CN101497884B

CN101497884B - A method for controlling the branching of Apocynum apocynum through gene silencing

Info

Publication number: CN101497884B
Application number: CN2008100571322A
Authority: CN
Inventors: 赵兵; 贾海燕; 王晓东
Original assignee: Institute of Process Engineering of CAS
Current assignee: Beijing Zhongke Joinyou Biotechnology Co ltd
Priority date: 2008-01-30
Filing date: 2008-01-30
Publication date: 2011-02-09
Anticipated expiration: 2028-01-30
Also published as: CN101497884A

Abstract

本发明涉及一种通过基因沉默控制罗布麻分枝的方法，属于生物技术领域。根据NCBI Genbank公布的已知CUC3基因cDNA序列，设计兼并引物，利用反转录PCR技术进行目的片段克隆；筛选的阳性克隆进行酶切、PCR鉴定、DNA测序以及核苷酸序列同源性比较，获得罗布麻CUC3基因的保守区基因片段；构建反义表达载体，通过发根农杆菌途径转化罗布麻；由罗布麻发根途径实现植株再生，以达到控制罗布麻分枝的目的。The invention relates to a method for controlling apocynum branching through gene silencing, and belongs to the field of biotechnology. According to the cDNA sequence of the known CUC3 gene published by NCBI Genbank, annexation primers were designed, and the target fragment was cloned by reverse transcription PCR technology; the screened positive clones were subjected to enzyme digestion, PCR identification, DNA sequencing and comparison of nucleotide sequence homology. Obtain the gene fragment of the conserved region of the Apocynum CUC3 gene; construct an antisense expression vector, transform Apocynum rhizogenes through the Agrobacterium rhizogenes pathway; realize plant regeneration through the hairy root pathway of Apocynum apocynum, so as to achieve the purpose of controlling the branches of Apocynum apocynum.

Description

A kind of method by gene silencing control kendir branch

Technical field

The present invention relates to a kind of method, belong to biological technical field by gene silencing control kendir branch.

Background technology

Kendir is a kind of perennial perennial root herbaceous plant of Apocynaceae (Apocynaceae) Apocynum (Apocynum), and it comprises spreads out bluish dogbane (Apocynum venetum Linn) and spread out hemp (Apocynumhendersonii Hook.f.).No matter kendir all has high comprehensive utilization value aspect fiber utilization, the medicinal still healthcare products exploitation.Domestic and international existing report mainly is the research about the apocynum fibre degumming technology, and to breeding, the growth characteristics in early stage and the research report that how to improve the flaxen fiber quality seldom.Because kendir branch is more, has both influenced planting density, also be difficult to realize mechanize stripping fiber crops, directly influenced staple length and output.Wild resource can't satisfy the ever-increasing market requirement far away.At present, though carried out some artificial growth tests on a small scale, branch is many, and fiber problem short and that yield poorly still can't solve.Therefore, pass through modern biotechnology, utilize the transgenic breeding method to reduce the branch of kendir, cultivate the new variety that branch is few, fiber production is high, improve the quality and the output of numb skin, not only significant aspect the research of controlling plant branch, and the popularization of institute's seed selection new variety will produce huge social and economic benefit.

Though for the branch developmental regulation existing more research both at home and abroad of other plant, branch control relates to very complicated phytohormone Regulation and signal transduction pathway at present.Along with the discovery of some branch regulatory genes and transcription factor, for we realize providing possibility to the control of plant branching by transgenic technology.The tblgene of corn be considered to the branch regulatory gene (J.Doebley et al.Nature, 1997,386,485-488), this gene itself has the branched effect that suppresses.In addition, also have research to contain the MADS-box expression carrier to realize increasing farm crop and the branched purpose of ornamental plant (United States Patent No.6995302) by structure.In the researchs to Arabidopis thaliana such as Ken-ichiro Hibara and Casper W.Vroemen, find CUC3 gene pairs collateral development play an important role (Ken-ichiro Hibara et al.The Plant Cell, 2006,18,2946-2957; Casper W.Vroemen et al.The plant cell, 2003,15,1563-1577).The CUC3 gene belongs to the NAC transcription factor family, and it brings into play main effect to the growth of side shoot, define and the formation of apical meristem on organ anlage border.In Arabidopis thaliana, suppress the CUC3 gene, the collateral generation of Arabidopis thaliana is suppressed.Nearest discovers that aspect collateral development, CUC3 plays positive regulating and controlling effect.The present invention is applied to kendir with Antisense RNA Technique, and the CUC3 gene of reticent kendir is realized its branched control.

