CN110452914B - Gene BnC04BIN2-like1 for regulating brassinolide signal transduction and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a gene BnC04BIN2-like1 for regulating brassinolide signal transduction and application thereof. The invention obtains the gene BnC04BIN2-like1 for negatively regulating and controlling the signal transduction of vegetable brassinolide by cloning and separating the brassica napus dwarf mutant material MB1501-1, and the gene and a reference gene have 11 base substitutions. The invention constructs a PBI121-BnC04BIN2-like1 overexpression vector and a CRISPR-BnC04BIN2-like1 vector, obtains a PBI121-BnC04BIN2-like1 overexpression transgenic plant, and has a dwarfing phenotype; and meanwhile, CRISPR-BnC04BIN2-like1 transgenic plants are obtained, and the transgenic plants recover the high-stem phenotype. The cloned BnC04BIN2-like1 gene can negatively regulate the signal transduction of brassinolide, thereby controlling the high character of rape.

Description

Gene BnC04BIN2-like1 for regulating brassinolide signal transduction and application thereof

Technical Field

The present invention belongs to the field of plant gene engineering technology. In particular to the separation and cloning, functional verification and application of a DNA fragment (gene) for regulating and controlling the synthesis of brassinolide of a cabbage type rape plant.

Background

Brassinolide (BRs) is a type of sterol plant hormone that promotes growth, also known as brassinolide, brassinosteroid. It is a natural plant hormone, widely exists in organs such as pollen, seeds, stems and leaves of plants, and has been internationally praised as the sixth hormone because its physiological activity greatly exceeds the existing five hormones. The functions of brassinolide include: plays an important role in regulating the elongation growth of stems, pollen tube growth, leaf extension, root growth, plant fertility, senescence and resistance. However, the known BR signal transduction components do not constitute a complete signal transduction pathway, and some problems remain to be elucidated.

With the development of applied disciplines such as genetics, molecular biology, biochemistry and the like, research related to BR signal transduction also benefits. Researchers screened the progeny population for bril mutants in 1996 among EMS mutagenesis progeny populations that appeared insensitive to BR and exhibited phenotypes such as dwarfing, dark green leaves, rolling under leaves, and the like. Next, the investigator cloned the BRI1 by map-based cloning, which encodes a leucine-rich repeat receptor protein kinase. The research group found the BR co-receptor BAK1 that can interact with BRI1 by yeast two-hybrid and using activation tagging. Although Russinova (2004) topic group demonstrated that BRI1 could form homodimers by means of fluorescence resonance energy transfer, new crystal structure data of Sheet et al (2011) showed that BRI1 might structurally form heterodimers with BAK1 to a greater extent, jointly accepting BR signals. By the yeast two-hybrid method, researchers screened BIK1, which interacted with BRI1 when BR levels were low in plants, to inhibit BRI1 recruitment to co-receptor BAK1, which in turn inhibits BR signaling downstream. When plants sense BR signals, BRI1 is activated by autophosphorylation, activated BRI1 in turn phosphorylates activation co-receptor BAK1, activated BAK1 in turn continues to phosphorylate sites not previously phosphorylated by BRI1, and all activated BRI1, together with active BAK1, direct BR signals downstream. BSU1 was screened by activation tagging and encodes a nuclear-localized serine/threonine protein kinase. BSU1 can be activated by phosphorylation of activated BSK1 and CDG1, and phosphorylation-activated BSU1 dephosphorylates BIN2, thereby inhibiting kinase activity thereof and promoting degradation process of BIN 2. So far, we can be briefly summarized as: after BR is combined with BRI1, BRI1 is released from the interaction state with BIK1 and combined with BAK1, the two comprehensively activate the activity of each other through a mutual phosphorylation mode, and then BSU1 is activated through BSK1 and CDG1, and the activated BSU1 can dephosphorize BIN2 to inhibit the activity of BIN 2.

In 2002 researchers found that bin2-1 exhibited a BR insensitive phenotype similar to bril. BIN2 encodes a GSK 3-like protein kinase that plays a major role in BR signaling. When the BR level in the plant is low, BIN2 is in an activated state, and active BIN2 can phosphorylate and inhibit the function of BZR1/BES 1. The binding site of BIN2 and BZR1 is 12 amino acids at the C terminal, and BZR1 phosphorylated by BIN2 is bound in cytoplasm by phosphorylated binding protein 14-3-3 and cannot enter into nucleus to be combined with DNA to block down stream conduction of BR signals. When the BR level is higher, the kinase activity of BIN2 is reduced firstly, and PP2A can rapidly dephosphorylate BZR1, so that dephosphorylated BZR1 enters into nucleus to regulate the expression of downstream genes, and BR signals are transmitted downwards.

