CN110452914A - One gene BnC04BIN2-like1 for regulating and controlling brassinosteroid signal transduction and its application - Google Patents

Abstract

The present invention is gene engineering technology field, and in particular to a gene BnC04BIN2-like1 for regulating and controlling brassinosteroid signal transduction and its application.The present invention obtains the gene BnC04BIN2-like1 of negative regulation plant brassinosteroid signal transduction, the gene and the replacement for having 11 bases with reference to gene from short bar mutant material " MB1501-1 " clone and separate of cabbage type rape.The present invention constructs PBI121-BnC04BIN2-like1 over-express vector and CRISPR-BnC04BIN2-like1 carrier, obtains PBI121-BnC04BIN2-like1 and is overexpressed transgenic plant, Dwarfing phenotypes occurs；CRISPR-BnC04BIN2-like1 transgenic plant is obtained simultaneously, and transgenic plant restores high bar phenotype.It clones obtained BnC04BIN2-like1 gene and is capable of the signal transduction of negative regulation brassinosteroid, to control rape plant height character.

Description

One regulation brassinosteroid signal transduction gene BnC04BIN2-like1 and its Using

Technical field

The invention belongs to field of plant genetic project technology.More particularly in a kind of regulation cabbage type rape plant rape element Separation clone, functional verification and its application of the DNA fragmentation (gene) of Lipase absobed.

Background technique

Brassinosteroid (BRs) is a kind of somatotrophic sterol plant hormone, also known as brassin lactones, rape element sterol. It is a kind of natural phytohormone, is widely present in the organs such as pollen, seed, stem and the leaf of plant, due to its physiological activity Existing five kinds of hormones are substantially exceeded, are described as the 6th hormone in the world.The function of brassinosteroid includes: to adjust stretching for stem Important adjustment effect is played in long growth, pollen tube growth, the stretching, extension of leaf, the growth of root, plant fertility, aging and resistance. But known BR signal transduction component can't form a complete signal transduction pathway, wait to illustrate there are also some problems.

Along with the development of the application branches of learning such as science of heredity, molecular biology and biochemistry, BR signal transduction is relevant Research is also benefited wherein.Researcher screened bril mutant in EMS mutagenic progeny group in 1996, the mutation body surface Now to be insensitive to BR, and dwarfing, leaf dark green, blade last volume isophenous is presented.And then, researcher passes through map based cloning Method clone to obtain BRI1, it encode one be rich in leucine repetitive sequence receptor protein kinase.Research group passes through ferment Female double cross and use activation tagging, have found can with BRI1 interact BR co-receptor BAK1.Although Russinova (2004) seminar proves that BRI1 can form homodimer by the method for fluorescence resonance energy transfer, but Sheet etc. (2011) new crystal structural data shows that BRI1 may form heterodimeric with BAK1 to a greater extent in structure Body receives BR signal jointly.By the method for yeast two-hybrid, researcher screens BIK1, when in plant BR level compared with When low, it and BRI1 interaction can inhibit BRI1 to recruit co-receptor BAK1, and then BR signal is inhibited downstream to export.When When plant perceives BR signal, BRI1 occurs autophosphorylation and is activated, the BRI1 being activated then phosphorylation activation co-receptor BAK1, the BAK1 being activated continue the site not being phosphorylated before phosphorylation BRI1 in turn, the BRI1 being all activated with Active BAK1 jointly downstream conducts BR signal.BSU1 is screened by Activation tagging, it encodes a seed nucleus The serine/threonine protein kitase of positioning.BSU1 can be activated after BSK1 and CDG1 phosphorylation and activate, phosphorylation swash BSU1 living can be by BIN2 dephosphorylation, and then inhibits its kinase activity, while promoting the degradation process of BIN2.So far, we It can simply conclude are as follows: after BR is in conjunction with BRI1, BRI1 is freed and BAK1 under the interacting state with BIK1 It is combined with each other, the two mutual activity of full activation by way of mutual phosphorylation, and then activated by BSK1 and CDG1 BSU1, the BSU1 of activation can inhibit the activity of BIN2 with dephosphorylation BIN2.