Antisense RNA Technique be by carrying out base pairing with RNA mode from duplicate, transcribe and level such as translation on to genetic expression performance regulating and controlling effect.At present, Antisense RNA Technique has been widely used in the plant genetic engineering, becomes an important tool of crop genetic improvement.Utilize at present Antisense RNA Technique regulation and control plant RNA technology be penetrated into phytology research every field (Meng Bo etc. foreign medical science genetics fascicle, 2001,24 (6), 289-292), as: the improvement of growth and development of plants gene expression regulation, economical character (Xu Benbo etc. Chinese agronomy circular, 2003,19 (3), 84-88), the regulation and control of secondary metabolism approach and disease-resistant transgenic plant breeding (Lou Hong etc., Liaoning University's journal, 2005,32 (4), 328-333), and obtained stable transgenosis new lines, be with a wide range of applications.Also do not control kendir and other crops and the branched relevant report of ornamental plant at present by the reticent CUC3 gene of Antisense RNA Technique.

Though the tissue culture of kendir has been carried out some researchs both at home and abroad, has not set up stable regeneration system as yet.The present invention infects the kendir explant by Agrobacterium rhizogenes, can obtain root of hair, can directly obtain regeneration plant from the root of hair approach, thereby set up the stable regeneration system of kendir.With kendir group training approach is realized that Study on Regeneration compared in the past, this method is difficult for producing the mosaic plant, therefore, is a kind of ideal and stable conversion and transgenic plant regeneration system.

The present invention utilizes the Agrobacterium rhizogenes approach kendir to be transformed and realizes the regeneration of transfer-gen plant by making up the antisense expression vector of CUC3 gene, thereby realizes the control to kendir branch.The present invention a kind ofly realizes the new way of branch regulation and control to plant, also provides new thinking for other crops and the control of ornamental plant branch proterties simultaneously.

Summary of the invention

The objective of the invention is to use the gene silent technology that sense-rna causes, the segmental antisense expression vector of branch regulatory gene CUC3 conserved region gene of design and structure kendir, the silence that causes this gene suppresses the branch of kendir and grows, and obtains subramose kendir new variety.The invention provides a kind of method of using sense-rna gene silent technology control kendir branch, main contents comprise:

1. the clone of kendir branch regulatory gene CUC3 conserved regions;

Using the RT-PCR method, extract the total RNA reverse transcription of kendir and produce cDNA, is template clone branch regulatory gene CUC3 conserved region gene fragment with cDNA, and makes up antisense expression vector according to sense-rna gene silencing principle.

1) degenerate primer of clone CUC3 conserved regions:

Primer1：5′-ATTACT/CTTT/CTACTTAGCTTCCAAAA/G-3′

Primer2：5′-CCTTTGT?AGAAC/A?ACC/AAA/G?C/TGTC/TT?TCTTC-3′

CUC3 conserved regions cDNA sequence of 2) being cloned into and homology compare:

The CUC3 conserved regions cDNA total length of being cloned into is 287bp, 95 amino acid of encoding, this aminoacid sequence compared with the nucleotide sequence of other plant code CUC3 gene among the NCBI Genbank show: kendir CUC3 gene conserved regions and petunia, Arabidopis thaliana, soybean and paddy rice respective regions homology of nucleotide sequence are respectively 76%, 75%, 75% and 74%, with the CUC3 that announces, CUC2, the homology of the corresponding conserved regions aminoacid sequence of CUC1 is 77%, 73%, 68%.This explanation utilizes the reverse transcription PCR technology to obtain coding kendir CUC3 gene conservative district cDNA.The sequence of this conserved regions is as follows:

