GmCYP75B1 gene and application thereof in regulation and control of plant seed size

Abstract

The invention discloses a GmCYP75B1 gene and application thereof in regulation and control of the size of plant seeds, the size of the plant seeds is increased by overexpressing the GmCYP75B1 gene in a plant, the gene can be fully utilized to improve soybean varieties subsequently, and the yield of soybeans can be increased.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, in particular to a GmCYP75B1 gene and an application thereof in regulating plant seed size. Background Art

[0004] Soybean seed size is an important economic trait that determines soybean yield and appearance quality, and has always been an important breeding goal. Seed size is mainly determined by the degree of cell division and cell elongation length of the seed coat, embryo, and endosperm. In the model plants Arabidopsis and rice, a series of key genes regulating seed size and detailed signal pathway regulatory networks have been reported, including the G protein-coupled signaling pathway, the ubiquitin-proteasome pathway, the mitogen-activated protein kinase cascade signal transduction pathway, and the plant hormone-mediated signal transduction pathway.

[0005] 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 genes that exactly regulate seed size and their mechanisms of action are mostly unknown, and their breeding potential remains to be verified. There are still few genes in soybean that have been clearly identified to regulate seed size and 100-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.

[0006] The soybean cytochrome P450 (CYP) family includes a large family of monooxygenases with 346 members, among which four members of the CYP78A subfamily can regulate grain size; homologous genes of this family member in Arabidopsis and wheat can also promote seed size. In addition, other subfamily members in the CYP family have been studied in plant stress resistance. For example, CYP99A1 and CYP709C1 are involved in the low temperature stress process in sorghum; overexpression of the CYP709B3 gene provides salt tolerance in transgenic Arabidopsis; and the CYP96A8 gene participates in drought tolerance in corn by participating in the biosynthesis of leaf lignin. However, except for the CYP78A subfamily members, whether other subfamily members regulate seed size and their mechanism of action have not been reported. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a GmCYP75B1 gene and an application thereof in regulating the size of plant seeds.

[0008] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0009] A kit for regulating the size of plant seeds, the kit comprising molecular biological elements capable of regulating the expression of specific genes; the specific genes are genes related to the ability of plants to regulate seed size.

[0010] As a preferred technical solution of the present invention, the molecular biological elements may optionally include: an element combination for overexpressing the specific gene, and / or an element combination for inhibiting or reducing the expression amount of the specific gene, and / or an element combination for silencing the expression of the specific gene, and / or an element combination for knocking out the expression of the specific gene.

[0011] As a preferred technical solution of the present invention, the molecular biological element is a combination of elements that overexpress the specific gene expression.

[0012] As a preferred technical solution of the present invention, the specific gene is the GmCYP75B1 gene, whose nucleotide sequence is shown in SEQ ID NO.1, or an equivalent gene having the same plant physiological function.

[0013] The present invention also includes the following technical solution: a recombinant expression vector comprising the GmCYP75B1 gene or a homologous gene thereof.

[0014] As a preferred technical solution of the present invention, the recombinant expression vector is an overexpression vector.

[0015] A method for increasing the seed size of a plant, comprising overexpressing the GmCYP75B1 gene in the plant to increase the seed size of the plant.

[0016] A method for increasing plant yield, overexpressing the GmCYP75B1 gene in the plant to increase the size of plant seeds, thereby increasing the yield of the corresponding plant.

[0017] The present invention also includes the use of the GmCYP75B1 gene for regulating the size of plant seeds.

[0018] As a preferred technical solution of the present invention, the plant is soybean.