Researcher in 2002 has found that bin2-1 is similar to bril and the insensitive phenotype of BR is presented.BIN2 encodes a kind of GSK3 Albuminoid kinases plays a major role in BR signal transduction.When BR is horizontal lower in plant, BIN2 is in activation shape State, active BIN2 is phosphorylatable and inhibits the function of BZR1/BES1.The binding site of BIN2 and BZR1 is at the end its C 12 amino acid at end, can be phosphorylated bindin 14-3-3 by the BZR1 after BIN2 phosphorylation and be bundled in cytoplasm, into And it cannot be introduced into core and the downstream of BR signal blocked to conduct in conjunction with DNA.When BR level is higher, the kinases of BIN2 first is living Property can reduce, while PP2A can be quickly by BZR1 dephosphorylation, and the BZR1 after dephosphorylation, which enters, adjusts downstream base in core The expression of cause conducts BR signal downwards.

In the 1990s, with the application of genomics technologies, researcher is confirmed in most of BR defect and unwise The elongation of cell in mutant strain is felt by serious influence, and hypocotyl length shortens and can then go out under illumination condition under dark condition The phenotype (salchert et al., 1998) now downgraded.AtCPD gene encodes Cytochrome P450 (CYP90) albumen, Conserved domain with sterols hydroxylase, enzyme missing will lead to the short and small of plant, apply BR outside or are overexpressed CPD's CDNA can make it restore the phenotype (Szekeres et al., 1996) of wild type.Dwf4 mutant strain also belongs to BR conjunction At deficiency Dwarf Mutant, any hormone cannot all restore its short and small phenotype (Azpiroz et other than brassinosteroid al.,1998).Rice BR defect mutant brd1 (BR-deficient dwarf1) have internode can hardly extend, leaf sheath Shorten, the phenotype that blade is short and small and curling is serious, tiller is few and sterile, is the prominent of first BR deficiency being found in rice Variant.The mutant can restore phenotype after external source applies BR.BRD1 gene encodes a C-6 oxidizing ferment, belongs to the C-6 of early stage Oxidative pathway, gene will lead to rice plant and downgrade (Hong et al., 2002) after mutating.Similar BR deficiency is prominent There are also bul1-1, the elongation of the mutant cells has been similarly subjected to inhibit mutant, carries out observation hair to its cell using microscope Now parallel micro-pipe tissue significantly reduces in mutant compared with wild type.The gene encodes a Δ 7- sterol-C- dehydrogenase, should The missing of enzyme causes short and small phenotype by influencing the content of brassinosteroid in plant body to affect the structure of cell (Catterou et al.,2001)。

The present invention has found one from rape and can regulate and control the gene of brassinosteroid signal transduction, this gene exists It can reduce the brassinosteroid signal transduction of its regulation after being overexpressed, so that the synthesis of plant tumor growth element is reduced, and Finally plant is caused to be downgraded, when this gene is interfered in expression, plant can restore the brassinosteroid signal of its regulation Transduction to restore the synthesis of plant tumor growth element, and makes dwarfing material grow tall.

Summary of the invention

The purpose of the present invention is to provide the genes that one regulates and controls brassinosteroid signal transduction.

The gene of regulation brassinosteroid signal transduction provided by the present invention, entitled BR-INSENSITIVE-2- Like1 (abbreviation BIN2-like1 or BnC04BIN2-like1) is derived from cabbage type rape (Brassica napus), from one The genetic fragment that clone obtains in a cabbage type rape Dwarf mutant material " MB1501-1 " was announced (2017 with 2014 Update) French rape database have the replacements of 11 bases.

The gene BnC04BIN2-like1 of one regulation rape brassinosteroid signal transduction, is following amino acid residues One of sequence has the replacement of 2 amino acid residues with the French rape database that 2014 announce and (update for 2017), is located at Aa-187 and aa-309:

(1) the SEQ ID NO:1 in sequence table；

(2) amino acid residue sequence of SEQ ID NO:1 in sequence table is passed through into taking for one to ten amino acid residue Generation and/or deletion and/or addition and the protein with regulation plant brassinosteroid signal transduction functionality.

SEQ ID NO:1 in sequence table is made of 410 amino acid residues, from aminoterminal (N-terminal) 65-357 ammonia Base acid residue is conserved sequence.

One to ten amino acid residue of the substitution and/or deletion and/or addition is that the amino acid in non-structural domain is residual Base, change will not have an impact the function of the albumen.