The information of SEQ.ID.NO 1

A) sequence signature

Length: 287 Nucleotide

Type: nucleic acid

Chain: two strands

B) molecule type: cDNA

C) suppose: not

D) antisense: not

E) initial source: kendir

F) sequence description: SEQ.ID.NO 1

1 ATTACCTTTT?ACTTAGCTTC?CAAAGTCTTA?TACGGACGCT?TCTGTGGACT?CGACATTGCT

61 GAAGTCGACC?TCAACAGATG?TGAGCCATGG?GAGCTTCCCG?ATGCAGCTAA?GATGGGAGAG

121?AGAGAGTGGT?ACCTTTTCAG?CTTAAGGGAC?AGGAAGTACC?CAACGGGGCT?GAGAACAAAT

181?AGAGCCACTT?GTGCCGGGTA?CTGGAAAGCA?ACGGGGAAAG?ATAGAGAAGT?CTACGGCAGT

241?GAAGGCGTTG?TTGTGGGCAT?GAAGAAGACA?TTTGTTTTCT?ACAAAGG

The information of SEQ.ID.NO 2

(a) sequence signature

Length: 95 amino acid

Type: amino acid

Chain: strand

(b) molecule type: protein

(c) sequence description: SEQ.ID.NO 2

1 ITFYLASKVL?YGRFCGLDIA?EVDLNRCEPW?ELPDAAKMGE?REWYLFSLRD?RKYPTGLRTN

61?RATCAGYWKA?TGKDREVYGS?EGVVVGMKKT?FVFYK

2. the structure of gene silencing carrier;

The purpose product fragment of RT-PCR is connected into cloning vector pEASY-T1, carries out dna sequence analysis then, sequence is carried out homology relatively among sequencing result and the Genbank; The correct CUC3 gene fragment of order-checking is downcut from pEASY-T1 with XbaI and SacI, oppositely insert the expression vector pCAMBIA-1301 that contains 35S promoter and NOS terminator, be built into antisense expression vector pCAMBIA-1301-CUC3.

3. carry out transgenosis by the method for Agrobacterium rhizogenes mediation;

Adopt freeze-thaw method that antisense expression vector pCAMBIA1301-CUC3 is imported Agrobacterium rhizogenes, coating contains card to be received behind the YEB flat board of mycin (50-100mg/L), at 28 ℃ of constant incubators, cultivates after 2 days, and picking list bacterium colony detects with PCR method.The clone of test positive is used to infect kendir true leaf, stem, cotyledonary node, cotyledon, hypocotyl, different explants positions such as root.

4. conversion of kendir and transgenic plant regeneration;

Infect the above-mentioned different explants of kendir position with the Agrobacterium rhizogenes that imports antisense expression vector.Metainfective explant was cultivated under dark condition 2 days altogether, went to the MS substratum that contains the 500mg/L cephamycin then, and 25 ± 1 ℃, 14h/10h light/dark the cultivation, every switching in 5-7 days once, corotation connects 3-5 time, infects the back and produces hairly root in about 7-10 days.Treat that hairly root grows to 2-3cm, detects with PCR method.Go to the enterprising row filter of bud inducing culture of 50mg/L kantlex and 300mg/L cephamycin under the undercut with test positive, bud to be screened grows to 2-3cm when high, change root media over to, through cultivating in 2-3 week, transformed plant is taken root and is grown up to complete regeneration and plants.

5. the detection of transfer-gen plant and screening;

Transfer-gen plant is taked the three-step approach screening.The first step detects the goal gene of transfer-gen plant by adopting PCR and Southern blot method.Second step, to the transgenic line of test positive, carry out hardening after, be transplanted to the land for growing field crops and cultivate.After a growth season, its branch amount is carried out individual plant statistics, with contemporaneously wild type seeds seedling relatively after, screening obtains the less transgenosis kendir strain system of branch.The 3rd step, many generation screenings, with the seed of last one year of results, behind the field planting, further repeated screening is to obtain stable phenotype and germ plasm resource.

