Application of BnCYP90A1 gene in regulation and control of rapeseed grain size and pod length

Abstract

The invention discloses application of a BnCYP90A1 gene in regulation and control of rapeseed grain size and pod length, and belongs to the technical field of plant molecular biology. The invention discloses the application of the BnCYP90A1 gene of the rape in regulating and controlling the pod length and the grain size of the rape for the first time. The nucleotide sequence of the BnCYP90A1 gene is as shown in SEQ ID NO. 1. According to the invention, the BnCYP90A1 gene is over-expressed in a cabbage type rape dm1-s1 mutant with reduced plant height, reduced grains and shortened pod length, a back-complementing strain is constructed, the pod length and thousand seed weight of each strain are investigated, and experimental results show that the BnCYP90A1 gene can obviously promote the extension of the pod and the enlargement of the grains of the rape plant. The invention provides a new application direction for the BnCYP90A1 gene, and provides a new technical support for breeding improvement of rapeseed grain yield and pod phenotype.

Description

Technical Field

[0001] The invention relates to the technical field of plant molecular biology, and in particular to application of a BnCYP90A1 gene in regulating rapeseed grain size and silique length. Background Art

[0002] Rapeseed, as one of the most important oil crops in my country, has important economic value, ornamental value and ecological value. With the growth of population, especially the continuous improvement of people's living standards, the supply and demand structure of edible vegetable oil in my country is unbalanced, the external dependence is getting higher and higher, and the oil security situation in my country is becoming increasingly severe. Limited by the influence of limiting factors such as land resources and planting costs in my country, the planting area of ​​rapeseed in my country cannot be expanded on a large scale. Therefore, the increase in the yield of rapeseed per unit area is an important way to increase the yield of Brassica napus. Seed size and grain weight are significantly positively correlated and directly affect the yield of rapeseed per plant. Rapeseed silique is the organ where seeds are attached. It not only provides sufficient growth space for seed development, but also, in the late stage of seed maturity, as the main photosynthetic organ, it continuously provides nutrients for seed development to ensure the normal development of seeds. Changing the silique traits will directly or indirectly lead to yield changes. Therefore, improving the seed size and silique traits of rapeseed is of great significance to improving the yield of rapeseed.

[0003] Grain weight and silique length of rapeseed are important yield components at the individual level of rapeseed, and are also hot topics in the study of rapeseed yield traits. A large number of studies have been conducted to explore related genes and segments through QTL, and some genes have been cloned. For example, Auxin Response Factor 18 (ARF18) is a negative regulatory gene for silique length and grain weight of rapeseed. This gene controls silique length and seed size by inhibiting the expression of downstream auxin-related genes. The gene BnaA9.CYP78A9 encodes a P450 protein. After overexpression of this gene, rapeseed with longer siliques and increased grain weight was obtained. Some related genes have also been screened through reverse genetics. For example, overexpression of the Arabidopsis ubiquitin receptor mutant gene AtDA1R358K in rapeseed will lead to downregulation of endogenous BnDA1 expression in rapeseed, which significantly increases the seed size of rapeseed. At the same time, combined transcriptomics was used to identify genetic variation in the regulation of BnaUPL3.C03 in Brassica napus. BnaUPL3.C03 encodes HECTE3 ubiquitin ligase. After reducing the expression of BnaUPL3.C03, rapeseed seeds became significantly larger. Although researchers have cloned some genes that regulate seed size and silique length, a large number of genes have not been reported, and the specific pathways involved in their regulation and the key factors are still unknown. It is urgent to explore genes that have the function of regulating rapeseed seed size and silique length. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the BnCYP90A1 gene in regulating rapeseed grain size and silique length to solve the problems existing in the above-mentioned prior art. The BnCYP90A1 gene provided by the present invention can significantly promote the elongation of siliques and the enlargement of seeds of rapeseed plants.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the use of the BnCYP90A1 gene in any of the following:

[0007] (1) Application in regulating rapeseed grain size;

[0008] (2) Application in breeding large-grain rapeseed varieties;

[0009] (3) Application in regulating the length of rapeseed siliques;

[0010] (4) Application in breeding silique rape varieties;

[0011] The nucleotide sequence of the BnCYP90A1 gene is shown in SEQ ID NO.1.

[0012] The present invention provides the use of a recombinant vector comprising the BnCYP90A1 gene in any of the following:

[0013] (1) Application in regulating rapeseed grain size;

[0014] (2) Application in breeding large-grain rapeseed varieties;

[0015] (3) Application in regulating the length of rapeseed siliques;

[0016] (4) Application in the breeding of silique rape varieties.