Gene (BR-INSENSITIVE-2-like1, the letter of coding present invention regulation plant brassinosteroid signal transduction Claim BIN2-like1 or BnC04BIN2-like1), cDNA is one of following nucleotide sequence, is announced (2017 with 2014 Update) French rape database have the replacements of 11 bases, be located at from 5 ' ends the 21st, 135,147,312,399,454,466, 559,615,618 and 1197 bit bases:

(1) in sequence table SEQ ID NO:2 DNA sequence dna；

(2) in polynucleotide SEQ ID NO:1 DNA sequence dna；

(3) DNA sequence dna limited with SEQ ID NO:2 in sequence table with 90% or more homology and has regulation plant The nucleotide sequence of brassinosteroid signal transduction；

(4) nucleotides sequence that can hybridize with the DNA sequence dna limited of SEQ ID NO:2 in sequence table under high high stringency conditions Column.

The high high stringency conditions be 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS solution in, it is miscellaneous under the conditions of 65 DEG C It hands over and washes film.

For SEQ ID NO:2 in sequence table by 1672 base compositions, coded sequence 5 ' holds 73-1305 alkali Base encodes the protein with the amino acid residue sequence of SEQ ID NO:1 in sequence table, from 5 ' end 265-1143 bit bases Encode conserved sequence.

Its genomic gene, is one of following nucleotide sequence:

(1) in sequence table SEQ ID NO:3 DNA sequence dna；

(2) DNA sequence dna limited with SEQ ID NO:3 in sequence table with 90% or more homology and has regulation plant The nucleotide sequence of brassinosteroid signal transduction；

(3) nucleotides sequence that can hybridize with the DNA sequence dna limited of SEQ ID NO:3 in sequence table under high high stringency conditions Column.

The high high stringency conditions be 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS solution in, it is miscellaneous under the conditions of 65 DEG C It hands over and washes film.

SEQ ID NO:3 in sequence table is the genome from 5 ' end 73-178 bit bases by 3314 base compositions First exon of gene is the First Intron of the genomic gene from 5 ' end 179-755 bit bases, from 5 ' ends the 756-846 bit base is second exon of the genomic gene, is the genome base from 5 ' end 847-1035 bit bases Second introne of cause is the third exon of the genomic gene from 5 ' end 1036-1097 bit bases, holds the from 5 ' 1098-1187 bit base is the third introne of the genomic gene, is the genome from 5 ' end 1188-1459 bit bases 4th exon of gene is the 4th introne of the genomic gene from 5 ' end 1460-1547 bit bases, from 5 ' ends 1548-1624 bit base is the 5th exon of the genomic gene, is the gene from 5 ' end 1625-1701 bit bases 5th introne of group gene is the 6th exon of the genomic gene from 5 ' end 1702-1758 bit bases, from 5 ' Holding 1759-1855 bit base is the 6th introne of the genomic gene, is the base from 5 ' end 1856-1994 bit bases It is the 7th introne of the genomic gene from 5 ' end 1995-2083 bit bases because of the 7th exon of group gene, from 5 ' end 2084-2136 bit bases are the 8th exon of the genomic gene, and being from 5 ' end 2137-2245 bit bases should 8th introne of genomic gene is the 9th exon of the genomic gene from 5 ' end 2246-2341 bit bases, It is the 9th introne of the genomic gene from 5 ' end 2342-2430 bit bases, is from 5 ' end 2431-2512 bit bases Tenth exon of the genomic gene is included from the tenth that 5 ' end 2513-2629 bit bases are the genomic gene Son is the 11st exon of the genomic gene from 5 ' end 2630-2731 bit bases, from 5 ' 2732-2851, ends Base is the 11st introne of the genomic gene, is the of the genomic gene from 5 ' end 2852-2947 bit bases 12 exons are 5 ' ends noncoding region (UTR) of the genomic gene from 5 ' end 1-72 bit bases, from 5 ' end 2948- 3314 bit bases are 3 ' end noncoding regions of the genomic gene.

The gene BnC04BIN2-like1 is related to brassinosteroid signal transduction.By the complete coding of the gene Sequence (Coding sequence) in conjunction with over-express vector PBI121 after be directly transferred to cabbage type rape routine excellent variety In in double 11 (ZS11), transgenic plant plant height is obviously shorter than the plant height of adjoining tree；The gene C RISPR/ that will be built Cas9 carrier is directly transferred in the short bar rape variety ' MB1501-1 ' of cabbage type rape, and the plant height of transgenic plant can be restored just Often.Show that BnC04BIN2-like1 gene of the present invention is capable of the signal transduction of negative regulation brassinosteroid, to control rape strain High character.

The albumen of expression vector, transgenic cell line, host strain and its coding containing gene of the present invention belongs to this hair Bright protection scope.

Expand BnC04BIN2-like1 gene in any segment primer and primer pair also protection scope of the present invention it It is interior.

