GmCYP75B1 gene and application thereof in regulating plant seed size

Abstract

The application discloses GmCYP75B1 Genes and their application in regulating plant seed size, by overexpressing genes in plants GmCYP75B1 GmCYP75B1 to increase the size of plant seeds, and the genes can be used for subsequent improvement of soybean varieties, which helps to improve the yield of soybeans.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and particularly relates to a GmCYP75B1 gene and application thereof in regulating plant seed size. BACKGROUND

[0002] Soybean seed size is an important economic trait determining soybean yield and appearance quality, and has been an important target for breeding. Seed size is mainly determined by the degree of tissue cell division and the length of cell elongation of seed coat, embryo and endosperm. In model plants Arabidopsis and rice, a series of key genes and detailed signal pathway regulation networks regulating seed size have been reported, including G protein-coupled signal pathway, ubiquitin-proteasome pathway, mitogen-activated protein kinase cascade signal transduction pathway, and plant hormone-mediated signal transduction pathway, etc.

[0003] Soybean yield is a quantitative trait (QTL) controlled by multiple genes. Through linkage analysis and genome-wide association analysis, hundreds of QTL intervals related to soybean seed size and weight have been found, but the exact genes regulating seed size and their mechanisms are mostly unknown, and their breeding potential remains to be verified. There are still few genes in soybean that have been clearly defined to regulate seed size and hundred-grain weight, such as protein phosphatase PP2C-1, GmCIF1, GmSSS1, GA synthesis-related genes GmGA3ox1 and GmGA20OX, BIG SEED 1, GmJAZ3, GmKIX8-1 and POWR1, etc.

[0004] The soybean cytochrome P450 (CYP) family includes a large family of monooxygenases with 346 members, four members of the CYP78A subfamily can regulate seed size; the homologous genes of the family members in Arabidopsis and wheat can also promote seed size. In addition, other subfamily members of the CYP family have certain research on plant resistance stress, for example, CYP99A1 and CYP709C1 in sorghum are involved in the process of low temperature stress; overexpression of CYP709B3 gene provides salt tolerance of transgenic Arabidopsis; CYP96A8 gene participates in the biosynthesis of leaf lignin and participates in drought tolerance of maize. However, whether other subfamily members except CYP78A subfamily members regulate seed size and their mechanisms have not been reported. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a GmCYP75B1 gene and application thereof in regulating plant seed size.

[0006] To solve the above technical problem, the technical scheme adopted by the present application is as follows.

[0007] A kit for regulating the size of a plant seed, the kit comprising a molecular biology element capable of regulating the expression level of a specific gene; the specific gene being a gene related to the ability of a plant to regulate seed size.

[0008] As a preferred technical solution of the present application, the molecular biology element can alternatively comprise: a combination of overexpression elements of the specific gene, and / or a combination of elements inhibiting or reducing the expression level of the specific gene, and / or a combination of elements silencing the expression of the specific gene, and / or a combination of elements knocking out the expression of the specific gene.

[0009] As a preferred technical solution of the present application, the molecular biology element is a combination of elements overexpressing the expression of the specific gene.

[0010] As a preferred technical solution of the present application, the specific gene is a GmCYP75B1 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1, or an equivalent gene having an equivalent plant physiological function.

[0011] The present application also comprises the following technical solution: a recombinant expression vector comprising a GmCYP75B1 gene or a homologous gene thereof.

[0012] As a preferred technical solution of the present application, the recombinant expression vector is an overexpression vector.

[0013] A method for increasing the size of a plant seed, the method comprising overexpressing the expression of a GmCYP75B1 gene in the plant to increase the size of the plant seed.

[0014] A method for increasing the yield of a plant, the method comprising overexpressing the expression of a GmCYP75B1 gene in the plant to increase the size of the plant seed, thereby increasing the yield of the corresponding plant.

[0015] The present application also comprises the use of a GmCYP75B1 gene for regulating the size of a plant seed.

[0016] As a preferred technical solution of the present application, the plant is soybean.

