATAQVESCDADGTSTTVASSEQGNDAVSSQVQHSELLLRNSEAFK QMRGNNFDMDLEEVMLMEAIWLSIQDQEALGNPGCVATTPPSIQRPYDGSMTTTAEAASSGGFACAVAALAEQQHIHGESSSTPTCQTTRFDTLSRSDRSYTEDLSIVGSSSSDSRVEEPSSSGTHRTVEGAESSNDQWSEIAEAGTSHAGSDVTAEAIAANSAASVGSSIASIPDSFEEQMMLAMALSLVDARTRANSPGLAWR.

[0032] The present invention also provides a biological material expressing the gene gl1, wherein the biological material includes but is not limited to recombinant DNA, an expression cassette, a recombinant plasmid, a recombinant bacterium or a recombinant cell.

[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, comprising expressing the gene gl1 in millet plants. In the present invention, the gene gl1 is preferably expressed in millet plants via Agrobacterium-mediated genetic transformation. The present invention does not specifically limit the Agrobacterium-mediated genetic transformation procedure; conventional Agrobacterium-mediated genetic transformation procedures in the art may be employed. In the present invention, the millet plant variety is preferably Jingu 21.

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

[0037] Example 1

[0038] Obtaining the sequence of the mutant gene gl1

[0039] Design and utilization of gene editing targets targetDesign-scau.edu.cn The wild-type gene sequence is shown in SEQ ID NO. 3, and the gene editing target is TCAGTGGTCAGAGATCGCCG.

[0040] Use Crispr / Cas9 technology and millet genetic transformation to generate mutant material. Refer to the genetic transformation steps in the article "Aminifoxtail millet with an Arabidopsis-like life cycle as a C4 model system" to obtain transgenic material.

[0041] After obtaining the transgenic gene-edited material, 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 sequencing 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) Generate gene-edited mutants of foxtail millet using Agrobacterium-mediated genetic transformation of wild-type JG21. Genetic transformation of foxtail millet: Follow the genetic transformation steps described in “A mini foxtail millet with an Arabidopsis-like lifecycle as a C4 model system” to obtain transgenic material.

[0046] The length and width of millet grains of mutant materials and wild-type materials were measured using the Wanshen SG-G automatic seed copy 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 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 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

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

2.

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

5.

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

4. The gene according to claim 1 gl1 , the use of the protein according to claim 2 or the biological material according to claim 3 in regulating the shape of foxtail millet grains, characterized in that Expressing the gene recited in claim 1 in a foxtail millet plant gl1 , the protein recited in claim 2, or introducing the biological material recited in claim 3 can increase the length and decrease the width of foxtail millet grains.

5. The gene according to claim 1 gl1 , the application of the protein according to claim 2 or the biological material according to claim 3 in foxtail millet breeding, characterized in that Expressing the gene recited in claim 1, the protein recited in claim 2, or introducing the biological material recited in claim 3 in a foxtail millet plant can increase the length of foxtail millet grains. gl1 ​ 6. A method for increasing the grain length of foxtail millet, characterized in that, Expressing the gene according to claim 1 in a foxtail millet plant gl1 .

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

8. The method according to claim 6, wherein The variety of the said foxtail millet plant is Jingu 21.

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