The gene BnC04BIN2-like1 that the present invention is cloned can be used on resistant to lodging and compact rapeseed breeding.

Specific steps are as follows:

(1) the gene BnC04BIN2-like1 is imported into Wild cabbage type oil using the transgenic method of mediated by agriculture bacillus Dish receptor obtains transformed plant；

(2) positive transgenic plant is analyzed and identified by PCR method；

(3) transgenic plant of step (2) is subjected to field planting and observes its character；

(4) it is analyzed by RT-PCR and participates in brassinosteroid signal transduction dependency basis in transgenic plant and WT lines Because of the expression of BnC04BIN2-like1.

BnC04BIN2-like1 the utility model has the advantages that

The gene BnC04BIN2-like1 is related to brassinosteroid signal transduction.By the complete coding of the gene Sequence (Coding sequence) in conjunction with over-express vector PBI121 after be directly transferred to cabbage type rape routine excellent variety In in double 11 (ZS11), the plant height of the obvious shorter plant double in of transgenic plant plant height；The gene C RISPR/ that will be built Cas9 carrier is directly transferred in the short bar rape variety ' MB1501-1 ' of cabbage type rape, and the plant height of transgenic plant can restore high Bar phenotype.Show that BnC04BIN2-like1 gene of the present invention is capable of the signal transduction of negative regulation brassinosteroid, to control oil Dish plant height character.

Detailed description of the invention

Fig. 1 is the frame structure of BnC04BIN2-like1 genomic gene；

Fig. 2 is the protein sequence analysis figure of BnC04BIN2-like1；

Fig. 3 is that plant over-express vector PBI121 constructs schematic diagram；

Fig. 4 is plant CRISPR/Cas9 vector construction schematic diagram；

Fig. 5 is BnC04BIN2-like1 gene overexpression transgenic plant phenotype；Wherein the left side is that wild type ZS11 is mono- Strain, the right are PBI121-BnC04BIN2-like1 transgenic plant phenotype.

Fig. 6 is BnC04BIN2-like1 gene C RISPR/Cas9 vector transgene plant phenotype；Wherein the left side is CRISPR-BnC04BIN2-like1 transgenic plant phenotype, centre are the short bar plant of heterozygosis, and the right is high bar plant.

Specific embodiment

Method therefor is conventional method unless otherwise instructed in following embodiments.

Embodiment 1

Regulate and control the clone of plant brassinosteroid signaling genes BnC04BIN2-like1

Cabbage type rape (Brassica napus) fresh blade is extracted with TRIZAL reagent and referring to kit specification Total serum IgE, then closed with the Reverse Transcription kit of Takara company and being inverted according to kit specification At cDNA, using synthesized cDNA as template, then using published French cabbage type rape genome as Reference Design primer, In Primer P1 (upstream primer): 5 '-ATAGCACATGACATCACTATC-3 ' and P2 (downstream primer): 5 '- The gene of regulation plant brassinosteroid signal transduction under the primer of TCTCCTCTTTTCCACAAG-3 ' in PCR amplification rape, After reaction, 1% agarose gel electrophoresis detection is carried out to pcr amplification product, is recycled and is tried using AXYGEN centrifugal column type glue Recovery product is connected into carrier Easy Blunt according to illustrating purpose band recycling and purify it by agent box In Simple, Escherichia coli (E.coli) DH5 α competent cell is converted through heat shock method.Blue hickie screening positive clone, is connect Kind in LB liquid medium containing kanamycins, 37 DEG C, cultivate under 200rpm, extraction plasmid is sequenced it, sequencing result Show that amplified fragments have the nucleotide sequence of the SEQ ID NO:2 in sequence table, by 1233 base compositions, coded sequence Site position is, from 5 ' end 1-1233 bit bases, coding has the egg of the amino acid residue sequence of SEQ ID NO:1 in sequence table White matter, wherein encode conserved sequence from 5 ' end 193-1071 bit bases.Its genomic gene has SEQ ID in sequence table The nucleotide sequence of NO:3, by 3314 base compositions, from first that 5 ' end 73-178 bit bases are the genomic gene Exon is the First Intron of the genomic gene from 5 ' end 179-755 bit bases, from 5 ' the 756-846 alkali in end Base is second exon of the genomic gene, is in second of the genomic gene from 5 ' end 847-1035 bit bases It is the third exon of the genomic gene from 5 ' end 1036-1097 bit bases, from 5 ' 1098-1187, ends containing son Base is the third introne of the genomic gene, is the 4th of the genomic gene from 5 ' end 1188-1459 bit bases A exon is the 4th introne of the genomic gene from 5 ' end 1460-1547 bit bases, from 5 ' end 1548- 1624 bit bases are the 5th exon of the genomic gene, are the genomic gene from 5 ' end 1625-1701 bit bases The 5th introne, be the 6th exon of the genomic gene from 5 ' end 1702-1758 bit bases, from 5 ' ends the 1759-1855 bit base is the 6th introne of the genomic gene, is the genome from 5 ' end 1856-1994 bit bases 7th exon of gene is the 7th introne of the genomic gene from 5 ' end 1995-2083 bit bases, from 5 ' ends 2084-2136 bit base is the 8th exon of the genomic gene, is the gene from 5 ' end 2137-2245 bit bases 8th introne of group gene is the 9th exon of the genomic gene from 5 ' end 2246-2341 bit bases, from 5 ' Holding 2342-2430 bit base is the 9th introne of the genomic gene, is the base from 5 ' end 2431-2512 bit bases It is the tenth introne of the genomic gene from 5 ' end 2513-2629 bit bases because of the tenth exon of group gene, from 5 ' end 2630-2731 bit bases are the 11st exon of the genomic gene, are from 5 ' end 2732-2851 bit bases 11st introne of the genomic gene, from the 12nd that 5 ' end 2852-2947 bit bases are the genomic gene Exon is 5 ' ends noncoding region (UTR) of the genomic gene from 5 ' end 1-72 bit bases, from 5 ' 2948-3314, ends Base is 3 ' end noncoding regions of the genomic gene.Framework junction part Fig. 1 of the gene.

