CN111118044B - Kenaf HcCLA1 gene VIGS silencing system - Google Patents
Kenaf HcCLA1 gene VIGS silencing system Download PDFInfo
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- CN111118044B CN111118044B CN202010073455.1A CN202010073455A CN111118044B CN 111118044 B CN111118044 B CN 111118044B CN 202010073455 A CN202010073455 A CN 202010073455A CN 111118044 B CN111118044 B CN 111118044B
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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- C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
- C12Y202/01—Transketolases and transaldolases (2.2.1)
- C12Y202/01007—1-Deoxy-D-xylulose-5-phosphate synthase (2.2.1.7)
Abstract
The invention belongs to the technical field of plant genetic engineering and discloses kenafHcCLA1Gene VIGS silencing system. By constructing a catalyst containingHcCLA1pTRV2 virus silencing expression vector plasmid of gene specific fragment, agrobacterium mediating method for transforming kenaf and inducing kenaf endogenous sourceHcCLA1The silencing occurs, and the kenaf is effectively reducedHcCLA1Expression level of the gene, resulting in a albino phenotype. The method establishes a system for silencing kenaf endogenous genes by TRV-VIGS so as to identify the gene functions, has the advantages of high speed, high flux and easy operation, and provides a new way for developing kenaf gene function research.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to kenafHcCLA1Gene VIGS silencing system.
Background
Virus-induced gene silencing (VIGS) belongs to post-transcriptional gene silencing (PTGS). Among them, the gene silencing (TRV-VIGS) induced by Tobacco Rattle Virus (TRV) is a widely used gene silencing system at present due to its advantages of long duration, high silencing efficiency, light host plant virus symptoms, no covering of silencing phenotype, and generation of gene silencing in various tissues. TRV-VIGS has been successfully applied to plants such as tomato, tobacco, pepper, Arabidopsis thaliana and the like.
The VIGS system is established in different plants, and 1-deoxyxylulose-5-phosphate synthase gene (cloroplasto alterados,CLA1) The gene serves as a reporter gene. Plant and method for producing the sameCLA1(cloroplasis alterados 1) gene is involved in the development process of chloroplast, and has high evolutionThe degree of the method is conservative,CLA1the gene mutant has obvious albino phenotype and is easy to identify marker character. The invention constructs a carrierHcCLA1The tobacco brittle fracture virus recombinant vector of the interference fragment successfully infects kenaf through agrobacterium tumefaciens to establish a kenaf efficient VIGS system, thereby providing technical support for kenaf functional genomics research.
Disclosure of Invention
The invention aims to provide kenafHcCLA1Gene VIGS silencing system. The method successfully establishes kenafHcCLA1The VIGS silencing system of the gene realizes simple, high-efficiency and low-cost identification of the kenafHcCLA1Gene function.
In order to achieve the purpose, the invention adopts the following technical scheme:
kenaf 1-deoxyxylulose-5-phosphate synthase geneHcCLA1The nucleotide sequence of the full-length gene of the gene is shown as SEQ ID NO. 1.
Silencing hibiscus cannabinus 1-deoxyxylulose-5-phosphate synthase geneHcCLA1Is/are as followsHcCLA1A gene-specific fragment ofHcCLA1The nucleotide sequence of the gene specificity fragment is shown as SEQ ID NO. 4.
Kenaf 1-deoxyxylulose-5-phosphate synthase geneHcCLA1The VIGS viral vector of (1), which is obtained by recombinant cloning using gateway technologyHcCLA1The gene specific fragment is connected to a VIGS virus framework vector pTRV2 to construct and obtain a recombinant virus vector pTRV2-HcCLA1。
KenafHcCLA11, the construction method of the gene VIGS silencing system comprises the following steps:
(1) the recombinant viral vector pTRV2-HcCLA11 is transformed into agrobacterium GV3101 competent cells, and PCR identification is positive;
(2) mixing the transformed positive agrobacterium liquid and pTRV1 liquid according to the volume ratio of the liquid to be 1:1 to prepare mixed liquid;
(3) soaking the kenaf seeds in the mixed bacterial liquid obtained in the step (2) for 24 hours at 24 ℃;
(4) dropping the seeds soaked in the step (3) in the soil thoroughly watered with clear water at 24 ℃/2Growing at 0 deg.C for 14 h/10 h light/dark period, observing phenotype change of newly grown kenaf true leaf, and detecting leafHcCLA11, constructing the expression of the kenafHcCLA11 gene sequence silencing system.