[0019] The beneficial effect of the above technical solution is that the coding sequence of the GmCYP75B1 gene is constructed into an overexpression binary vector, and its overexpression is found to promote the enlargement of Arabidopsis seeds through the floral dipping transgenic technology, and at the same time, the cotyledon node stable transformation technology is used to find that its overexpression can also promote the enlargement of soybean seeds. Therefore, overexpression of the GmCYP75B1 gene can achieve the purpose of promoting the enlargement of plant seeds, and the gene can be fully utilized to improve soybean varieties in the future, which helps to increase soybean yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram of the tissue expression pattern of soybean GmCYP75B1. 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;

[0021] Figure 2 The figure is a diagram of the construction of the p35S:GmCYP75B1-GFP overexpression vector, in which A is a schematic diagram of the p35S:GmCYP75B1-GFP vector; the left figure in B is an electrophoretic diagram of p35S:GmCYP75B1-GFP after being digested with Xba I and BamH I, and the target band is 1545 bp; the right figure is an electrophoretic diagram of p35S:GmCYP75B1-GFP after being digested with Hind III and BamH I, and the target band is 2410 bp; "+" represents the addition of restriction endonucleases, and "-" represents the absence of restriction endonucleases;

[0022] Figure 3 The diagram is an observation diagram of seed size phenotype of Col wild-type control strain and p35S:GmCYP75B1-GFP overexpression strain in Arabidopsis thaliana. In the diagram, A is the mature dry seed size phenotype of Col wild-type control and p35S:GmCYP75B1-GFP overexpression strain (2-18 and 8-14), and the scale bar is 1 mm; B is the seed area statistical diagram of Col wild-type control and p35S:GmCYP75B1-GFP overexpression strain (2-18 and 8-14), ***, p<0.001, t-test, there is an extremely significant difference between the seed size of the transgenic strain and the Col wild-type control;

[0023] Figure 4 The figure shows the construction of pUbi:GmCYP75B1-3FLAG overexpression vector. In the figure, A is a schematic diagram of pUbi:GmCYP75B1-3FLAG vector; B is an electrophoresis diagram of pUbi:GmCYP75B1-3FLAG after restriction digestion with Pst I and Nco I, and the target band is 1274 bp; "+" represents the addition of restriction endonuclease, and "-" represents the absence of restriction endonuclease;

[0024] Figure 5The diagram shows the seed size phenotype of the WT wild-type control strain and the pUBI:GmCYP75B1-3FLAG overexpression strain in soybean. In the diagram, A shows the phenotype of the length, width and thickness of mature dry seeds of the WT wild-type control strain and the pUBI:GmCYP75B1-3FLAG overexpression strain (6#, 28# and 10#), and the scale bar is 1 cm; B shows the statistical diagram of the length, width, thickness and 100-grain weight of the pUBI:GmCYP75B1-3FLAG overexpression strain (6#, 28# and 10#), ****, p<0.0001, t-test, the seed size of the transgenic strain is extremely significantly different from that of the wild-type control WT. DETAILED DESCRIPTION

[0025] The following examples describe the present invention in detail. Various raw materials and equipment used in the present invention are conventional commercial products, and can be directly obtained by market purchase. In the description of the following examples, for the purpose of illustration rather than limitation, specific details such as specific system structures, technologies, etc. are proposed, so as to thoroughly understand the present application embodiments. However, it should be clear to those skilled in the art that the present application can also be realized in other embodiments without these specific details. In other cases, the detailed description of well-known systems, devices and methods is omitted to prevent unnecessary details from hindering the description of the present application.

[0026] It should be understood that when used in the present specification and the appended claims, the term "comprising" indicates the presence of the described features, wholes, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or their collections. It should also be understood that the term "and / or" used in the present specification and the appended claims refers to any combination of one or more of the items listed in association and all possible combinations, and includes these combinations. As used in the present specification and the appended claims, the term "if" can be interpreted as "when..." or "once" or "in response to determination" or "in response to detection" according to the context. Similarly, the phrase "if it is determined" or "if [described condition or event] is detected" can be interpreted as meaning "once it is determined" or "in response to determination" or "once [described condition or event] is detected" or "in response to detection [described condition or event]" according to the context.

[0027] In addition, in the description of the present application specification and the attached claims, the terms "first", "second", "third", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance. The reference to "one embodiment" or "some embodiments" described in the present application specification means that one or more embodiments of the present application include specific features, structures or characteristics described in conjunction with the embodiment. Therefore, the statements "in one embodiment", "in some embodiments", "in some other embodiments", "in some other embodiments", etc. that appear in the different parts of this specification do not necessarily refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "including", "comprising", "having" and their variations all mean "including but not limited to", unless otherwise specifically emphasized in other ways.