Embodiment 2

The acquisition of BnC04BIN2-like1 overexpression transgenic plant

One, the building of the plant over-express vector of the gene containing BnC04BIN2-like1

Xba I restriction endonuclease sites, upstream primer are added in the CDS Sequences upstream of BnC04BIN2-like1 gene P3:5 '-GCTCTAGAATGACATCACTATC-3 ' is limited in the CDS sequence downstream of BnC04BIN2-like1 gene plus Sma I Property restriction enzyme site downstream primer P4:5 '-TCCCCCGGGCTAATGACCAGGCT-3 ' processed.With primer P3 and P4 to embodiment 1 The nucleotide sequence for cloning SEQ ID NO:2 in obtained sequence table carries out PCR amplification, after reaction, to pcr amplification product 1% agarose gel electrophoresis detection is carried out, using AXYGEN centrifugal column type plastic recovery kit, according to illustrating to carry out purpose band It recycles and it is purified, recovery product is connected into carrier Easy BluntSimple, converts Escherichia coli through heat shock method (E.coli) DH5 α competent cell.Blue hickie screening positive clone, is inoculated in LB liquid medium containing kanamycins, 37 DEG C, cultivate under 200rpm, extract plasmid, it be sequenced, sequencing result shows that amplified fragments have the SEQ in sequence table ID NO:2 adds the nucleotide sequence of restriction enzyme Xba I and Sma I site, and with restriction enzyme Xba I and Sma I carries out digestion to the plasmid containing BnC04BIN2-like1 gene built, carries out 1% Ago-Gel to digestion products Electrophoresis detection recycles the BnC04BIN2-like1 genetic fragment of length about 1233 and purifies to it, and recycling segment is used T4DNA ligase (Takara) is linked with the carrier PBI121 through identical digestion, then connection product heat shock method is converted large intestine Bacillus (E.coli) DH5 α competent cell, screening positive clone are inoculated in the LB liquid medium containing kanamycins, It is cultivated at 37 DEG C, 200rpm, extracts plasmid, digestion mirror is carried out with restriction enzyme Xba I and Sma I to recombinant plasmid It is fixed, it is consistent with expected results, then be further PCR with primer P3 and P4 and identify, has as a result obtained 1233bp's through PCR amplification DNA fragmentation, it is consistent with expected results, show to have obtained insetion sequence and position correctly containing the plant of BnC04BIN2-like1 Object expression vector, is named as PBI121-BnC04BIN2-like1.