As described aboveHcCLA1Silencing 1-deoxyxylulose-5-phosphate synthase gene of kenaf by gene specific fragmentHcCLA1The use of (1).
The kenaf 1-deoxyxylulose-5-phosphate synthase geneHcCLA1The VIGS viral vector is used for silencing 1-deoxyxylulose-5-phosphate synthase gene of kenafHcCLA1The use of (1).
The kenafHcCLA1VIGS silencing system of 1 gene for silencing 1-deoxyxylulose-5-phosphate synthase gene of kenafHcCLA1The use of (1).
The above kenaf 1-deoxyxylulose-5-phosphate synthase geneHcCLA1Acquisition of the full-length gene sequence: arabidopsis thaliana was obtained at the TAIR website (https:// www.arabidopsis.org /)AtCLA1(AT 4G 15560) gene protein sequence. Then, at the NCBI website (https:// www.ncbi.nlm.nih.gov/, Sequence Read Archive (SRA) accession number PRJNA 596386), the 1-deoxyxylulose-5-phosphate synthase gene was obtained by searching for the kenaf homologous gene by BLASTP using the reference genome of the kenaf fine variety Fuhong 952 (Zhang et al, 2020, Plant Biotechnology Journal, DOI: 10.1111/pbi.13341)HcCLA1The nucleotide sequence of the full-length gene sequence is shown as SEQ ID NO. 1.
As described aboveHcCLA1Cloning of gene-specific fragments: with a 1-deoxyxylulose-5-phosphate synthase geneHcCLA1The full-length gene sequence of (a) is a template designed for amplificationHcCLA1Primers for a 250bp specific fragment on the CDS domain of a geneHcCLA1-F andHcCLA1-R(ii) a The nucleotide sequences are respectively:
HcCLA1-F：SEQ ID NO.2：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATTATGTCTTCAATGCTCCTAGAG-3’
HcCLA1-R：SEQ ID NO.3：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCACAACATTATTCCTTTCACCTTTC-3’
taking young leaves of the kenaf ford 952, freezing the young leaves in a mortar by using liquid nitrogen, grinding the young leaves into powder, extracting RNA by using an OMEGA kit operation step, and freezing and storing the RNA at minus 80 ℃ for later use. Taking out RNA frozen at-80 deg.C, synthesizing cDNA first strand according to TaKaRa reverse transcription kit operation procedure, and storing at-20 deg.C. Using cDNA as template and specific primerHcCLA1-F andHcCLA1- RPCR amplificationHcCLA1The size of the gene specific fragment is 250bp, and the nucleotide sequence is shown in SEQ ID NO. 4.
The invention has the beneficial effects that:
1. the invention constructs the kenaf for the first timeHcCLA1A gene VIGS silencing system, obtains the gene capable of leading kenafHcCLA1A system of gene silencing.
2. The invention constructs the kenaf by adopting the tobacco brittle fracture virusHcCLA1The gene silencing system can perform systematic diffusion and transmission in kenaf plants.
3. The silencing system can effectively reduce the kenafHcCLA1The expression level of the gene, chlorophyll, was bleached.
Drawings
FIG. 1 recombinant viral vector pTRV2 constructed in example 1 of the present inventionHcCLA1Agarose gel electrophoresis picture of agrobacterium liquid PCR. M is DNA ladder DL2000, 1-8 is Agrobacterium clone.
FIG. 2 the albino phenotype profile of new leaves that appears after the silencing vehicle in example 1 of the present invention has infected kenaf seedlings.
FIG. 3 detection of kenaf by real-time fluorescent quantitative PCR (qRT-PCR) in example 2 of the present inventionHcCLA1The silencing effect of (3). CK: kenaf seedlings infected by the empty vector pTRV 2;HcCLA1: recombinant viral vector pTRV2-HcCLA1Infected kenaf seedlings.
FIG. 4 plasmid pTRV2-HcCLA1Structure diagram. The invention adopts the gateway cloning method to construct the vector plasmid pTRV2-HcCLA1. Wherein ccdB is lethal gene and Cm is target geneHcCLA1。
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
The present invention will be described in further detail below with reference to specific production examples and application examples, and with reference to the data. It will be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. The primers used are indicated for the first time and the same primers used thereafter are indicated for the first time.