In the 90 s of the 20 th century, with the application of genomics, researchers demonstrated that cell elongation was severely affected in most BR-deficient and insensitive mutants, with the hypocotyl length shortening in the dark and dwarfing phenotype in the light (salchert et al, 1998). The AtCPD gene encodes a cytochrome P450(CYP90) protein with a conserved domain of sterol hydroxylase, deletion of which results in dwarfing of plants, and exo-application of BR or overexpression of cDNA of CPD all restore wild-type phenotype (Szekeres et al, 1996). Dwf4 mutant strains also belong to BR synthesis-deficient dwarf mutants, which do not restore the dwarf phenotype by any hormone other than brassinolide (Azpiroz et al, 1998). The rice BR-deficient mutant brd1 (BR-specific dwarf1) has a phenotype of almost no internode elongation, shortened leaf sheath, short and short leaf blade with severe curling, less tillering and sterility, and is the first BR-deficient mutant found in rice. The mutant can recover phenotype after exogenous application of BR. The BRD1 gene encodes a C-6 oxidase, belongs to the early C-6 oxidation pathway, and can cause rice plant dwarfing after mutation (Hong et al, 2002). A similar BR deficient mutant is also bul1-1, which is also inhibited in cell elongation and observed microscopically to show a significant reduction in parallel microtubule organization compared to the wild type. The gene encodes a Δ 7-sterol-C-dehydrogenase whose deletion affects the structure of the cell by affecting the amount of brassinolide in the plant, resulting in a dwarf phenotype (cotterou et al, 2001).

The invention finds a gene which is derived from rape and can regulate brassinolide signal transduction, the gene can reduce the regulated brassinolide signal transduction after overexpression, thereby reducing the synthesis of auxin in plants and finally causing the dwarfing of the plants, and when the expression of the gene is interfered, the plants can recover the regulated brassinolide signal transduction, thereby recovering the synthesis of the auxin in the plants and ensuring the growth of dwarfing materials.

Disclosure of Invention

The invention aims to provide a gene for regulating brassinolide signal transduction.

The gene for regulating and controlling brassinolide signal transduction, which is provided by the invention, is named as BR-INSENSITIVE-2-like1 (BIN 2-like1 or BnC04BIN2-like1 for short), is derived from Brassica napus (Brassica napus), is cloned from a Brassica napus dwarf mutant material MB1501-1 to obtain a gene fragment, and is replaced by 11 bases with a French rape database published in 2014 (updated in 2017).

A gene BnC04BIN2-like1 for regulating brassinolide signal transduction is one of the following amino acid residue sequences, has 2 amino acid residue substitutions with a French rape database published in 2014 (updated in 2017), and is located in aa-187 and aa-309:

(1) SEQ ID NO: 1;

(2) and (3) mixing the amino acid sequence shown in SEQ ID NO: 1 through substitution and/or deletion and/or addition of one to ten amino acid residues, and has the function of regulating and controlling signal transduction of plant brassinolide.

SEQ ID NO: 1 consists of 410 amino acid residues, and amino acid residues 65-357 from the amino terminal (N terminal) are conserved sequences.

The one to ten amino acid residues substituted and/or deleted and/or added are amino acid residues in the non-structural domain, and the change thereof does not affect the function of the protein.

The cDNA of the gene (BR-INSENSITIVE-2-like1, BIN2-like1 or BnC04BIN2-like1) for regulating and controlling the signal transduction of the vegetable brassinolide is one of the following nucleotide sequences, has 11-base substitution with a French rape database published in 2014 (updated in 2017), and is positioned at the 21,135,147,312,399,454,466,559,615,618 and 1197 bases from the 5' end:

(1) SEQ ID NO: 2;

(2) encoding the amino acid sequence shown in SEQ ID NO: 1;

(3) and SEQ ID NO: 2 the DNA sequence has more than 90% of homology and has a nucleotide sequence for regulating and controlling the signal transduction of the vegetable brassinolide;

(4) can be combined with the sequence shown in SEQ ID NO: 2 to the DNA sequence defined in the specification.

The high stringency conditions are hybridization and membrane washing in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃.

SEQ ID NO: 2 consists of 1672 bases, the coding sequence of the gene is 73 th to 1305 th bases on the 5' end, and the gene codes a nucleotide sequence with SEQ ID NO: 1, and the conserved sequence is encoded by the 265 rd and 1143 rd bases from the 5' end.

The genome gene is one of the following nucleotide sequences:

(1) SEQ ID NO: 3 in the sequence listing;

(2) and SEQ ID NO: 3, the DNA sequence has more than 90 percent of homology and has a nucleotide sequence for regulating and controlling the signal transduction of the vegetable brassinolide;

(3) can be combined with the sequence shown in SEQ ID NO: 3 to the DNA sequence defined in the specification.

The high stringency conditions are hybridization and membrane washing in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃.

SEQ ID NO: 3 consists of 3314 bases, the 73-178 bases from the 5' end are the first exon of the genome gene, the 179-755 bases from the 5' end are the first intron of the genome gene, the 756-846 bases from the 5' end are the second exon of the genome gene, the 847-1035 bases from the 5' end are the second intron of the genome gene, the 1036-1097 bases from the 5' end are the third exon of the genome gene, the 1098-1187 bases from the 5' end are the third intron of the genome gene, the 1188-1459 bases from the 5' end are the fourth exon of the genome gene, the 1460-1547 bases from the 5' end are the fourth intron of the genome gene, and the 1548-1624 bases from the 5' end are the fifth exon of the genome gene, the 1625-position 1701 base from the 5' end is the fifth intron of the genome gene, the 1702-position 1758 base from the 5' end is the sixth exon of the genome gene, the 1759-position 1855 base from the 5' end is the sixth intron of the genome gene, the 1856-position 1994 base from the 5' end is the seventh exon of the genome gene, the 1995-position 2083 base from the 5' end is the seventh intron of the genome gene, the 2084-position 2136 base from the 5' end is the eighth exon of the genome gene, the 2137-position 2245 base from the 5' end is the eighth intron of the genome gene, the 2341-position 2341 base from the 5' end is the ninth exon of the genome gene, the 2430-position 2342-position 2430 base from the 5' end is the ninth intron of the genome gene, and the tenth 2512-position 2512 base from the 2431-position of the genome gene, the 2513-2629 th base from the 5 'end is the tenth intron of the genomic gene, the 2630-2731 th base from the 5' end is the eleventh exon of the genomic gene, the 2732-2851 th base from the 5 'end is the eleventh intron of the genomic gene, the 2852-2947 th base from the 5' end is the twelfth exon of the genomic gene, the 1-72 th base from the 5 'end is the 5' end non-coding region (UTR) of the genomic gene, and the 2948-3314 th base from the 5 'end is the 3' end non-coding region of the genomic gene.