[0017] The present invention provides the use of an engineered bacterium comprising the recombinant vector in any of the following:

[0018] (1) Application in regulating rapeseed grain size;

[0019] (2) Application in breeding large-grain rapeseed varieties;

[0020] (3) Application in regulating the length of rapeseed siliques;

[0021] (4) Application in the breeding of silique rape varieties.

[0022] The present invention also provides the use of BnCYP90A1 protein in any of the following:

[0023] (1) Application in regulating rapeseed grain size;

[0024] (2) Application in breeding large-grain rapeseed varieties;

[0025] (3) Application in regulating the length of rapeseed siliques;

[0026] (4) Application in breeding silique rape varieties;

[0027] The amino acid sequence of the BnCYP90A1 protein is shown in SEQ ID NO.2.

[0028] Preferably, the expression level of the BnCYP90A1 gene is up-regulated to promote the increase of the rapeseed grains and / or the elongation of the rapeseed siliques.

[0029] The present invention also provides a method for promoting the enlargement of rapeseed grains, comprising the step of overexpressing the BnCYP90A1 gene in rapeseed, wherein the nucleotide sequence of the BnCYP90A1 gene is shown in SEQ ID NO.1.

[0030] The present invention also provides a method for promoting the elongation of rapeseed siliques, comprising the step of overexpressing the BnCYP90A1 gene in rapeseed, wherein the nucleotide sequence of the BnCYP90A1 gene is shown in SEQ ID NO.1.

[0031] Preferably, the overexpression of the BnCYP90A1 gene in rapeseed comprises the following steps: constructing a recombinant plasmid by combining the BnCYP90A1 gene with an expression vector; and transferring the recombinant plasmid into rapeseed by Agrobacterium-mediated method to overexpress the BnCYP90A1 gene.

[0032] The present invention also provides a method for cultivating large-grain rapeseed, comprising the step of overexpressing the BnCYP90A1 gene in rapeseed, wherein the nucleotide sequence of the BnCYP90A1 gene is shown in SEQ ID NO.1.

[0033] The present invention also provides a method for cultivating silique rape, comprising the step of overexpressing the BnCYP90A1 gene in rape, wherein the nucleotide sequence of the BnCYP90A1 gene is shown in SEQ ID NO.1.

[0034] The present invention discloses the following technical effects:

[0035] The present invention discloses for the first time the application of the rapeseed BnCYP90A1 gene in regulating the length of rapeseed siliques and the size of seeds. The present invention overexpresses the BnCYP90A1 gene in the dm1-s1 mutant of the Brassica napus type with reduced plant height, reduced seeds and shortened siliques, constructs a complementation strain, and investigates the silique length and 1000-grain weight of each strain. The experimental results show that the BnCYP90A1 gene can significantly promote the elongation of siliques and the enlargement of seeds of rapeseed plants. The present invention provides a new application direction for the BnCYP90A1 gene, and provides new technical support for the breeding improvement of rapeseed seed yield and silique phenotype. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 The phenotypes of dm1-s1 mutant plants and Brassica napus ZS11 are shown in Figure 1. a is the overall appearance phenotype of the plant, with a scale of 10 cm; b is the silique phenotype, with a scale of 1 cm; c is the grain weight statistics, Thousand grain weight is the thousand-grain weight, dm1 is the dm1-s1 mutant plant, and *** indicates P < 0.001.

[0038] Figure 2 The phenotypes of the dm1-s1 mutant, Brassica napus ZS11, and BnCYP90A1 transgenic positive plants com.#1 and com.#2 at the seedling stage; the scale bar in the figure is 10 cm;

[0039] Figure 3 The morphological phenotypes of siliques of the dm1-s1 mutant, Brassica napus ZS11, and BnCYP90A1 transgenic positive plants com.#1 and com.#2 are shown in the figure; the scale bar is 5 cm;

[0040] Figure 4 The figure shows the statistical analysis of silique length of dm1-s1 mutant plants, Brassica napus ZS11, and BnCYP90A1 transgenic positive plants com.#1 and com.#2; Silique length refers to silique length, ns means no significant difference, * means P<0.05, **** means P<0.0001;

[0041] Figure 5The statistical analysis chart of thousand-grain weight of dm1-s1 mutant plants, Brassica napus ZS11, and BnCYP90A1 transgenic positive plants com.#1 and com.#2; Thousand grain weight refers to thousand-grain weight, and *** indicates P<0.001. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0045] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] The overall experimental concept of the embodiment of the present invention is as follows:

[0048] Cloning of BnCYP90A1 gene: DNA and RNA of rapeseed ZS11 were used as templates, primers BnCYP90A1-F / BnCYP90A1-R and primers pBnCYP90A1-F / pBnCYP90A1-R were used for amplification, and the promoter and coding region of BnCYP90A1 were obtained. The above fragments were then fused with pCAMBIA1300 vector by homologous recombination and transferred into Escherichia coli DH5α to screen positive clones.