[0017] The beneficial effects of the above technical solution are as follows: the present application constructs the coding sequence of a GmCYP75B1 gene into an overexpression binary vector, and finds that overexpression of the gene promotes the increase in the size of seeds of Arabidopsis thaliana through flower immersion transgenic technology, and also finds that overexpression of the gene can promote the increase in the size of seeds of soybean using cotyledon node stable transformation technology. Therefore, overexpression of a GmCYP75B1 gene can achieve the purpose of promoting the increase in the size of plant seeds, and the gene can be fully utilized for the improvement of soybean varieties in the future, which helps to increase the yield of soybean. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure 9 is a diagram of the tissue expression pattern analysis of soybean GmCYP75B1, wherein the expression level of GmCYP75B1 in roots, stems, leaves, flowers, apical meristems, hypocotyls, cotyledons and seeds 16 days after fertilization was detected by RT-qPCR technology;

[0019] Figure 2 Figure 13 is a diagram of the p35S:GmCYP75B1-GFP overexpression vector construction, wherein A is a schematic diagram of the p35S:GmCYP75B1-GFP vector; B is an electrophoresis diagram of p35S:GmCYP75B1-GFP after enzyme digestion using Xba I and BamH I, the target band is 1545 bp; B is an electrophoresis diagram of p35S:GmCYP75B1-GFP after enzyme digestion using Hind III and BamH I, the target band is 2410 bp; "+" represents the addition of restriction endonuclease, "-" represents no addition of restriction endonuclease;

[0020] Figure 3 Figure 15 is a diagram of the seed size phenotype observation of Col wild type control lines and p35S:GmCYP75B1-GFP overexpression lines in Arabidopsis, wherein A is the size phenotype of mature dry seeds of Col wild type control and p35S:GmCYP75B1-GFP overexpression lines (2-18 and 8-14), the scale is 1 mm; B is a statistical diagram of seed area of Col wild type control and p35S:GmCYP75B1-GFP overexpression lines (2-18 and 8-14), ***, p<0.001, t-test, there is a very significant difference in seed size between the transgenic lines and the Col wild type control;

[0021] Figure 4 Figure 17 is a diagram of the pUbi:GmCYP75B1-3FLAG overexpression vector construction, wherein A is a schematic diagram of the pUbi:GmCYP75B1-3FLAG vector; B is an electrophoresis diagram of pUbi:GmCYP75B1-3FLAG after enzyme digestion using Pst I and Nco I, the target band is 1274 bp; "+" represents the addition of restriction endonuclease, "-" represents no addition of restriction endonuclease;

[0022] Figure 5Figure A is the phenotype of seed size of WT wild type control and pUBI:GmCYP75B1-3FLAG overexpression lines (6#, 28# and 10#) mature dry seeds of grain length, grain width and grain thickness, scale bar is 1 cm; Figure B is the statistical chart of grain length, grain width, grain thickness and hundred-grain weight of pUBI:GmCYP75B1-3FLAG overexpression lines (6#, 28# and 10#), ****, p<0.0001, t-test, there is extremely significant difference in seed size of transgenic lines and relative wild type control WT. DETAILED DESCRIPTION

[0023] The following examples illustrate the present application. The various raw materials and equipment used in the present application are all conventional commercially available products, which can be directly obtained by market purchase. In the description of the following examples, specific details such as specific system structures, techniques, etc. are presented for the purpose of illustration but not for the purpose of limitation, so as to thoroughly understand the embodiments of the present application. However, it should be clear to those skilled in the art that the present application can also be implemented in other embodiments without these specific details. In other cases, detailed descriptions of well-known systems, devices and methods are omitted to avoid unnecessary details that hinder the description of the present application.

[0024] It should be understood that the term "comprises" as used in the specification and the appended claims indicates the presence of the described features, integers, steps, operations, elements, and / or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. It should also be understood that the term "and / or" as used in the specification and the appended claims indicates any combination of one or more of the associated listed items and all possible combinations thereof. As used in the specification and the appended claims, the term "if" can be interpreted as meaning "when" or "once" or "in response to a determination" or "in response to detecting" depending on the context. Similarly, the phrase "if determined" or "if detected [the described condition or event]" can be interpreted as meaning "once determined" or "in response to a determination" or "once detected [the described condition or event]" or "in response to detecting [the described condition or event]" depending on the context.