Two, the acquisition of PBI121-BnC04BIN2-like1 transgene rape

The plant expression vector PBI121-BnC04BIN2-like1 heat shock method of step 1 building is converted into Agrobacterium EHA105 competent cell is coated in the LB resistant panel containing kanamycins and rifampin, is trained at 28 DEG C, 150rpm It supports, the Agrobacterium single colonie that picking is grown is inoculated in the 20ml LB liquid medium containing kanamycins and rifampin, 28 DEG C, cultivate 2 days under 150rpm, then, then by 2% inoculum concentration bacterium bacterium solution is inoculated in containing kanamycins and rifampin In 300ml LB liquid medium, cultivated 16-18 hours at 28 DEG C, 150rpm.After culture, 5000rpm is centrifuged 20 points It collects thallus after clock, then thallus is suspended in 250ml and contains 5% sucrose, in the solution of 0.1%Silwetl-77.Finally, by bacterium Liquid turns in 250ml beaker, the cabbage type rape for end of having pollinated has been spent, then plant is inverted in beaker, has made its flower Sequence invades in bacterium solution completely, to improve transformation efficiency, repeats after a week primary.Transformed plant is subjected to routine culture, is harvested Seed, gained seed obtain BnC04BIN2-like1 transgenic plant after kanamycins and PCR evaluation and screening.

Three, PBI121-BnC04BIN2-like1 transgenic plant Phenotypic Observation

The seed plantation of the positive transgenic plant for the overexpression BnC04BIN2-like1 that step 3 is obtained and crop field item Under part, using cabbage type rape wild type as control.Observe the life that BnC04BIN2-like1 is overexpressed plant and WT lines Long situation, the phenotype that BnC04BIN2-like1 overexpression plant grows under field conditions (factors) is as shown in figure 5, BnC04BIN2- Like1 is overexpressed plant and downgrades, is shaky.

Embodiment 3

The acquisition of CRISPR-BnC04BIN2-like1 transgenic plant

One, the building of the plant CRISPR/Cas9 carrier of the gene containing BNC04BIN2-LIKE1

2 target spots are designed to BnC04BIN2-like1 gene, one in the area ORF-5 ', design of primers is P5 (5'gRT1 +): 5 '-CTCCGTCGCGAAGCTGCGGGTTTTAGAGCTAGAAAT-3 ', P6 (5'U3dT1-): 5 '-CAAAGCACCATTGG TCACTCCGTCGCGAAGCTGCGG-3'；One in conserved sequence region, design of primers is P7 (conservative gRT2+): 5 '-ACCG AGCCTGCGTTACACTGGTTTTAGAGCTAGAAAT-3 ', P8 (conservative U3dT2-): 5 '-CAAAGCACCATTGGTCAACCG AGCCTGCGTTACACTG-3'.SgRNA expression cassette adapter-primer reverse primer P9:5 '-CTCCGTTTTACCTGTGGAATCG- 3 ' and 5 '-CGGAGGAAAATTCCATCCAC-3 ' of connector forward primer.4 kinds are used in the building reaction of each sgRNA expression cassette Primer: each 0.2 μM of P9 and P10, each 0.1 μM of (P5 or P7) and (P6 or P8).25-28 circulation: 94 DEG C of 10s, 58 DEG C of 15s, 68 DEG C 20s.In amplification procedure, P9/ (P6 or P8) expands P9- (P6 or P8) sequence, P10/ (P5 or P7) when several circulations of beginning Expand (P5 or P7)-sgRNA sequence.The sgRNA that 2 segments merge is generated by overlapping PCR in the circulation in later period Expression cassette segment.Take 1 μ l H of PCR product₂O dilutes 10 times, and taking 1 μ l is template, each expression cassette 20-50 μ l PCR (1 target spot 50μl；Each 30 μ l of 2 target spots).Every kind of primer combination working solution (0.15 μM of ultimate density) of 1/10 amount is added.Use appropriate KOD- Plus High fidelity PCR enzyme.20 circulation of amplification: 95 DEG C of 10s, 58 DEG C of 15s, 68 DEG C of 20s.Taking 2-3 μ l electrophoretic examinations product length is It is no to meet, and estimate the substantially concentration of sample.Binary vector is reacted with digestion-connection of sgRNA expression cassette: 10 × CutSmart Buffer (1.5 μ l), 10mM ATP (1.5 μ l), pYLCRISPR/Cas9 plasmid (80ng), sgRNA expression cassette mixture (15ng), Bsa I-HF (10U), T4DNA ligase (35U), H₂O is mended to 15 μ l.Digestion connection is carried out about with temperature varied cyclical 10-15 circulation: 37 DEG C of 5min；10 DEG C of 5min, 20 DEG C of 5min；Last 37 DEG C of 5min.Take 1-1.5 μ l connection product heat-shock transformed 1ml SOC, 37 DEG C of culture 1.5h is added in E.coli DH10B competent cell after heat shock.Coated plate culture medium is LB+25 μ g/ml Kan, picking positive monoclonal are cultivated with 37 DEG C of culture medium of μ g/ml Kan of liquid LB+25, and utilize the special of each target spot primer Property, particular target point is sequenced.Correct bacterium solution is sequenced, shows to have obtained insetion sequence and correctly contains BnC04BIN2- The CRISPR/Cas9 carrier of like1, is named as CRISPR-BnC04BIN2-like1.