Example 1 construction of recombinant viral vector pTRV2-HcCLA1
（1）HcCLA1Obtaining full-Length Gene sequences
First, Arabidopsis thaliana was obtained at the TAIR website (https:// www.arabidopsis.org /)AtCLA1(AT 4G 15560) gene protein sequence. Then, at the NCBI website (https:// www.ncbi.nlm.nih.gov/, Sequence Read Archive (SRA) accession number PRJNA 596386), the 1-deoxyxylulose-5-phosphate synthase gene was obtained by searching for the kenaf homologous gene by BLASTP using the reference genome of the kenaf fine variety Fuhong 952 (Zhang et al, 2020, Plant Biotechnology Journal, DOI: 10.1111/pbi.13341)HcCLA1The nucleotide sequence of the full-length gene sequence is shown as SEQ ID NO. 1.
（2）HcCLA1Cloning of Gene-specific fragments
With a 1-deoxyxylulose-5-phosphate synthase geneHcCLA1The full-length gene sequence of (a) is a template designed for amplificationHcCLA1Primers for a 250bp specific fragment on the CDS domain of a geneHcCLA1-F andHcCLA1-R(ii) a The nucleotide sequences are respectively:
HcCLA1-F：SEQ ID NO.2：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATTATGTCTTCAATGCTCCTAGAG-3’
HcCLA1-R：SEQ ID NO.3：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCACAACATTATTCCTTTCACCTTTC-3’
taking young leaf of flos Rhododendri Simsii 952, freezing with liquid nitrogen in mortar, and grinding into powderThen using OMEGA kit operation steps to extract RNA, and freezing and storing at-80 ℃ for later use. Taking out RNA frozen at-80 deg.C, synthesizing cDNA first strand according to TaKaRa reverse transcription kit operation procedure, and storing at-20 deg.C. Using cDNA as template and specific primerHcCLA1-F andHcCLA1- RPCR amplificationHcCLA1A gene-specific fragment.
The PCR reaction system is as follows: mu.L of cDNA template, 5. mu.L of upstream and downstream primers (10. mu.M) each 1.5. mu. L, dNTP (2mM), and 10 XKOD PCR buffer 5. mu. L, MgSO 42. mu.L (25mM), 1. mu.L of KOD Plus, Plus ddH2O to 50. mu.L. The amplification conditions were: denaturation at 94 ℃ for 3min, denaturation at 55 ℃ for 30s, denaturation at 68 ℃ for 2min for 32 cycles, and extension at 72 ℃ for 10 min. To obtainHcCLA1A total of 250bp of specific sequence (SEQ ID NO. 4) is sent to a company for sequencing analysis to ensure that the specific fragment is positionedHcCLA1The gene domain of (a). After the silencing of the fragment(s) is complete,HcCLA1the function of the gene is lost.
(3) Vector construction and VIGS infection
The vector was constructed using Gateway technology. The operation steps are as follows:
will be provided withHcCLA1The specific fragment (SEQ ID NO. 4), the vector pDONR207 and the BP enzyme are mixed uniformly according to the system and then placed in a PCR instrument at 25 ℃ for overnight connection. The resulting ligation products were then heat shock transformed into DB3.1 competent cells, plated on LB plates containing gentamicin (50. mu.g/mL), cultured overnight at 37 ℃ and the positive monoclonal selected for expanded culture. Extraction of plasmid pDONR207-HcCLA1Thereafter, the detection was carried out by PCR (SEQ ID NO. 6) amplification using the universal primer pDONR207-F (SEQ ID NO. 5) of pDONR207 and pDONR 207-R. The plasmid pDONR207-HcCLA1、The pTRV2 vector and LR enzyme were mixed well and ligated overnight in a 25 ℃ PCR instrument. The resulting ligation products were then heat shock transformed into TOP10 competent cells, plated on LB plates (50. mu.g/mL) containing kanamycin, incubated overnight at 37 ℃, and positive single colonies were picked for expansion. Extraction of plasmid pTRV2-HcCLA1By usingHcCLA1-F andHcCLA1the-R carries out PCR amplification detection, and the sequence is verified by sequencingHcCLA1The specific fragment of SEQ ID NO. 4. Plasmid pTRV2-HcCLA1(structural diagram, FIG. 4) transfer of G by freeze-thawV3101 competent cells, containing 50. mu.g mL-1Kanamycin, 50. mu.g mL-1Gentamicin and 50. mu.g mL-1Screening rifampicin to obtain target gene containing segmentHcCLA1Agrobacterium strains of the specific fragment.