[0025] In addition, in the description of the present application and the appended claims, the terms "first", "second", "third", etc. are used only to distinguish descriptions, and cannot be understood as indicating or implying relative importance. In the present application, the reference "one embodiment" or "some embodiments" means that the specific features, structures or characteristics described in connection with the embodiment are included in one or more embodiments of the present application. Therefore, the statements "in one embodiment", "in some embodiments", "in other some embodiments", "in further some embodiments" and the like appearing in different places in the specification are not necessarily all referring to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized. The terms "include", "contain", "have" and their variants mean "include but not limited to", unless otherwise specifically emphasized.

[0026] The soybean GmCYP75B1 gene (Glyam.06G202300) provided by the present application encodes a flavonoid 3'-hydroxylase related to flavonoid synthesis. The nucleotide sequence is shown in SEQ ID NO. 1. The expression amount of the gene in seeds is high.

[0027] Example 1, cloning of soybean GmCYP75B1 gene

[0028] Primer design: The upstream primer required for amplifying the target gene needs to add 15 bp of upstream vector sequence and 6 bp of target enzyme cutting site Xba I recognition sequence at the 5' end of ATG. The downstream primer needs to add 15 bp of downstream vector sequence and 6 bp of target enzyme cutting site BamH I recognition sequence at the 5' end of the original downstream primer sequence. The specific primer sequence is:

[0029] Table 1 primer pair sequence list

[0030]

[0031]

[0032] The high-fidelity KOD FX polymerase was used to amplify according to the system shown in Table 2.

[0033] Table 2 PCR amplification system

[0034]

[0035] Table 3 PCR amplification program

[0036]

[0037] After amplification, the product was recovered using an agarose gel product recovery kit. The specific steps are referred to the kit instruction manual of Jet Bio Technology Co., Ltd.

[0038] Example 2, obtaining of overexpression transgenic lines

[0039] Using pCAMBIA 130035S:GFP binary vector as the backbone, the vector was linearized with Xba I and BamH I restriction enzymes, and then a one-step cloning (One Step Cloning) ligation reaction was performed using the system shown in Table 4:

[0040] Table 4 Cloning ligation reaction system

[0041]

[0042] Note: Optimal linearized vector addition amount (b) = [0.02 x number of base pairs of cloning vector] ng

[0043] Optimal insert fragment usage amount (a) = [0.04 x number of base pairs of insert fragment] ng

[0044] The above ligation system was placed in a 37°C metal bath, and after 15 min of reaction, DH5a was transformed, and after enzyme digestion identification (attached Figure 2 ), Sanger sequencing was performed, and the correct sequencing p35S:GmCYP75B1-GFP plant expression vector was obtained, and p35S:GmCYP75B1-GFP overexpression transgenic Arabidopsis lines were obtained by Arabidopsis flower immersion transgenesis.

[0045] Using pUBI:3FLAG binary vector as the backbone, the vector was linearized with Kpn I restriction enzyme, and then a one-step cloning (One Step Cloning) ligation reaction was performed using the system shown in Table 5:

[0046] Table 5 Cloning ligation reaction system

[0047]

[0048] Note: Optimal linearized vector addition amount (b) = [0.02 x number of base pairs of cloning vector] ng

[0049] Optimal insert fragment usage amount (a) = [0.04 x number of base pairs of insert fragment] ng

[0050] The above ligation system was placed in a 37°C metal bath, and after 15 min of reaction, DH5a was transformed, and after enzyme digestion identification (attached Figure 4 ), Sanger sequencing was performed, and the correct sequencing pUBI:GmCYP75B1-3FLAG plant expression vector was obtained, and pUBI:GmCYP75B1-3FLAG overexpression transgenic soybean lines were obtained by cotyledon node stable transformation.

[0051] Example 3 Seed size phenotype observation of Arabidopsis thaliana GmCYP75B1 heterologous overexpression lines

[0052] The seeds of the Col wild type control and p35S:GmCYP75B1-GFP overexpression lines to be detected were as evenly as possible scattered on white paper, and the seeds were simply arranged with the needle of a syringe, and then collected under a body microscope (LEICAM250 FA). Under the same setting conditions, a ruler was also photographed as a scale, as shown in Figure 3 -A. After collection, the area of the seeds was measured using ImageJ software, and the measurement results were statistically analyzed using Prism software, as shown in Tables 6, 7 and Figure 3 -B.

[0053] Table 6 Seed area size statistics of Col wild type control lines and p35S:GmCYP75B1-GFP overexpression lines in Arabidopsis thaliana

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] Note: / is an error value, which has been removed.