[0049] Agrobacterium-mediated genetic transformation: The obtained positive plasmid was transferred into Agrobacterium GV3101, and the dm1-s1 hypocotyl was used as the infection site. BnCYP90A1 was transferred into the mutant material. After hygromycin selection, independent resistant callus was obtained, which was further differentiated and regenerated into a complete transgenic strain.

[0050] Application of BnCYP90A1 gene: By comparing the silique length and 1000-grain weight of dm1-s1 and complement materials, it was found that compared with dm1-s1, the silique length and 1000-grain weight of complement materials were much greater than those of dm1-s1.

[0051] The culture medium configuration method used in the embodiment of the present invention is as follows:

[0052] M1 solid culture medium: weigh 4.4 g MS, 30 g sucrose, and dilute to 1 L aqueous solution with 18 g mannitol and 5.5 g plant gel. After high-temperature sterilization, add filter-sterilized 2,4-dichlorophenoxyacetic acid 1 mg, kinetin 0.3 mg, plant gel 5.5 g, and 100 μmol acetosyringone. Mix well and prepare a plate.

[0053] M2 solid medium: weigh 4.4 g MS, 30 g sucrose, 18 g mannitol, and 5.5 g plant gel, dilute to 1 L, sterilize at high temperature, add 1 mg of 2,4-dichlorophenoxyacetic acid, 0.3 mg of kinetin, 300 mg of timentin, 150 μmol of sodium thiosulfate, and 50 mg of hygromycin, mix well, and prepare the plate

[0054] M3 solid culture medium: weigh 4.4 g MS, 10 g glucose, 0.25 g xylose, and 0.6 g 2-morpholineethanesulfonic acid, dilute to 1 L, add 5.5 g plant gel, sterilize at high temperature for 20 min, then add 2 mg of filter-sterilized zeatin, 0.1 mg of indoleacetic acid, 300 mg of timentin, and 150 μmol of silver nitrate in sequence after the culture medium cools down, mix well, and then prepare a plate.

[0055] M4 medium: Weigh 4.4 g of MS and 10 g of sucrose and dissolve them in water, and make up to 1 L. Finally, add 5.5 g of plant gel and sterilize.

[0056] DM liquid culture medium: Weigh 4.4 g of MS and 30 g of sucrose and dissolve them in 1 L of culture medium. After sterilization, add 100 μmol of filter-sterilized acetosyringone.

[0057] Table 1 Sequences of primers used in the examples of the present invention

[0058]

[0059] The Brassica napus ZS11 (Zhongshuang No. 11) used in the embodiments of the present invention was bred by the Wuhan Oils and Fertilizers Research Institute of the Chinese Academy of Agricultural Sciences and has been disclosed in the document "Wu Yongcheng, Ma Ni, Huang Xiaoming, et al. Effect of nitrogen application rate on agronomic traits, yield quality and nitrogen fertilizer utilization rate of Zhongshuang No. 11 rapeseed [J]. Journal of Sichuan Agricultural University, 201, 32(03): 260-264+282." The applicant promises to distribute the above-mentioned biological materials to the public within 20 years from the date of application of the present invention.

[0060] The Brassica napus dm1-s1 mutant material used in the embodiments of the present invention is obtained by EMS mutagenesis of the Brassica napus cultivar ZS11. The applicant promises to release the above biological material to the public within 20 years from the filing date of the present invention.

[0061] like Figure 1 As shown, compared with rapeseed ZS11, the dm1-s1 mutant showed shorter plant height, shorter silique length and significantly smaller grains.

[0062] Example 1 Cloning of the promoter of rapeseed BnCYP90A1 and the coding region fragment of BnCYP90A1

[0063] 1. Extract RNA and DNA from Brassica napus ZS11 using Trizol and CTAB methods respectively

[0064] RNA extraction by Trizol method: The seedlings of rapeseed ZS11 were collected, and after being fully broken by liquid nitrogen, Trizol solution (Invitrogen, 15596026CN) was added. After vortexing and mixing evenly, chloroform was added and reacted on ice for 5-10 min. Subsequently, the supernatant was aspirated and mixed with an equal volume of isopropanol to precipitate RNA. Finally, the RNA was rinsed with anhydrous ethanol, dried at room temperature, and RNase-free water was added to obtain ZS11 rapeseed RNA.