The present invention can improve the expression efficiency of goal gene by the structure of the segmental antisense expression vector of CUC3 gene conserved region gene; By setting up efficient, the stable root of hair genetic conversion system of kendir, improved the regeneration efficiency of seed selection transgenosis kendir strain system; Can shorten breeding cycle, improve breeding efficiency, obtain subramose kendir new variety, this will promote the development of the kendir industry of China, and provide a new approach to the branch regulation and control of other crop and ornamental plant.

Technological line

1. degenerate primer design

At present, only the CUC3 gene in Arabidopis thaliana, paddy rice, petunia, the soybean isotype plant is obtained total length by the clone.Therefore to clone CUC3 gene conservative district, at first to retrieve NCBI and obtain CUC3 known cDNA sequence in plant, use sequence alignment tools DNAMAN that nucleotide sequence is compared then, obtain the conservative region of goal gene, in conserved regions, design degenerate primer.Sequence is as follows:

Primer1：5′-ATTACT/CTTT/CTACTTAGCTTCCAAAA/G-3′

Primer2：5′-CCTTTGT?AGAAC/AACC/AAA/G?C/TGTC/TT?TCTTC-3′

2. kendir CUC3 gene conserved regions is cloned

Extract the total RNA of kendir seedling plant.(referring to J. Sa nurse Brooker, " molecular cloning experiment guide ", (second edition), Jin Dongyan etc. translate, and 1992, Science Press).With total RNA is template, uses First-StandcDNA Synthesis Kit (Shanghai Shenergy Biocolor BioScience ﹠ Technology Company), and reverse transcription generates cDNA first chain, carries out RT-PCR afterwards, uses above-mentioned degenerate primer.The cloning vector pEASY-T1 that reclaims target DNA fragment and TransGen Biotech company behind the PCR product agarose gel electrophoresis connects into recombinant plasmid p-EASY-CUC3, and heat shock transforms the Top10 competent cell, blue hickie screening.Select hickie, shake bacterium, extract plasmid, carry out PCR and detect and restriction analysis.Detect correct transformant, by the order-checking of the handsome Bioisystech Co., Ltd in Shanghai.Sequencing result carries out Nucleotide and protein sequence comparison in gene library, and homology analysis.Select the higher clone of CUC3 gene order homology for use with other plant.

3. the structure of antisense expression vector

By DNAMAN known array is carried out the restriction enzyme mapping analysis.CUC3 conserved region gene fragment will oppositely be inserted the expression vector pCAMBIA-1301 that contains 35S promoter and terminator.On cloning vector and expression vector, choose proper restriction site.Chosen XbaI and Sacl respectively and carried out the double digestion reaction, reclaimed the purpose fragment and connect construction recombination plasmid pCAMBIA1301-CUC3 with the T4DNA ligase enzyme.Transform the Top10 competent cell and screen positive connexon, carry out PCR and detect and restriction analysis.

4. antisense expression vector transforming agrobacterium rhizogenes

Connect correct clone with detecting, extract plasmid.Adopt freeze-thaw method that antisense expression vector is imported the Agrobacterium rhizogenes competent cell, cultivated 2-3 days in 28 ℃ of incubators.Select mono-clonal and carry out bacterium colony PCR detection (figure

1)。Be used to infect the kendir explant after detecting correct clone's cultivation, carry out genetic transformation.

5. the foundation of transformation system of kendir and transgenosis regeneration system

The Agrobacterium rhizogenes that imports antisense expression vector infects kendir different explants position by leaf dish method, produces hairly root (Fig. 2).Positive root system by obtaining after cephamycin sterilization and the kalamycin resistance screening extracts genomic dna with the CTAB method, carries out PCR and detects.

Detection to agrobatcerium T-DNA: according to Ri plasmid T _LRolC gene design primer on the-DNA.The purpose product is 574bp (Fig. 3).