Refer to the preparation of Vel squez et al. The pTRV1 plasmid, pTRV2 plasmid and pTRV2-HcCLA1The plasmids were transformed into Agrobacterium GV3101, respectively. Selecting freshly cultured pTRV 1-containing plasmid, pTRV2 plasmid and pTRV2-HcCLA1Single colonies of Agrobacterium GV3101 transformant of plasmid (FIG. 1) were inoculated into 1 mLLB liquid medium (Kan, 50. mu.g mL)-1; Gen, 50 μg mL-1;Rif, 50 μg mL-1) Middle, 28 ℃ 180 r min-1Culturing for 24h, transferring into 150 mL LB liquid medium (Kan, 50. mu.g mL)–1; Gen, 50 μg mL–1;Rif, 50 μg mL–1) Middle, 28 ℃ 180 r min-1Culturing for 12 h; the cells were collected by centrifugation at 5000rpm for 10min at 28 ℃ and resuspended in an appropriate volume (10 mmol L)-1MgCl2, 10 mmol L-1MES and 200. mu. mol L-1Acetosyringone) was resuspended to a final concentration of 1.5 (OD)600) (ii) a Standing the re-suspension at room temperature for 3 h, and respectively mixing Agrobacterium GV3101 bacterial liquid carrying pTRV1 vector with Agrobacterium GV3101 bacterial liquid carrying pTRV2 and Agrobacterium GV3101 bacterial liquid carrying pTRV2-HcCLA1The heavy suspension of the plasmid agrobacterium GV3101 bacteria liquid is mixed in the volume ratio of 1 to prepare 2 kinds of mixed bacteria liquid for infecting kenaf seeds. And (3) immersing the kenaf seeds in the mixed bacterial liquid for 24h at 24 ℃, and growing the kenaf seed experimental material under the conditions of 24 ℃/20 ℃ and 14 h/10 h of light/dark cycle.
Example 2HcLMI1Expression analysis in kenaf
(1) After infection, kenafHcLMI1Gene expression level measurement
To be coveredpTRV: CLA1When the infected seeds grow seedlings with white leaf phenotype (FIG. 2), the seedlings are pickedpTRV: CLA1Infected leaves, extracting total RNA from leaves with RNA kit to obtainActinDetecting the expression level of the silenced target gene by RT-PCR as an internal reference gene. The primers used were:HcCLA1-RT-F (SEQ ID NO.7); HcCLA1-RT-R (SEQ ID NO.8)；Actin-RT-F (SEQ ID NO.9)；Actin-RT-R (SEQ ID NO.10)。HcCLA1the results of the gene expression amount measurement are shown in FIG. 3, and the results in FIG. 3 show that: compared with the control CK, is subjected to the reaction containing pTRV2-HcCLA1In kenaf leaves infected with vector AgrobacteriumHcCLA1The gene expression level is obviously reduced.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> hibiscus cannabinus HcCLA1 gene VIGS silencing system
<130> 10
<160> 10
<170> PatentIn version 3.3
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ttgactcaaa ggtttgcaac tcagtcctcc acttaccatt tgagaaataa acagtgccaa 60
aaacagtggc acagtcaata accaattttg tttataaatc tacatttcat tggttccacc 120
atctctcatt cttgcactca cttggtgggg atactttatt ttttttctgg gttcttctca 180
ttttgagctc ttggccaata attttataaa aatacttcat tgttctttgt agattgttta 240
gtattttcta gccatggctc tttgtgcatg ttcatttcct gccattgtta gctgctgtgc 300