The gene BnC04BIN2-like1 is related to brassinolide signal transduction. After the complete Coding sequence (Coding sequence) of the gene is combined with an over-expression vector PBI121, the gene is directly transferred into a conventional good variety of the brassica napus, namely double 11(ZS11), and the height of a transgenic plant is obviously shorter than that of a control plant; the constructed gene CRISPR/Cas9 vector is directly transferred into a brassica napus dwarf variety 'MB 1501-1', and the plant height of the transgenic plant can be recovered to be normal. The BnC04BIN2-like1 gene can negatively regulate the signal transduction of brassinolide, so that the high character of rape is controlled.

The expression vector, the transgenic cell line, the host bacterium containing the gene and the protein coded by the host bacterium belong to the protection scope of the invention.

The primer and the primer pair for amplifying any fragment of BnC04BIN2-like1 gene are also within the protection scope of the invention.

The cloned gene BnC04BIN2-like1 can be used for lodging-resistant and compact rape breeding.

The specific operation steps are as follows:

(1) introducing the gene BnC04BIN2-like1 into a cabbage type rape receptor by utilizing an agrobacterium-mediated transgenic method to obtain a transformed plant;

(2) analyzing and identifying the positive transgenic plant by means of a PCR method;

(3) carrying out field planting on the transgenic plant in the step (2) and observing the character of the transgenic plant;

(4) the expression of the gene BnC04BIN2-like1 related to brassinolide signal transduction in the transgenic plants and wild plants is analyzed by RT-PCR.

BnC04BIN2-like1 has the following beneficial effects:

the gene BnC04BIN2-like1 is related to brassinolide signal transduction. After the complete Coding sequence (Coding sequence) of the gene is combined with an over-expression vector PBI121, the gene is directly transferred into the conventional good variety of the cabbage type rape double 11(ZS11), and the height of a transgenic plant is obviously higher than that of the plant of the conventional good variety of the cabbage type rape double 11(ZS 11); the constructed gene CRISPR/Cas9 vector is directly transferred into a brassica napus dwarf rape variety 'MB 1501-1', and the plant height of a transgenic plant can restore the high-stalk phenotype. The BnC04BIN2-like1 gene can negatively regulate the signal transduction of brassinolide, so that the high character of rape is controlled.

Drawings

FIG. 1 is a frame structure of BnC04BIN2-like1 genomic gene;

FIG. 2 is a diagram of the protein sequence analysis of BnC04BIN2-like 1;

FIG. 3 is a schematic diagram of the construction of a plant overexpression vector PBI 121;

FIG. 4 is a schematic diagram of plant CRISPR/Cas9 vector construction;

FIG. 5 shows phenotype of BnC04BIN2-like1 gene over-expressed transgenic plants; the left side is a wild type ZS11 individual plant, and the right side is a PBI121-BnC04BIN2-like1 transgenic plant phenotype.

FIG. 6 shows BnC04BIN2-like1 gene CRISPR/Cas9 vector transgenic plant phenotype; wherein the left side is CRISPR-BnC04BIN2-like1 transgenic plant phenotype, the middle is heterozygous dwarf plant, and the right side is high-stem plant.

Detailed Description

The methods used in the following examples are conventional methods unless otherwise specified.

Example 1

Clone for regulating and controlling vegetable brassinolide signal transduction gene BnC04BIN2-like1