[0065] CTAB method for DNA extraction: Collect rapeseed ZS11 seedlings, add CTAB solution to break them, and place them in a 65℃ constant temperature box for 1 hour. Then add chloroform / isoamyl alcohol mixture, mix evenly, centrifuge and transfer the supernatant to mix with an equal volume of anhydrous ethanol. After the white precipitate is precipitated, keep the precipitate, rinse twice, add double distilled water, and obtain DNA.

[0066] 2. PCR amplification to obtain the promoter and coding region fragment of BnCYP90A1

[0067] The rapeseed ZS11 seedling RNA was reverse transcribed using the reverse transcription kit (R423-01, Novozymes) according to the instructions to obtain ZS11 cDNA, and the obtained cDNA product was diluted 3 times as a PCR amplification template. Regarding the amplification of the BnCYP90A1 promoter, 1 μl of ZS11 DNA was taken as a template, and amplification primers pBnCYP90A1-F / pBnCYP90A1-R (primer sequences are shown in Table 1, and lowercase letters are homology arm primers) were added to perform PCR reaction. Regarding the amplification of the BnCYP90A1 coding region fragment, 1 μl of ZS11 cDNA was taken as a template, and primers BnCYP90A1-F / BnCYP90A1-R (primer sequences are shown in Table 1, and lowercase letters are homology arm primers) were added to perform amplification reaction. After the reaction was completed, the amplification products of the BnCYP90A1 promoter and the BnCYP90A1 coding region fragment were recovered by agarose gel electrophoresis; the amplification products were collected, purified, and sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing to obtain the gene coding region nucleotide sequence (CDS sequence) of the BnCYP90A1 gene, as shown in SEQ ID NO.1; the amino acid sequence of the BnCYP90A1 protein can be obtained as shown in SEQ ID NO.2; the nucleotide sequence of the BnCYP90A1 promoter is shown in SEQ ID NO.3.

[0068] SEQ ID NO.1:

[0069]

[0070] SEQ ID NO.2:

[0071] MAFSFSSTALLLLLSSITAGFLLLLRRTRYRRMGLPPGSLGLPLIGETLQLIAAYKTENPEPFVDARVARYGSVFMTHIFGEPTVFSADPETNRFVLQNEGKLFECSYPASICNLLGKHSLLLMKGSLHKRMHSLTSLTMSFANSSIIKDHLMLDIDRLVRFNLDSWSSRVLLMEEAKKITFELTVKQLMSFDPGEWSESLRKEYLLVIEGFFSIPLPLFSTTYRKAIKARTKVAEALTVVVMKRREEEEEGEEERKKDMLAALLAAEERFSDEEIVDFLVALLVAGYETTSTIMTLAVKFLTETPSALAQLKEEHENIRAMKSDSYSLEWSDYKSMPFTQCVVSETLRVANIIGGVFRRATTDVEIKGYKIPKGWKVFASFRAVHLDPNHFKDARTFNPWRWQGNSVTTCPSNVFTPFGGGPRLCPGYELARVALSVFLHRLVTGFSWVPAEKDKLVFFPTTRTQKRYPIIVTRRDDGGLSAT.

[0072] SEQ ID NO.3:

[0073]

[0074] Example 2 Overexpression vector construction

[0075] 1. Homologous recombination of overexpression vector

[0076] The plasmid expression vector pCAMBIA1300-pUBQ10-flag (the sequence of the expression vector pCAMBIA1300-pUBQ10-flag has been disclosed in patent CN118127038A) was extracted, and the vector was linearized using restriction endonucleases EcoRI and SpeI. Then, the homologous recombination kit (C116-01, Novezan) was used to perform homologous recombination according to the steps in the instruction manual to obtain the recombinant product.

[0077] 2. E. coli transformation of recombinant products and screening of positive clones

[0078] The recombinant product was added to the competent cells of E. coli DH5α, and the recombinant product was transferred into E. coli by heat shock propagation. The next day, the single clones grown were identified one by one using identification primers (BnCYP90A1-GT-F and BnCYP90A1-GT-R), the positive clones were propagated, and the plasmid was extracted and sequenced using the Thermo Fisher plasmid extraction kit (K0503, Thermo Fisher), and finally the pCAMBIA1300-pBnCYP90A1-BnCYP90A1-flag plasmid was obtained.