[0028] The soybean GmCYP75B1 gene (Glyam.06G202300) provided by the present invention encodes a flavonoid 3'-hydroxylase, which is related to flavonoid synthesis. Its nucleotide sequence is shown in SEQ ID NO.1. The expression level of the gene in seeds is relatively high.

[0029] Example 1: Cloning of soybean GmCYP75B1 gene

[0030] Primer design: The upstream primer required to amplify the target gene needs to add 15bp of upstream vector sequence and 6bp of target restriction site Xba I recognition sequence at the 5' end of ATG, and the downstream primer needs to add 15bp of downstream vector sequence and 6bp of target restriction site BamH I recognition sequence at the 5' end of the original downstream primer sequence. The specific primer sequences are:

[0031] Table 1 Primer pair sequence list

[0032]

[0033] Amplification was performed using the high-fidelity KOD FX polymerase according to the system shown in Table 2.

[0034] Table 2 PCR amplification system

[0035]

[0036] Table 3 PCR amplification program

[0037]

[0038] After amplification, the product was recovered using an agarose gel product recovery kit. For specific steps, refer to the kit instructions of Jerri Biotech.

[0039] Example 2: Obtaining overexpression transgenic strains

[0040] Using pCAMBIA 130035S:GFP binary vector as the backbone, the vector was linearized with Xba I and BamH I restriction endonucleases, and then a one-step cloning (One Step Cloning) ligation reaction was performed using the system shown in Table 4:

[0041] Table 4 Cloning and ligation reaction system

[0042]

[0043] Note: The most suitable amount of linearized vector added (b) = [0.02 × number of base pairs of cloning vector] ng

[0044] Optimal amount of insert fragment used (a) = [0.04 × number of base pairs of insert fragment] ng

[0045] The above ligation system was placed in a 37°C metal bath and reacted for 15 min to transform DH5α. Figure 2 ), Sanger sequencing was performed to obtain the correctly sequenced p35S:GmCYP75B1-GFP plant expression vector, and the p35S:GmCYP75B1-GFP overexpressing transgenic Arabidopsis strains were obtained by Arabidopsis floral dipping method.

[0046] Using the pUBI:3FLAG binary vector as the backbone, the vector was linearized with Kpn I restriction endonuclease, and then a one-step cloning (One Step Cloning) ligation reaction was performed using the system shown in Table 5:

[0047] Table 5 Cloning ligation reaction system

[0048]

[0049] Note: The most suitable amount of linearized vector added (b) = [0.02 × number of base pairs of cloning vector] ng

[0050] Optimal amount of insert fragment used (a) = [0.04 × number of base pairs of insert fragment] ng

[0051] The above ligation system was placed in a 37°C metal bath and reacted for 15 min to transform DH5α. Figure 4 ), Sanger sequencing was performed to obtain the correctly sequenced pUBI:GmCYP75B1-3FLAG plant expression vector, and the pUBI:GmCYP75B1-3FLAG overexpressing transgenic soybean strain was obtained by the cotyledon node stable transformation method.

[0052] Example 3 Observation of seed size phenotype of Arabidopsis thaliana GmCYP75B1 heterologous overexpression strains

[0053] The seeds of the Col wild-type control and the p35S:GmCYP75B1-GFP overexpressing strain to be tested were spread as evenly as possible on white paper, and the seeds were simply arranged with the needle of a syringe. After that, the seeds were collected under a stereo microscope (LEICAM250 FA). Under the same setting conditions, a ruler was taken as a scale, as shown in Fig. Figure 3 After the collection was completed, the area of ​​the seeds was measured using ImageJ software, and the measurement results were statistically analyzed using Prism software. The results are shown in Tables 6, 7 and Figure 3 -B.

[0054] Table 6 Seed area statistics of Col wild-type control strain and p35S:GmCYP75B1-GFP overexpression strain in Arabidopsis

[0055]

[0056]

[0057]

[0058]

[0059]

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

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

[0062]

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

[0064] The present invention constructs a p35S:GmCYP75B1-GFP vector and overexpresses it in Arabidopsis, and statistically analyzes the seed size phenotype of the overexpression strain. The results fully show that overexpression of the GmCYP75B1 gene has the effect of promoting the seed size of Arabidopsis. According to the above content, the GmCYP75B1 gene has application value in studying and transforming the seed size of Arabidopsis.