[0061] Table 7 Statistical analysis of seed area size of Col wild type control lines and p35S:GmCYP75B1-GFP overexpression lines in Arabidopsis thaliana

[0062]

[0063] From Tables 7 and Figure 3 It can be seen that the differences in seed area size of the Col wild type control lines and the p35S:GmCYP75B1-GFP overexpression lines shown in Table 6 all reached a significant level (P<0.05). Among them, the seed area of the p35S:GmCYP75B1-GFP overexpression lines was larger than that of the Col wild type control lines.

[0064] The application fully proves that GmCYP75B1 gene overexpression has the effect of promoting the seed size of Arabidopsis thaliana by constructing p35S:GmCYP75B1-GFP vector and overexpressing in Arabidopsis thaliana, and statistically analyzing the seed size phenotype of the overexpression strain. According to the above content, the GmCYP75B1 gene has application value in the research and modification of Arabidopsis thaliana seed size.

[0065] Example 4 Seed size phenotype observation of soybean pUBI:GmCYP75B1-3FLAG overexpression strain

[0066] The mature soybean seeds of the WT wild type control strain and the pUBI:GmCYP75B1-3FLAG overexpression strain to be detected were randomly weighed 100 seeds respectively, and then placed in a thousandth balance to statistically analyze the hundred-grain weight. The soybean seeds to be detected were collected under a body microscope (LEICAM250 FA), and a ruler was photographed again under the same setting condition as a scale, as shown in Figure 5 -A. After the collection, the grain length, grain width and grain thickness of the seeds were measured by using Image J software, and the measurement results were statistically analyzed by using Prism software, as shown in Tables 8, 9, 10 and Figure 5 -B.

[0067] Table 8 Statistical table of seed grain length, grain width and grain thickness of WT wild type control strain and pUBI:GmCYP75B1-3FLAG overexpression strain in soybean

[0068]

[0069]

[0070] Table 9 Statistical results of hundred-grain weight of WT wild type control strain and pUBI:GmCYP75B1-3FLAG overexpression strain in soybean

[0071]

[0072] Table 10 Statistical analysis results of seed grain length, grain width, grain thickness and hundred-grain weight of WT wild type control strain and pUBI:GmCYP75B1-3FLAG overexpression strain in soybean

[0073]

[0074] Table 10 and Figure 3It can be known that the differences of seed length, width, thickness and 100-seed weight of the WT wild type control strain and the pUBI:GmCYP75B1-3FLAG overexpression strain shown in Table 8 and Table 9 all reach a significant level (P<0.05). Among them, the seed length, width, thickness and 100-seed weight of the pUBI:GmCYP75B1-3FLAG overexpression strain are all greater than those of the WT wild type control strain.

[0075] The present application constructs the pUBI:GmCYP75B1-3FLAG vector, overexpresses in soybean, and statistically analyzes the seed length, width, thickness and 100-seed weight of the overexpression strain, and the results fully show that the overexpression of the GmCYP75B1 gene has the effect of promoting the size of soybean seeds. According to the above content, the GmCYP75B1 gene has application value in the research and modification of soybean seed size.

[0076] In summary of the above examples, the coding sequence of the GmCYP75B1 gene is constructed into the overexpression binary vector, and it is found by the flower immersion method that the overexpression of the gene promotes the seed enlargement of Arabidopsis thaliana, and at the same time, it is found by the cotyledon node stable transformation technology that the overexpression of the gene also promotes the seed enlargement of soybean. Therefore, the overexpression of the GmCYP75B1 gene can achieve the purpose of promoting the seed enlargement of plants, and the gene can be fully utilized for the improvement of soybean varieties in the future, which is helpful to improve the yield of soybean.

[0077] In the above examples, the description of each example has its own emphasis, and the parts not described or recorded in a certain example can be referred to the related description of other examples.

[0078] The above examples are only used to illustrate the technical solutions of the present application, rather than limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application, and should be included in the protection scope of the present application.

Claims

1. A method for increasing the size of soybean seeds, characterized in that: Overexpression of soybean GmCYP75B1 Genes to increase soybean seed size, the GmCYP75B1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A method for increasing soybean yield, characterized in that: Overexpression of soybean GmCYP75B1 Genes designed to increase soybean seed size, thereby increasing the yield of the corresponding soybeans, are described. GmCYP75B1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

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