Two, the acquisition of CRISPR-BnC04BIN2-like1 transgene rape

The plant CRISPR-BnC04BIN2-like1 carrier heat shock method of step 1 building is converted into Agrobacterium EHA105 Competent cell is coated in the LB resistant panel containing kanamycins and rifampin, cultivates, choose at 28 DEG C, 150rpm Take the Agrobacterium single colonie grown to be inoculated in the 20ml LB liquid medium containing kanamycins and rifampin, 28 DEG C, It cultivates 2 days under 150rpm, then, then by 2% inoculum concentration bacterium bacterium solution is inoculated in the 300ml containing kanamycins and rifampin In LB liquid medium, cultivated 16-18 hours at 28 DEG C, 150rpm.After culture, 5000rpm centrifugation is received after twenty minutes Collect thallus, then thallus is suspended in 250ml and contains 5% sucrose, in the solution of 0.1%Silwetl-77.Finally, by bacterium solution turn in In 250ml beaker, the cabbage type rape for end of having pollinated is spent, then plant is inverted in beaker, has kept its inflorescence complete It invades in bacterium solution, to improve transformation efficiency, repeats after a week primary.Transformed plant is subjected to routine culture, harvests seed, institute It obtains seed and obtains BnC04BIN2-like1 transgenic plant after kanamycins and PCR evaluation and screening.

Three, CRISPR-BnC04BIN2-like1 transgenic plant Phenotypic Observation

By the seed plantation of the CRISPR-BnC04BIN2-like1 carrier positive transgenic plant of step 2 acquisition and greatly Under the conditions of field, using cabbage type rape wild type as control.It observes CRISPR-BnC04BIN2-like1 carrier and wild type is planted The growing state of strain, phenotype that CRISPR-BnC04BIN2-like1 transgenic plant is grown under field conditions (factors) as shown in fig. 6, Short bar plant restores the phenotype of high bar.

Sequence table

<110>Agricultural University Of Nanjing

<120>gene BnC04BIN2-like1 for regulating and controlling brassinosteroid signal transduction and its application

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 410

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Ser Ser Ser Gln Arg Met Pro Ile Phe Tyr Val Lys Leu Tyr Thr Tyr

165 170 175

Gln Ile Phe Arg Gly Leu Ala Tyr Ile His Ser Val Pro Gly Val Cys

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Tyr Ser Pro Ser Leu Arg Tyr Thr Ala Leu Glu Ala Cys Ala His Pro

340 345 350

Phe Phe Asn Glu Leu Arg Glu Pro Asn Ala Arg Leu Pro Asn Gly Arg

355 360 365

Pro Leu Pro Ala Leu Phe Asn Phe Lys Gln Glu Leu Ala Gly Ala Ser

370 375 380

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

385 390 395 400

Ser Gly Gly Phe Pro Ser Gln Pro Gly His

405 410

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<213>artificial sequence (Artificial Sequence)

<400> 2

gggaaagata gtctttactc ttcagtggtg ggtagagagc gaaagttaga gaaagagaga 60

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 (10)

1. the gene of a regulation brassinosteroid signal transduction, which is characterized in that the gene is following amino acid residue sequences One of column:

(1) the SEQ ID NO:1 in sequence table；

(2) by the amino acid residue sequence of SEQ ID NO:1 in sequence table by one to ten amino acid residue substitution and/ Or it is deleted and/or added and has the protein of regulation plant brassinosteroid signal transduction functionality.

SEQ ID NO:1 in sequence table is made of 410 amino acid residues, from aminoterminal (N-terminal) 65-357 amino acids Residue is conserved sequence.

2. the gene of regulation brassinosteroid signal transduction according to claim 1, which is characterized in that the amino acid is residual Base is the amino acid residue in non-structural domain, and change will not have an impact the function of the albumen.