Extracting total RNA of fresh leaves of Brassica napus (Brassica napus) by using TRIZAL reagent and referring to kit instructions, then using Reverse Transcription kit of Takara company and according to kit instructions to reversely synthesize cDNA, using the synthesized cDNA as a template, and using the disclosed genome of the Brassica napus as a reference to design a primer, and adding the primer P1 (upstream primer): 5'-ATAGCACATGACATCACTATC-3' and P2 (downstream primer): 5'-TCTCCTCTTTTCCACAAG-3', PCR amplifying the gene regulating the signal transduction of the vegetable brassinolide in the rape, after the reaction, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, adopting an AXYGEN centrifugal column type gel recovery kit, recovering and purifying the target band according to the instructions, connecting the recovered product into an Easy Blunt Simple vector, and transforming the escherichia coli (E.coli) DH5 alpha competent cell by a heat shock method. And (3) screening positive clones by using the blue-white spots, inoculating the positive clones into a kanamycin-containing LB liquid culture medium, culturing at 37 ℃ and 200rpm, extracting plasmids, sequencing the plasmids, and indicating that the amplified fragments have the nucleotide sequences shown in SEQ ID NO: 2 consisting of 1233 bases, the site of the coding sequence is the 1 st to 1233 rd bases from the 5' end, and the coding sequence has the sequence shown in SEQ ID NO: 1, wherein the base at position 193-1071 from the 5' end encodes a conserved sequence. The genome gene of the gene has the nucleotide sequence shown in SEQ ID NO: 3 consisting of 3314 bases, the 73-178 bases from the 5' end being the first exon of the genomic gene, the 179-755 bases from the 5' end being the first intron of the genomic gene, the 756-846 bases from the 5' end being the second exon of the genomic gene, the 847-1035 bases from the 5' end being the second intron of the genomic gene, the 1036-1097 bases from the 5' end being the third exon of the genomic gene, the 1098-1187 bases from the 5' end being the third intron of the genomic gene, the 1188-1459 bases from the 5' end being the fourth exon of the genomic gene, the 1460-1547 bases from the 5' end being the fourth intron of the genomic gene, the 1544 bases from the 5' end being the fifth exon of the genomic gene, the 1625-position 1701 base from the 5' end is the fifth intron of the genome gene, the 1702-position 1758 base from the 5' end is the sixth exon of the genome gene, the 1759-position 1855 base from the 5' end is the sixth intron of the genome gene, the 1856-position 1994 base from the 5' end is the seventh exon of the genome gene, the 1995-position 2083 base from the 5' end is the seventh intron of the genome gene, the 2084-position 2136 base from the 5' end is the eighth exon of the genome gene, the 2137-position 2245 base from the 5' end is the eighth intron of the genome gene, the 2341-position 2341 base from the 5' end is the ninth exon of the genome gene, the 2430-position 2342-position 2430 base from the 5' end is the ninth intron of the genome gene, and the tenth 2512-position 2512 base from the 2431-position of the genome gene, the 2513-2629 th base from the 5 'end is the tenth intron of the genomic gene, the 2630-2731 th base from the 5' end is the eleventh exon of the genomic gene, the 2732-2851 th base from the 5 'end is the eleventh intron of the genomic gene, the 2852-2947 th base from the 5' end is the twelfth exon of the genomic gene, the 1-72 th base from the 5 'end is the 5' end non-coding region (UTR) of the genomic gene, and the 2948-3314 th base from the 5 'end is the 3' end non-coding region of the genomic gene. The structural frame of the gene is shown in FIG. 1.

Example 2

BnC04 acquisition of BIN2-like1 overexpression transgenic plant

Construction of plant over-expression vector containing BnC04BIN2-like1 gene

Adding Xba I restriction endonuclease site in CDS sequence upstream of BnC04BIN2-like1 gene, and adding upstream primer P3: 5'-GCTCTAGAATGACATCACTATC-3', adding a Sma I restriction endonuclease site downstream primer P4 to the CDS sequence of BnC04BIN2-like1 gene: 5'-TCCCCCGGGCTAATGACCAGGCT-3' are provided. Primers P3 and P4 were used to determine the nucleotide sequence of SEQ ID NO: 2, carrying out PCR amplification, carrying out 1% agarose gel electrophoresis detection on a PCR amplification product after the reaction is finished, adopting an AXYGEN centrifugal column type gel recovery kit, recovering a target band according to instructions, purifying the target band, connecting the recovered product into a carrier Easy BluntSimple, and transforming an escherichia coli (E.coli) DH5 alpha competent cell by a heat shock method. And (3) screening positive clones by using the blue-white spots, inoculating the positive clones into a kanamycin-containing LB liquid culture medium, culturing at 37 ℃ and 200rpm, extracting plasmids, sequencing the plasmids, and indicating that the amplified fragments have the nucleotide sequences shown in SEQ ID NO: 2 adding nucleotide sequences of restriction endonucleases Xba I and Sma I sites, carrying out restriction enzyme digestion on the constructed plasmid containing BnC04BIN2-like1 gene by using the restriction endonucleases Xba I and Sma I, carrying out 1% agarose gel electrophoresis detection on the digestion product, recovering BnC04BIN2-like1 gene fragment with the length of about 1233, purifying the gene fragment, linking the recovered fragment with a vector PBI121 subjected to the same restriction enzyme digestion by using T4DNA ligase (Takara), transforming the ligation product into escherichia coli (E.coli) DH5 alpha competent cells by using a heat shock method, screening positive clones, inoculating the obtained product into an LB plasmid culture medium containing kanamycin, culturing at 37 ℃ and 200rpm, extracting, carrying out restriction enzyme digestion identification on recombinant plasmids by using the restriction endonucleases Xba I and Sma I, conforming to the expected result, further carrying out PCR identification by using primers P3 and P4, and obtaining a 1233bp DNA fragment amplified by PCR, consistent with the expected results, the plant expression vector containing BnC04BIN2-like1 with correct insertion sequence and position is obtained and named PBI121-BnC04BIN2-like 1.

Secondly, obtaining PBI121-BnC04BIN2-like1 transgenic rape

Transforming Agrobacterium EHA105 competent cells by a heat shock method with the plant expression vector PBI121-BnC04BIN2-like1 constructed in the first step, spreading the competent cells on LB resistant plates containing kanamycin and rifampicin, culturing at 28 ℃ and 150rpm, picking out single colonies of the Agrobacterium grown out, inoculating the single colonies into 20ml LB liquid culture medium containing kanamycin and rifampicin, culturing at 28 ℃ and 150rpm for 2 days, inoculating the single colonies into 300ml LB liquid culture medium containing kanamycin and rifampicin according to 2% of inoculum concentration, and culturing at 28 ℃ and 150rpm for 16-18 hours. After completion of the culture, the cells were centrifuged at 5000rpm for 20 minutes, and the cells were collected and suspended in 250ml of a solution containing 5% sucrose and 0.1% Silwetl-77. And finally, transferring the bacterial liquid into a 250ml beaker, removing the cabbage type rape flowers after pollination, and pouring the plants into the beaker to ensure that the inflorescences of the plants completely invade the bacterial liquid, wherein the steps are repeated after one week in order to improve the transformation efficiency. And (3) carrying out conventional culture on the transformed plant, harvesting seeds, and identifying and screening the obtained seeds by kanamycin and PCR to obtain BnC04BIN2-like1 transgenic plants.