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

[0066] Randomly weigh 100 seeds of the mature WT wild-type control strain and the pUBI:GmCYP75B1-3FLAG overexpression strain to be tested, and then place them on a 1 / 1000 scale to count the weight of 100 seeds. Collect the soybean seeds to be tested under a stereo microscope (LEICAM250 FA), and take a picture of a ruler as a scale under the same setting conditions, such as Figure 5 After the collection was completed, the seed length, width and thickness were measured using Image J software, and the measurement results were statistically analyzed using Prism software. The results are shown in Tables 8, 9, 10 and Figure 5 -B.

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

[0068]

[0069]

[0070] Table 9 Statistical results of 100-grain weight of seeds of WT wild-type control strain and pUBI:GmCYP75B1-3FLAG overexpression strain in soybean

[0071]

[0072]

[0073] Table 10 Statistical analysis results of seed length, width, thickness and 100-grain weight of WT wild-type control strain and pUBI:GmCYP75B1-3FLAG overexpression strain in soybean

[0074]

[0075] From Table 10 and Figure 3It can be seen that the differences in seed length, width, thickness and 100-grain weight between the WT wild-type control strain and the pUBI:GmCYP75B1-3FLAG overexpression strain shown in Tables 8 and 9 all reached significant levels (P<0.05). Among them, the seed length, width, thickness and 100-grain weight of the pUBI:GmCYP75B1-3FLAG overexpression strain were greater than those of the WT wild-type control strain.

[0076] The present invention constructs a pUBI:GmCYP75B1-3FLAG vector, overexpresses it in soybean, and counts the grain length, grain width, grain thickness, and 100-grain weight of the seeds of the overexpression strain. 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 studying and modifying the size of soybean seeds.

[0077] In summary, the present invention constructs the coding sequence of the GmCYP75B1 gene into an overexpression binary vector, and finds that its overexpression promotes the enlargement of Arabidopsis seeds through floral dipping transgenic technology, and at the same time, finds that its overexpression can also promote the enlargement of soybean seeds through cotyledon node stable transformation technology. Therefore, overexpression of GmCYP75B1 can achieve the purpose of promoting the enlargement of plant seeds, and the gene can be fully utilized to improve soybean varieties in the future, which helps to increase soybean yield.

[0078] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0079] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included in the protection scope of the present invention.

Claims

1. A kit for regulating the size of plant seeds, characterized in that: The kit contains molecular biological elements capable of regulating the expression of specific genes; the specific genes are genes related to the ability of plants to regulate seed size.

2. The kit according to claim 1, characterized in that: The molecular biological elements may optionally include: an element combination for overexpressing the specific gene, and / or an element combination for inhibiting or reducing the expression level of the specific gene, and / or an element combination for silencing the expression of the specific gene, and / or an element combination for knocking out the expression of the specific gene.

3. The kit according to claim 1, characterized in that: The molecular biological element is a combination of elements that overexpress the specific gene.

4. The kit according to claim 1-3, characterized in that: The specific gene is GmCYP75B1 A gene, whose nucleotide sequence is shown in SEQ ID NO.1, or an equivalent gene having the same plant physiological function.

5. Inclusion GmCYP75B1 A recombinant expression vector of a gene or its homologous gene.

6. The recombinant expression vector according to claim 5, characterized in that: The recombinant expression vector is an overexpression vector.

7. A method for increasing the size of plant seeds, characterized in that: Overexpression in the plant GmCYP75B1 Expression of genes to increase seed size in plants.

8. A method for increasing plant yield, characterized in that: Overexpression in the plant GmCYP75B1 The expression of genes to increase the size of plant seeds and thus increase the yield of the corresponding plants.

9. GmCYP75B1 The use of genes to regulate the size of plant seeds.

10. The kit according to claims 1 to 4, the method according to claim 7, the method according to claim 8, or the use according to claim 9, characterized in that: The plant is soybean.

Images