RolC gene F primer: 5 '-GATATATGCCAAATTTACACTAG-3 '

RolC gene R primer: 5 '-GTTAACAAAGTAGGAAACAGG-3 '

The root of hair of test positive is cultivated on the bud inducing culture and is sprouted, and takes root on the root media, obtains kendir transgenic regenerated plant (Fig. 4).

6. the detection of transfer-gen plant goal gene

By the genomic dna of CTAB method extraction transfer-gen plant, adopt PCR and Southern blot method to detect the goal gene of transfer-gen plant.

PCR detects: adopt the segmental degenerate primer of above-mentioned clone CUC3 conserved region gene, purpose fragment 287bp.

Southern blot detects: the total DNA of regeneration plant (10 μ g) carries out Southern hybridization behind the complete enzymolysis of HindIII.Dna probe is 35S promoter-segmental full sequence of CUC3 conserved region gene, carries out mark by the random priming digoxigenin labeled test kit specification sheets of TAKaRa company.

7. the screening of the less transgenic line of branch

Transfer-gen plant is taked the three-step approach screening.The first step detects the goal gene of transfer-gen plant by adopting PCR and Southern blot method.Second step, to the transgenic line of test positive, carry out hardening after, be transplanted to the land for growing field crops and cultivate.After a growth season, its branch amount is carried out individual plant statistics, with contemporaneously wild type seeds seedling relatively after, screening obtains the less transgenosis kendir strain system of branch.The 3rd step, many generation screenings.With the seed of last one year of results, behind the field planting, further repeated screening is to obtain stable phenotype and germ plasm resource.

Description of drawings

Fig. 1: PCR detects behind the recombinant plasmid pCAMBIA1301-CUC3 transforming agrobacterium rhizogenes R1601.

1: negative control 2-10: positive colony

Fig. 2: Agrobacterium rhizogenes transforms the root of hair that generates behind the kendir explant

Fig. 3: root of hair PCR detects

1-2: Agrobacterium rhizogenes plasmid positive control PCR result;

3-4: root of hair genomic dna PCR result

Fig. 4: by root of hair approach regeneration of transgenic seedling

Embodiment

Embodiment 1: the clone of kendir CUC3 gene conserved regions

1. the extraction of the total RNA of kendir: the kendir seedling that will organize training 6-8 week is extracted the total RNA of plant according to the Trizol method of " molecular cloning experiment guide ".Agarose gel electrophoresis detects.

2. reverse transcription and RT-PCR: use the First-StandcDNASynthesis Kit of Shanghai Shenergy Biocolor BioScience ﹠ Technology Company, reverse transcription generates cDNA first chain, is template with cDNA first chain, carries out RT-PCR with the degenerate primer of design and increases.

3. the purpose fragment is connected with cloning vector, screening positive clone: PCR electrophoresis product reclaims the back and is connected with the pEASY-T1 cloning vector of TransGen Biotech company, after transforming the Top10 competent cell, cultivate at the LB solid medium that contains penbritin and kantlex/IPTG/X-Gal, be inverted for 37 ℃ and cultivated 12-20 hour, the bacterium colony that has is white, and what have becomes blueness.Choose white colony, extract plasmid DNA, carry out enzyme and cut with PCR and identify.

4. sequencing: the handsome Bioisystech Co., Ltd in Shanghai finishes order-checking.

Embodiment 2: kendir CUC3 gene conserved region gene fragment sequence information