Thirdly, PBI121-BnC04BIN2-like1 transgenic plant phenotype observation

And (3) planting seeds of the positive transgenic plants which overexpress BnC04BIN2-like1 and are obtained in the step three, and using wild type brassica napus as a control under field conditions. The growth conditions of BnC04BIN2-like1 overexpression plants and wild plants are observed, the phenotype of BnC04BIN2-like1 overexpression plants growing under natural conditions is shown in figure 5, and BnC04BIN2-like1 overexpression plants are dwarfed and weak.

Example 3

Acquisition of CRISPR-BnC04BIN2-like1 transgenic plant

Construction of plant CRISPR/Cas9 vector containing BNC04BIN2-LIKE1 gene

2 targets were designed for BnC04BIN2-like1 gene, one in ORF-5 'region, primers designed as P5(5' gRT1 +): 5'-CTCCGTCGCGAAGCTGCGGGTTTTAGAGCTAGAAAT-3', P6(5' U3dT 1-): 5'-CAAAGCACCATTGGTCACTCCGTCGCGAAGCTGCGG-3', respectively; one in the conserved sequence region, the primer was designed as P7 (conserved gRT2 +): 5'-ACCGAGCCTGCGTTACACTGGTTTTAGAGCTAGAAAT-3', P8 (conserved U3dT 2-): 5'-CAAAGCACCATTGGTCAACCGAGCCTGCGTTACACTG-3' are provided. sgRNA expression cassette adaptor primer reverse primer P9: 5'-CTCCGTTTTACCTGTGGAATCG-3' and adaptor forward primer 5'-CGGAGGAAAATTCCATCCAC-3'. 4 primers were used in each sgRNA expression cassette construction reaction: p9 and P10 each at 0.2. mu.M, (P5 or P7) and (P6 or P8) each at 0.1. mu.M. And (2) 25-28 circulation: 94 ℃ for 10s, 58 ℃ for 15s and 68 ℃ for 20 s. During the amplification process, P9/(P6 or P8) expands P9- (P6 or P8) sequence and P10/(P5 or P7) expands (P5 or P7) -sgRNA sequence at the first few cycles. Later cycles generated 2 fragment-pooled sgRNA expression cassette fragments by overlapping PCR. Taking 1. mu.l of PCR product and using H₂O dilution 10 times, 1. mu.l of each expression cassette was taken as a template, and 20-50. mu.l of each PCR (1 target 50. mu.l; 2 targets 30. mu.l each) was performed. 1/10 amounts of each primer combination working solution (final concentration 0.15. mu.M) were added. The appropriate amount of KOD-Plus high fidelity PCR enzyme was used. Amplification for 20 cycles: 95 ℃ for 10s, 58 ℃ for 15s and 68 ℃ for 20 s. 2-3. mu.l of the electrophoresis was taken to check for product length agreement and to estimate the approximate concentration of the sample. Enzyme digestion-ligation reaction of binary vector and sgRNA expression cassette 10 XCutSmart Buffer (1.5. mu.l), 10mM ATP (1.5. mu.l), pYRCISPR/Cas 9 plasmid (80ng), sgRNA expression cassette mixture (15ng), Bsa I-HF (10U), T4DNA ligase (35U), H₂O was added to 15. mu.l. Performing enzyme digestion connection by temperature-variable circulation at 37 deg.C for 5min for about 10-15 cycles; 5min at 10 ℃ and 5min at 20 ℃; finally 5min at 37 ℃. E.coli DH10B competent cells were heat shock transformed with 1-1.5. mu.l of the ligation product, and 1ml of SOC was added after heat shock and cultured at 37 ℃ for 1.5 h. And selecting a positive monoclonal, culturing by using a liquid LB +25 mu g/ml Kan culture medium at 37 ℃, and sequencing specific targets by using the specificity of each target primer. Sequencing the correct bacterial liquid, and showing that the CRISPR/Cas9 vector containing BnC04BIN2-like1 and having the correct insertion sequence is obtained and is named as CRISPR-BnC04BIN2-like1。

II, obtaining CRISPR-BnC04BIN2-like1 transgenic rape

Transforming agrobacterium EHA105 competent cells by a heat shock method by using the plant CRISPR-BnC04BIN2-like1 vector constructed in the step one, coating the competent cells on LB resistant plates containing kanamycin and rifampicin, culturing at 28 ℃ and 150rpm, picking out a single colony of the grown agrobacterium, inoculating the single colony into 20ml of LB liquid culture medium containing kanamycin and rifampicin, culturing at 28 ℃ and 150rpm for 2 days, then inoculating the bacterial liquid into 300ml of LB liquid culture medium containing kanamycin and rifampicin according to 2% of inoculum concentration, and culturing at 28 ℃ and 150rpm for 16-18 hours. After completion of the culture, the cells were centrifuged at 5000rpm for 20 minutes, and the cells were collected and suspended in 250ml of a solution containing 5% sucrose and 0.1% Silwetl-77. And finally, transferring the bacterial liquid into a 250ml beaker, removing the cabbage type rape flowers after pollination, and pouring the plants into the beaker to ensure that the inflorescences of the plants completely invade the bacterial liquid, wherein the steps are repeated after one week in order to improve the transformation efficiency. And (3) carrying out conventional culture on the transformed plant, harvesting seeds, and identifying and screening the obtained seeds by kanamycin and PCR to obtain BnC04BIN2-like1 transgenic plants.