3. the gene of regulation brassinosteroid signal transduction according to claim 1, which is characterized in that its cDNA is following One of nucleotide sequence:

(1) in sequence table SEQ ID NO:2 DNA sequence dna；

(2) in polynucleotide SEQ ID NO:1 DNA sequence dna；

(3) DNA sequence dna limited with SEQ ID NO:2 in sequence table with 90% or more homology and has regulation plant rape The nucleotide sequence of plain lactone signal transduction；

(4) nucleotide sequence that can hybridize with the DNA sequence dna limited of SEQ ID NO:2 in sequence table under high high stringency conditions；

The high high stringency conditions be 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS solution in, hybridize simultaneously under the conditions of 65 DEG C Wash film.

4. the gene of regulation brassinosteroid signal transduction according to claim 3, which is characterized in that in sequence table For SEQ ID NO:2 by 1672 base compositions, coded sequence 5 ' holds 73-1305 bit base, and coding has in sequence table The protein of the amino acid residue sequence of SEQ ID NO:1 encodes conserved sequence from 5 ' end 265-1143 bit bases.

5. the gene of regulation brassinosteroid signal transduction according to claim 1, which is characterized in that its genome base Cause is one of following nucleotide sequence:

(1) in sequence table SEQ ID NO:3 DNA sequence dna；

(2) DNA sequence dna limited with SEQ ID NO:3 in sequence table with 90% or more homology and has regulation plant rape The nucleotide sequence of plain lactone signal transduction；

(3) nucleotide sequence that can hybridize with the DNA sequence dna limited of SEQ ID NO:3 in sequence table under high high stringency conditions.

The high high stringency conditions be 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS solution in, hybridize simultaneously under the conditions of 65 DEG C Wash film.

6. the gene of regulation brassinosteroid signal transduction according to claim 5, which is characterized in that in sequence table SEQ ID NO:3 is first exon of the genomic gene from 5 ' end 73-178 bit bases by 3314 base compositions, It is the First Intron of the genomic gene from 5 ' end 179-755 bit bases, is the base from 5 ' end 756-846 bit bases It is second introne of the genomic gene from 5 ' end 847-1035 bit bases because of second exon of group gene, from 5 ' end 1036-1097 bit bases are the third exon of the genomic gene, and being from 5 ' end 1098-1187 bit bases should The third introne of genomic gene is the 4th exon of the genomic gene from 5 ' end 1188-1459 bit bases, It is the 4th introne of the genomic gene from 5 ' end 1460-1547 bit bases, is from 5 ' end 1548-1624 bit bases 5th exon of the genomic gene is included from the 5th that 5 ' end 1625-1701 bit bases are the genomic gene Son is the 6th exon of the genomic gene from 5 ' end 1702-1758 bit bases, from 5 ' the 1759-1855 alkali in end Base is the 6th introne of the genomic gene, from the 7th that 5 ' end 1856-1994 bit bases are the genomic gene Exon is the 7th introne of the genomic gene from 5 ' end 1995-2083 bit bases, from 5 ' end 2084-2136 Bit base is the 8th exon of the genomic gene, is the of the genomic gene from 5 ' end 2137-2245 bit bases Eight intrones are the 9th exon of the genomic gene from 5 ' end 2246-2341 bit bases, from 5 ' end 2342- 2430 bit bases are the 9th introne of the genomic gene, are the genomic gene from 5 ' end 2431-2512 bit bases The tenth exon, be the tenth introne of the genomic gene from 5 ' end 2513-2629 bit bases, from 5 ' ends the 2630-2731 bit base is the 11st exon of the genomic gene, is the gene from 5 ' end 2732-2851 bit bases 11st introne of group gene is the 12nd exon of the genomic gene from 5 ' end 2852-2947 bit bases, It is 5 ' ends noncoding region (UTR) of the genomic gene from 5 ' end 1-72 bit bases, being should from 5 ' end 2948-3314 bit bases 3 ' end noncoding regions of genomic gene.

7. the egg of expression vector, transgenic cell line, host strain and its coding comprising any one of the claim 1-6 gene It is white.

8. the primer and primer pair of any segment in any one of the claim 1-6 gene.

9. application of any one of the claim 1-6 gene on resistant to lodging and compact rapeseed breeding.

10. application according to claim 9, operating procedure are as follows:

(1) channel genes cabbage type rape receptor described in claim 1 is obtained using the transgenic method of mediated by agriculture bacillus Transformed plant；

(2) positive transgenic plant is analyzed and identified by PCR method；

(3) transgenic plant of step (2) is subjected to field planting and observes its character；

(4) it is analyzed by RT-PCR and participates in brassinosteroid signal transduction related gene in transgenic plant and WT lines Expression.