The information of SEQ.ID.NO 1

G) sequence signature

Length: 287 Nucleotide

Type: nucleic acid

Chain: two strands

H) molecule type: cDNA

I) suppose: not

I) antisense: not

K) initial source: kendir

L) sequence description: SEQ.ID.NO 1

1 ATTACCTTTT?ACTTAGCTTC?CAAAGTCTTA?TACGGACGCT?TCTGTGGACT?CGACATTGCT

61 GAAGTCGACC?TCAACAGATG?TGAGCCATGG?GAGCTTCCCG?ATGCAGCTAA?GATGGGAGAG

121?AGAGAGTGGT?ACCTTTTCAG?CTTAAGGGAC?AGGAAGTACC?CAACGGGGCT?GAGAACAAAT

181 AGAGCCACTT?GTGCCGGGTA?CTGGAAAGCA?ACGGGGAAAG?ATAGAGAAGT?CTACGGCAGT

241 GAAGGCGTTG?TTGTGGGCAT?GAAGAAGACA?TTTGTTTTCT?ACAAAGG

The information of SEQ.ID.NO 2

(d) sequence signature

Length: 95 amino acid

Type: amino acid

Chain: strand

(e) molecule type: protein

(f) sequence description: SEQ.ID.NO 2

1 ITFYLASKVL?YGRFCGLDIA?EVDLNRCEPW?ELPDAAKMGE?REWYLFSLRD?RKYPTGLRTN

61 RATCAGYWKA?TGKDREVYGS?EGVVVGMKKT?FVFYK

Embodiment 3: the structure of antisense expression vector and conversion

1. the structure of antisense expression vector

With restriction enzyme XbaI and SacI CUC3 conserved regions sequence is downcut from the pEASY-T1 cloning vector,, reclaim the purpose fragment respectively with same restrictions endonuclease digestion expression vector pCAMBIA1301.Connect 16h with the T4DNA ligase enzyme in 16 ℃.Connect product and transform the Top10 competent cell, screen with kalamycin resistance.Select mono-clonal and carry out PCR detection and restriction analysis.

2. antisense expression vector transforming agrobacterium rhizogenes competent cell

The plasmid transforming agrobacterium rhizogenes uses freeze-thaw method.Get the plasmid pCAMBIA-1301-CUC3 that 6 μ l build and be transferred in the Agrobacterium rhizogenes R1601 competent cell that has just made, place 30min on ice.Immerse 5min in the liquid nitrogen, 37 ℃ of water-bath 5min add the empty YEB of 500 μ l, and 28 ℃, 150-160rpm shakes 3-5h.Containing on the YEB solid plate of kantlex, cultivating 2-3 days in 28 ℃ of incubators.The picking mono-clonal carries out bacterium colony PCR to be identified.

Embodiment 4: kendir root of hair culture system is set up

To change the Agrobacterium rhizogenes R1601 of antisense expression vector over to, R1000, LBA9402 activation 2-3 time, the picking mono-clonal is in the liquid nutrient medium of the YEB that contains kantlex (50mg/L), 28 ℃, 200r/min, incubated overnight.Next day, at logarithmic phase, the OD600 value was used for infecting for 0.5-1.0 by 1: 50 volume ratio enlarged culturing 3-5h.3500r/min collects thalline, after the MS substratum dilution with the equal volume of pH 5.80, respectively different sites explants such as the true leaf of kendir, stem, cotyledonary node is infected 20-30min, after 7-10 days successively hairiness shape root grow.Changing over to behind the aseptic water washing on the MS solid medium that contains cephamycin 500mg/L, 25 ± 1 ℃, 14h/10h light/dark the cultivation.Transferred once in 5-7 days, corotation connects 3-5 time, takes off bacterium to aseptic.

3 kinds of Agrobacterium rhizogenes are to the induction of hairy roots rate of kendir cotyledonary node and induce density as shown in table 1.

The explant number of the explant number/inoculation of hairly root inductivity=generation hairly root

Hairly root is induced the explant number of the sum/generation hairly root of density=generation hairly root

Table 1 different strains is to the inductivity of kendir cotyledonary node hairly root

Agrobacterium rhizogenes	Inoculation explant number	Produce hairly root explant number	The hairly root sum	Hairly root inductivity (%)	Hairly root is induced density
						R1000?	30?	26?	242?	86.7?	8.07?
R1601?	30?	28?	225?	93.3?	7.50?
						LBA9402?	30?	30?	216?	100?	7.20?