Thirdly, the observation of the phenotype of the CRISPR-BnC04BIN2-like1 transgenic plant

And (3) planting seeds of the CRISPR-BnC04BIN2-like1 vector positive transgenic plant obtained in the step two and taking the wild type of the brassica napus as a control under field conditions. Observing the growth conditions of the CRISPR-BnC04BIN2-like1 vector and wild plants, the phenotype of the CRISPR-BnC04BIN2-like1 transgenic plants grown under natural conditions is shown in figure 6, and the dwarf plants recover the high-stalk phenotype.

Sequence listing

<110> Nanjing university of agriculture

<120> gene BnC04BIN2-like1 for regulating brassinolide signal transduction and application thereof

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Met Thr Ser Leu Ser Leu Gly Pro Gln Pro Pro Ala Thr Ala Gln Pro

1 5 10 15

Pro Gln Leu Arg Asp Gly Asp Ala Ser Arg Arg Arg Ser Asp Met Asp

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Thr Asp Lys Asp Met Ser Ala Ala Val Ile Glu Gly Asn Asp Ala Val

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Thr Gly His Ile Ile Ser Thr Thr Ile Gly Gly Lys Asn Gly Glu Pro

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Lys Gln Thr Ile Ser Tyr Met Ala Glu Arg Val Val Gly Gln Gly Ser

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Phe Gly Ile Val Phe Gln Ala Lys Cys Leu Glu Thr Gly Glu Ser Val

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Ala Ile Lys Lys Val Leu Gln Asp Arg Arg Tyr Lys Asn Arg Glu Leu

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Gln Leu Met Arg Leu Met Asp His Pro Asn Val Val Ser Leu Lys His

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Cys Phe Phe Ser Thr Thr Ser Arg Asp Glu Leu Phe Leu Asn Leu Val

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Met Glu Tyr Val Pro Glu Thr Leu Tyr Arg Val Leu Lys His Tyr Thr

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His Arg Asp Val Lys Pro Gln Asn Leu Leu Val Asp Pro Leu Thr His

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Gln Cys Lys Leu Cys Asp Phe Gly Ser Ala Lys Val Leu Val Lys Gly

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Glu Ala Asn Ile Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro Glu

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Leu Ile Phe Gly Ala Thr Glu Tyr Thr Ser Ser Ile Asp Ile Trp Ser

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Ala Gly Cys Val Leu Ala Glu Leu Leu Leu Gly Gln Pro Leu Phe Pro

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Gly Glu Asn Ser Val Asp Gln Leu Val Glu Ile Ile Lys Val Leu Gly

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Thr Pro Thr Arg Glu Glu Ile Arg Cys Met Asn Pro Asn Tyr Thr Asp

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Phe Arg Phe Pro Gln Ile Lys Ala His Pro Trp His Lys Val Phe His

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Lys Arg Met Pro Pro Glu Ala Ile Asp Leu Ala Ser Arg Leu Leu Gln

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Pro Glu Leu Ile Asn Arg Leu Ile Pro Glu His Ile Arg Arg His Met

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Ser Gly Gly Phe Pro Ser Gln Pro Gly His

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gaagaatagc acatgacatc actatcattg ggccctcagc ctccggctac tgctcagccg 120