[0079] Example 3 Genetic transformation of rapeseed

[0080] Place the disinfected and sterilized ZS11 in a germination box for germination. During the seed germination period, transfer the target plasmid into Agrobacterium GV3101 to obtain the Agrobacterium required for transformation. After the seedlings of ZS11 germinate with hypocotyls of appropriate length, cut the hypocotyls and incubate them with Agrobacterium solution resuspended in DNA liquid. After the incubation is completed, transfer the hypocotyls to M1 medium for dark culture. After 2 days, transfer the hypocotyls to M2 medium and culture for 15 days. Continue to culture on M3 medium until germination. After germination, transfer to M4 medium until rooting occurs. When the seedlings grow to an appropriate size, move them into the soil for continued culture. The transgenic positive plants (complementary strains) com.#1 and com.#2 obtained are as follows Figure 2 shown.

[0081] Example 4 Identification of positive plants

[0082] Leaves from one oilseed rape plant were collected, frozen in liquid nitrogen, and then DNA was extracted using the CTAB method. PCR primers BnCYP90A1-GT-F and BnCYP90A1-GT-R (primer sequences are shown in Table 1) were designed using Primer Design software. The extracted transgenic rapeseed DNA was used as a template for amplification, and then analyzed by DNA gel electrophoresis to confirm the positive plants.

[0083] Example 5 Determination of rapeseed silique length and thousand-grain weight

[0084] The above transgenic materials were transplanted to the transgenic nursery for cultivation. There were 5 holes per row in the nursery, with a hole spacing of 20 cm, 3 seedlings per hole, and 5 rows of each transgenic line with a row spacing of 33 cm. After the transgenic materials grew naturally to maturity, 30 siliques of the main sequence were collected and the silique length was measured. At the same time, the thousand-grain weight of the harvested rapeseed grains was measured.

[0085] The results are as follows Figure 3-Figure 5 As shown in the figure, compared with the mutant line dm1-s1, the silique length of the plants of the complemented lines com.#1 and com.#2 increased significantly, and the 1000-grain weight increased significantly, indicating that BnCYP90A1 can significantly promote the development of rapeseed siliques and the enlargement of grains.

[0086] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of BnCYP90A1 gene in any of the following: (1) Application in regulating rapeseed grain size; (2) Application in breeding large-grain rapeseed varieties; (3) Application in regulating the length of rapeseed siliques; (4) Application in breeding silique rape varieties; The nucleotide sequence of the BnCYP90A1 gene is shown in SEQ ID NO.

1.

2. Use of the recombinant vector comprising the BnCYP90A1 gene according to claim 1 in any of the following: (1) Application in regulating rapeseed grain size; (2) Application in breeding large-grain rapeseed varieties; (3) Application in regulating the length of rapeseed siliques; (4) Application in the breeding of silique rape varieties.

3. Use of the engineered bacteria comprising the recombinant vector described in claim 2 in any of the following: (1) Application in regulating rapeseed grain size; (2) Application in breeding large-grain rapeseed varieties; (3) Application in regulating the length of rapeseed siliques; (4) Application in the breeding of silique rape varieties.

4. Use of BnCYP90A1 protein in any of the following: (1) Application in regulating rapeseed grain size; (2) Application in breeding large-grain rapeseed varieties; (3) Application in regulating the length of rapeseed siliques; (4) Application in breeding silique rape varieties; The amino acid sequence of the BnCYP90A1 protein is shown in SEQ ID NO.

2.

5. The use according to any one of claims 1 to 4, characterized in that: The expression level of the BnCYP90A1 gene is up-regulated to promote the increase of the rapeseed grains and / or the elongation of the rapeseed siliques.

6. A method for promoting the enlargement of rapeseed grains, characterized in that: The method comprises the step of overexpressing the BnCYP90A1 gene in rapeseed, wherein the nucleotide sequence of the BnCYP90A1 gene is shown as SEQ ID NO.

1.

7. A method for promoting the elongation of rapeseed siliques, characterized in that: The method comprises the step of overexpressing the BnCYP90A1 gene in rapeseed, wherein the nucleotide sequence of the BnCYP90A1 gene is shown as SEQ ID NO.

1.

8. The method according to claim 6 or 7, characterized in that The method of overexpressing the BnCYP90A1 gene in rapeseed comprises the following steps: constructing a recombinant plasmid by combining the BnCYP90A1 gene with an expression vector; and transferring the recombinant plasmid into rapeseed through Agrobacterium-mediated method to overexpress the BnCYP90A1 gene.

9. A method for cultivating large-grain rapeseed, characterized in that: The method comprises the step of overexpressing the BnCYP90A1 gene in rapeseed, wherein the nucleotide sequence of the BnCYP90A1 gene is shown as SEQ ID NO.

1.

10. A method for cultivating silique rape, characterized in that: The method comprises the step of overexpressing the BnCYP90A1 gene in rapeseed, wherein the nucleotide sequence of the BnCYP90A1 gene is shown as SEQ ID NO.1.

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