Embodiment 5: the regeneration of kendir transfer-gen plant root of hair approach

It is long to treat that hairly root grows to 2-3cm, downcuts, and extracts genomic dna with the CTAB method, carries out PCR and detects.The root of hair of test positive changes on the no hormone solid 1/2MS bud inducing culture and cultivates, and the bud point appears in 2-3 after week, and indefinite bud is long to 2-4 centimetre, downcuts and changes the 1/MS root induction substratum that contains IBA0.2-0.4mg/l over to, and 2-3 week sends out roots.Above-mentioned three kinds of Agrobacterium rhizogenes induce the regeneration plant inductivity of root of hair of generation as shown in table 2.

Regeneration plant inductivity: the root of hair number that produces regeneration plant sum/inoculation

The regeneration plant of the root of hair in table 2 different strains source inductivity

The root of hair source	The root of hair number of inoculation	Generation regeneration plant sum	Regeneration plant inductivity (%)
				R1000?	50?	5?	10?
R1601?	50?	15?	30?
				LBA9402?	50?	8?	16?

Embodiment 6: the detection of transfer-gen plant goal gene

Transgenosis kendir PCR detects: adopt the segmental degenerate primer of above-mentioned clone CUC3 conserved region gene, purpose fragment 287bp.PCR detected result shows, in 64 strains detection plant 50 strains being arranged is the PCR positive, amplified the molecular weight band identical, and adjoining tree does not amplify respective strap with target gene fragment.

Transgenosis kendir Southern blot detects: with the DNA hybridization of 35S promoter-target gene sequences as probe and transgenic regenerated plant, in the PCR positive plant, 80% plant transgenosis verity is confirmed through the Southern hybridization analysis, contains one to multiple hybrid belt.

Embodiment 7: the screening of the transgenic line that branch is less

With the 40 strain transgenosis kendir strains that obtain system, carry out hardening after, be transplanted to the land for growing field crops and cultivate.After a growth season, its branch amount is carried out individual plant statistics, with the wild type seeds seedling of contemporaneouslys 1000 strain field planting relatively after, screening obtains the less transgenosis kendir strain system of branch, carries out many generation screenings afterwards.With the seed of last one year of results, behind the field planting, further repeated screening is to obtain stable phenotype and germ plasm resource.

Claims

1. a method for controlling Apocynum apocynum branch by gene silencing, is characterized in that:

(1). Cloning of the conserved region of CUC3, a branch regulation gene of Apocynum apocynum;

The RT-PCR method was used to extract the total RNA of Apocynum apocynum and reverse transcribe to generate cDNA. The cDNA was used as a template to clone the gene fragment of the conservative region of the branching regulatory gene CUC3, and the antisense expression vector was constructed according to the principle of antisense RNA gene silencing;

Cloning of CUC3 gene fragments: Total RNA was prepared from aseptic seedlings of Apocynum apocynum, the first strand of cDNA was generated by reverse transcription, and then the first strand of cDNA was used as a template for RT-PCR reaction. According to the CUC3 gene included in Genbank The sequence of the conserved region was designed to design a pair of degenerate primers, the sequence of which is:

Primer1: 5′-ATTACT/CTTT/CTACTTAGCTTCCAAAA/G-3′;

Primer2: 5′-CCTTTGTAGAAC/AACC/AAA/GC/TGTC/TTTCTTC-3′

(2). Construction of gene silencing vector;

The target product fragment of RT-PCR was inserted into the cloning vector pEASY-T1, and then the DNA sequence was analyzed, and the sequence result was compared with the sequence in Genbank for homology;

The correctly sequenced CUC3 gene fragment was excised from pEASY-T1 with XbaI and SacI, and reversely inserted into the expression vector pCAMBIA-1301 containing the 35S promoter and NOS terminator to construct the antisense expression vector pCAMBIA-1301-CUC3;

(3). Gene transfer by the method mediated by Agrobacterium rhizogenes;

Introduce the antisense expression vector pCAMBIA1301-CUC3 into Agrobacterium rhizogenes by freeze-thaw method, coat the YEB plate containing 50-100 mg/L kanamycin, culture it in a constant temperature incubator at 28°C for 2 days, and pick a single colony , detect with PCR method, detect as positive clone and be used for infecting apocynum true leaf, stem, cotyledon node, cotyledon, hypocotyl, different explant parts of root;