ccgcagcttc gcgacggaga tgcttccagg cgtcgttccg atatggatac agacaaggat 180

atgtctgctg ctgtgataga gggaaacgat gctgttacag gccacatcat ttctactaca 240

attggaggca aaaacggtga acctaaacag accattagtt acatggcgga acgggttgtt 300

ggacaaggat cattcggaat cgtgttccag gccaagtgct tggaaactgg agaatctgta 360

gccattaaga aggtcttgca agaccggcgc tacaagaatc gtgagctgca gttgatgcga 420

ctaatggacc acccaaatgt ggtttccttg aagcattgtt tcttctctac tacgagtaga 480

gatgagctct tcctcaatct cgttatggag tatgtacccg agactttgta ccgggttctg 540

aagcactata ctagttcaag ccagagaatg cctattttct atgtcaaact ctacacatac 600

caaatcttca gaggcttggc ttatatccat actgttcctg gtgtctgtca cagagatgtg 660

aaaccacaaa atcttttggt tgatcccctt actcatcagt gtaagctgtg tgattttgga 720

agtgcaaaag tattggtgaa aggtgaagca aacatatcat acatctgctc tcggtattac 780

cgagctccag agctgatctt tggggccaca gagtatacat cctccataga catatggtct 840

gctggttgtg ttttggcaga gctccttctt ggccagccgt tgttcccggg agaaaattct 900

gtggaccagc tggtggagat catcaaggtt cttggtactc caacccgaga agaaatccga 960

tgcatgaatc caaactacac agacttcaga ttcccgcaaa tcaaagcaca cccgtggcat 1020

aaggttttcc ataagaggat gcctcctgaa gccattgacc tcgcatctcg gcttcttcag 1080

tattcaccga gcctgcgtta cactgcgctt gaagcatgtg cacatccgtt tttcaatgaa 1140

ctccgtgagc ccaatgctcg tcttccaaac ggccgacctc taccagcctt gttcaacttc 1200

aaacaagagt tagctggggc ttcaccagag ctgataaaca ggctcatacc ggagcacata 1260

aggcgacaga tgagtggagg cttcccatca cagcctggtc attagaaaag gaatatggaa 1320

actgagatgc ttttgcggag caaatgcctt gtggaaaaga ggagagaaga ttttacttgt 1380

ctctgatttt tcagtgggtt taactaaaat atcagcttat gagtagagag atgattggcc 1440

aattaagctt tttgagaaat caggaagtga tgatgattgt gtctattata cattctctct 1500

ctctcttttt atgttataat tcgcttttga cttgtagaga gatacctttt tctcgttgta 1560

ttatttgtat atgtttttgt tcgtaagaca gcaaaccgcg atgatggaag aatggaatga 1620

acgatgtcta aaacttaagc ctaatagcaa ggtcggagct tatacttaca ca 1672

<210> 3

<211> 3314

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gggaaagata gtctttactc ttcagtggtg ggtagagagc gaaagttaga gaaagagaga 60

gaagaatagc acatgacatc actatcattg ggccctcagc ctccggctac tgctcagccg 120

ccgcagcttc gcgacggaga tgcttccagg cgtcgttccg atatggatac agacaaggtt 180

gctctctccc tctctctctc tctctctcta tccactttaa cgtttggtga acaaattgca 240

tttcgattgg ctattgtaga tctcgcttag atctagcttc gatttcactt tgttttgcgg 300

tttctcagcg aatcgatctg tgttttctct tgctatcgtt tcgcgttcct tgatttgagc 360

tcactcgtcg gagttcgtag tagctatagc tagtcttact attcagctga atgtttcaac 420

caatcatagt gaagatcttg agctagattt tgattactag tattagggtg aaattcgatt 480

tcacaggagt atggagacga tttagatctt aattactaga ttgtttaact aatcacacgc 540

ttgttccatg actgtaagtg atttggtgta tttgatttac atttgtttgt tatttacttg 600

attggactct gaactaggcc tttgactgtt cttggatttg aagatttcat atgtttaaag 660

aatgattttg tctactgatt gttgtcatat cgtaatctca tgtttattgt ttacagaaga 720

atagtttaat gatatggtga ggttttggtt gcaggatatg tctgctgctg tgatagaggg 780

aaacgatgct gttacaggcc acatcatttc tactacaatt ggaggcaaaa acggtgaacc 840

taaacaggtt tgagttcctt tctttgtttg aaatcttcaa ttgtccttat tagtagtcat 900

tgctaatgat tatatagctt ttccacttat agcttaaaac aataactaaa cagagactct 960

ttgtggttca tttattacca actttaagta ggctacagct actcacttat gttttactca 1020

ttctgttttt ttacagacca ttagttacat ggcggaacgg gttgttggac aaggatcatt 1080

cggaatcgtg ttccaggtac ctttgtgctt ttcactcgct gttatcattt gtaggcaata 1140

gctttcttct ttcttttctg atcggattat taccttacca ttgtaggcca agtgcttgga 1200

aactggagaa tctgtagcca ttaagaaggt cttgcaagac cggcgctaca agaatcgtga 1260

gctgcagttg atgcgactaa tggaccaccc aaatgtggtt tccttgaagc attgtttctt 1320

ctctactacg agtagagatg agctcttcct caatctcgtt atggagtatg tacccgagac 1380

tttgtaccgg gttctgaagc actatactag ttcaagccag agaatgccta ttttctatgt 1440

caaactctac acataccaag tatgcattgt tatttatctg tttccctttc aggcagtatc 1500

tctctttgtt gattctaaaa cgggtaagaa tacttttttt tctgcagatc ttcagaggct 1560

tggcttatat ccatactgtt cctggtgtct gtcacagaga tgtgaaacca caaaatcttt 1620

tggtatgtta attctgtttt gggtttgtct tcggcgatct ttactagatt gtaatctaat 1680

aatttggtat gttctgtagg ttgatcccct tactcatcag tgtaagctgt gtgattttgg 1740

aagtgcaaaa gtattggtaa ggagctttac ctttaatatc ctgctttgct tatttcaact 1800

gtgtatgtgt tctgtctcat gaaatcattg caacacatga ttattcggat taggtgaaag 1860

gtgaagcaaa catatcatac atctgctctc ggtattaccg agctccagag ctgatctttg 1920

gggccacaga gtatacatcc tccatagaca tatggtctgc tggttgtgtt ttggcagagc 1980

tccttcttgg ccaggttagt gtaaactatt ttatctgttt aaatataact ctagaatgtt 2040

ccgctatcat ttttgatata tatataattt ttttatctgt cagccgttgt tcccgggaga 2100

aaattctgtg gaccagctgg tggagatcat caaggtgaag tttcattttg atcagatgtt 2160

accttactgt cgtattctgt tttgtatata aaactcatat aatcttatag atttgtaatg 2220

atatatgtgc tgcgtttgtt taggttcttg gtactccaac ccgagaagaa atccgatgca 2280

tgaatccaaa ctacacagac ttcagattcc cgcaaatcaa agcacacccg tggcataagg 2340

tatctaatat gcttgtcact ttctaacatg tcggataata caatggctta atagttggct 2400

cgctacctaa ttcctctatg acatccaggt tttccataag aggatgcctc ctgaagccat 2460

tgacctcgca tctcggcttc ttcagtattc accgagcctg cgttacactg cggtcagtat 2520

atttaagcca cttagtactc ttacttgtta gagtgattct ctctggattc ttcagtgatg 2580

ctgatgtttt ctttttaact gacatttgtt tgttttatgt gtgtaaaagc ttgaagcatg 2640

tgcacatccg tttttcaatg aactccgtga gcccaatgct cgtcttccaa acggccgacc 2700

tctaccagcc ttgttcaact tcaaacaaga ggtacgtcaa tcacagcaaa aaaaaggaag 2760

taatatagct ccaaaccata actagaatgt tcagttttaa acagttacct aatctgtaat 2820

ctctctctct ctattcgaat gttcataaca gttagctggg gcttcaccag agctgataaa 2880

caggctcata ccggagcaca taaggcgaca gatgagtgga ggcttcccat cacagcctgg 2940

tcattagaaa aggaatatgg aaactgagat gcttttgcgg agcaaatgcc ttgtggaaaa 3000

gaggagagaa gattttactt gtctctgatt tttcagtggg tttaactaaa atatcagctt 3060

atgagtagag agatgattgg ccaattaagc tttttgagaa atcaggaagt gatgatgatt 3120

gtgtctatta tacattctct ctctctcttt ttatgttata attcgctttt gacttgtaga 3180

gagatacctt tttctcgttg tattatttgt atatgttttt gttcgtaaga cagcaaaccg 3240

cgatgatgga agaatggaat gaacgatgtc taaaacttaa gcctaatagc aaggtcggag 3300

cttatactta caca 3314

Claims (9)

1. A gene regulating brassinolide signal transduction, wherein the protein sequence encoded by the gene is as shown in SEQ ID NO: 1 is shown in the specification;

SEQ ID NO: 1 consists of 410 amino acid residues, and amino acid residues 65-357 from the amino terminal (N terminal) are conserved sequences.

2. The gene of claim 1, having the sequence of SEQ ID NO: 2, respectively.

3. The gene of claim 2, wherein the amino acid sequence of SEQ ID NO: 2 consists of 1672 bases, the coding sequence of the gene is 73 th to 1305 th bases on the 5' end, and the gene codes a nucleotide sequence with SEQ ID NO: 1, and the conserved sequence is encoded by the 265 rd and 1143 rd bases from the 5' end.

4. The gene of claim 1, wherein the sequence of the gene is as shown in SEQ ID NO: 3, respectively.

5. The gene of claim 4, wherein SEQ ID NO: 3 consists of 3314 bases, the 73-178 bases from the 5' end are the first exon of the genome gene, the 179-755 bases from the 5' end are the first intron of the genome gene, the 756-846 bases from the 5' end are the second exon of the genome gene, the 847-1035 bases from the 5' end are the second intron of the genome gene, the 1036-1097 bases from the 5' end are the third exon of the genome gene, the 1098-1187 bases from the 5' end are the third intron of the genome gene, the 1188-1459 bases from the 5' end are the fourth exon of the genome gene, the 1460-1547 bases from the 5' end are the fourth intron of the genome gene, and the 1548-1624 bases from the 5' end are the fifth exon of the genome gene, the 1625-position 1701 base from the 5' end is the fifth intron of the genome gene, the 1702-position 1758 base from the 5' end is the sixth exon of the genome gene, the 1759-position 1855 base from the 5' end is the sixth intron of the genome gene, the 1856-position 1994 base from the 5' end is the seventh exon of the genome gene, the 1995-position 2083 base from the 5' end is the seventh intron of the genome gene, the 2084-position 2136 base from the 5' end is the eighth exon of the genome gene, the 2137-position 2245 base from the 5' end is the eighth intron of the genome gene, the 2341-position 2341 base from the 5' end is the ninth exon of the genome gene, the 2430-position 2342-position 2430 base from the 5' end is the ninth intron of the genome gene, and the tenth 2512-position 2512 base from the 2431-position of the genome gene, the 2513-2629 th base from the 5 'end is the tenth intron of the genomic gene, the 2630-2731 th base from the 5' end is the eleventh exon of the genomic gene, the 2732-2851 th base from the 5 'end is the eleventh intron of the genomic gene, the 2852-2947 th base from the 5' end is the twelfth exon of the genomic gene, the 1-72 th base from the 5 'end is the 5' end non-coding region (UTR) of the genomic gene, and the 2948-3314 th base from the 5 'end is the 3' end non-coding region of the genomic gene.

6. An expression vector, transgenic cell line or host bacterium comprising the gene of any one of claims 1 to 5.

7. The protein encoded by the gene of claim 1.

8. Use of the gene of any one of claims 1 to 5 for lodging resistance and compact rape breeding.

9. The application of claim 8, the operation steps are as follows:

(1) introducing the gene into a cabbage type rape receptor by utilizing an agrobacterium-mediated transgenic method to obtain a transformed plant;

(2) analyzing and identifying the positive transgenic plant by means of a PCR method;

(3) carrying out field planting on the transgenic plant in the step (2) and observing the character of the transgenic plant;

(4) the expression of the gene related to brassinolide signal transduction in the transgenic plants and wild plants is analyzed by means of RT-PCR.

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