(4). Transformation of apocynum and regeneration of transgenic plants;

Infect the above-mentioned different explant parts of Apocynum rhizogenes with the Agrobacterium rhizogenes that introduce the antisense expression vector, and the explants after infection are co-cultured for 2 days under dark conditions, and then transferred to MS medium containing 500mg/L cephalosporin , 25±1°C, 14h/10h light/dark culture, transfer once every 5-7 days, transfer 3-5 times in total, produce hairy roots about 7-10 days after infection, wait for the hairy roots to grow to 2-3cm, detect with the PCR method, cut off the positive root and transfer it to the bud induction medium of 50mg/L kanamycin and 300mg/L cephalosporin for screening, and the buds to be screened grow to When the height is 2-3cm, it is transferred to the rooting medium, and after 2-3 weeks of cultivation, the transformed plant takes root and grows into a complete regeneration plant;

(5). Detection and screening of transgenic plants;

The transgenic plants are screened in three steps. The first step is to detect the target gene of the transgenic plants by using PCR and Southern blot method. The PCR primers are the merger primers for the cloned CUC3 conserved region gene fragment described in step (1), Southern blot The DNA probe is the entire sequence of the 35S promoter-CUC3 conserved region gene segment. In the second step, the positive transgenic lines are hardened and transplanted to the field for cultivation. After one growing season, the The number of branches is counted per plant. After comparing with the wild-type seedlings of the same period, the transgenic Apocynum line with fewer branches is screened. The third step is multi-generation screening. The seeds harvested in the previous year are planted in the field. , further repeated screening to obtain stable phenotypes and germplasm resources.

2. a kind of method for controlling Apocynum apocynum branch by gene silencing according to claim 1, is characterized in that the nucleotide sequence of the CUC3 gene conservative region gene fragment that clone obtains is:

1ATTACCTTTT ACTTAGCTTC CAAAGTCTTA TACGGACGCT TCTGTGGACT CGACATTGCT

61GAAGTCGACC TCAACAGATG TGAGCCATGG GAGCTTCCCG ATGCAGCTAA GATGGGAGAG

121AGAGAGTGGT ACCTTTTCAG CTTAAGGGAC AGGAAGTACC CAACGGGGCT GAGAACAAAT

181AGAGCCACTT GTGCCGGGTA CTGGAAAGCA ACGGGGAAAG ATAGAGAAGT CTACGGCAGT

241GAAGGCGTTG TTGTGGGCAT GAAGAAGACA TTTGTTTTCT ACAAAGG.

3. a kind of method for controlling Apocynum apocynum branch by gene silencing according to claim 1, is characterized in that the aminoacid sequence encoded by the gene fragment that this clone obtains is:

1ITFYLASKVL YGRFCGLDIA EVDLNRCEPW ELPDAAKMGE REWYLFSLRD RKYPTGLRTN

61 RATCAGYWKA TGKDREVYGS EGVVVGMKKT FVFYK.

4. a kind of method for controlling apocynum branch by gene silencing according to claim 1, is characterized in that: the culture condition of described Agrobacterium rhizogenes is that basic medium is the YEB medium of pH=7.0, and temperature is At 28°C, the rotation speed is 160-200r/min, and the optimum concentration range for inducing hair roots is OD600=0.5-1.0.

5. a kind of method for controlling apocynum branch by gene silencing according to claim 1, is characterized in that: the formula of described bud induction medium is 1/2MS, and the formula of rooting medium is IBA 0.2-0.4 mg/L+1/2MS.

6. a kind of method for controlling Apocynum venetum branch by gene silencing according to claim 1, is characterized in that: described Apocynum venetum kind comprises Apocynum venetum Linn (Apocynum venetum Linn) and Apocynum hemp (Apocynum hendersonii Hook.f .).

7. A method for controlling apocynum branching by gene silencing according to claim 1, characterized in that: said Agrobacterium rhizogenes is R1601, R1000 or LBA9402.