tgctgcttca gaacctcaaa agtccactcc ttttgcttcc catttgcttg gtggttcaga 360
tctgggtttc cacccattgc ttaagctcaa tcaggttcat atgaagaata aaacagcaga 420
aaaaaaaaaa ctgggttttg ttttttttgg ggggttagat tttaaagtta ttatctgttt 480
gaataaagtt tagttttaag aaagggatag cctacgaaaa attctctctt tttttggctt 540
tctttctttc tttctcattt tctataatct agtatttgtt ttgtgttttc ttttgacttg 600
gatacaatta agtctttgat tccagtttaa aaatatgtat aaattgaaaa aaagggttgt 660
tgtttttttt gttttgattt gtttaaggtg aagaaaaggc catgtggggt ttatgcatca 720
ctatcagaag gagctgagta ttactcctca agaccagcaa cgccgctctt ggacactata 780
aactatccaa ttcatatgaa aaatctctct gtaaaggtat agaaacatta gaaaagaaca 840
tgtttttttt ttgttgtaat ttattatgag atgttcatct cttttatcat atatgtatgt 900
atgagaactt ttcatgcagg aactgaaaca actagctgat gaactgcggt ctgatgtgat 960
tttcaacgtt tcaaaaactg gtggtcactt ggggtcaagc cttggcgtgg ttgaacttac 1020
tgtggctctt cattatgtct tcaatgctcc tagagacaag atattatggg atgttggaca 1080
tcaggttcgg cttggttcta gttttgcttg gtttcctttt attttcttat gtgaattggc 1140
cctaatttgt tatagtaact catttggttt gtagtcttac cctcataaaa tcttgactgg 1200
gagaagagat aagatgcata ccatgaggca aactaatgga ttggccggat tcacgaaacg 1260
agctgagagc gaatatgatt gctttgggac tggtcacagt tcaaccacaa tctctgctgg 1320
cttgggtatt agtctggtct tcttgcattc attttgcata tatgttttag gttagaaagt 1380
tgaaatagat aaatattatc gaaccatcat cgagttcttt gatgttgctt ttagaaccgg 1440
gttggtttgt atggctaatc taaaccttgt tcatctgcta acatgtgatt tttgcttaat 1500
atgggatgaa gttttagttt tcgatatggt ggaattttat ttttgattct gttctgcaaa 1560
tcgagttcca cttgcccttt ggagttttaa ttgtctcatg ttttgggtgg tcaggaatgg 1620
ctgtgggaag ggatctgaaa ggtgaaagga ataatgttgt tgctgtcata ggcgatggtg 1680
caatgaccgc gggacaagct tacgaagcaa tgaataatgc cggatacctg gattccgata 1740
tgattgttat tcttaatgac aataaacaag tttctctgcc aactgccaat cttgatggac 1800
ctataccgcc cgtgggagct ttgagcagtg ctctcagtag gctgcaatcg aacaggcctc 1860
ttagagaact aagagaggtt gctaaggtaa atagaaagga acagagccgg agacgctaat 1920
ataattttgc ccgccaacta aaattaactt gaactgagtt tggttttatt gcagggagtt 1980
accaagcaaa tcggtgggcc catgcatgaa ctggctgcaa aagttgatga gtatgctcgt 2040
gggatgataa gtggttccgg ttcaacactt tttgaagaac ttggactgta ttatattgga 2100
cctgttgatg gccacaacat cgatgattta gtttctattc tcaaagaggt taagactact 2160
aaaacaacgg gtcctgtcct gatccatgtt gtcaccgaga aaggccgggg ttatccatat 2220
gcagagagag ctgatgacaa gtaccatggt aacatgcaca ataagccata tttagaagag 2280
atgtcatttt gtatgattta attcactgac ctcgatttat gaacttagtt tagggaacaa 2340
aagagaataa attatgtctg gctttctttt cacaggagtg gtgaagttcg atccggcaac 2400
tggaaagcaa ttcaaaggca gttctgttac ccagtcttac actacatatt ttgctgaggc 2460
tttgattgca gaagccgagg ccgacaaaaa tatcgttgcc atccatgctg caatgggagg 2520
tggaactgga ttaaacctct tcctccgccg tttcccacaa agatgttttg atgttgggat 2580
tgccgaacaa catgccgtaa cctttgctgc aggtttggcc tgtgaaggct tgaaaccttt 2640
ttgtgcaatc tactcatcat tcatgcaaag ggcttatgac caggtaagct accaaagtta 2700
ccattgtttt tacgatctgt aaaagattaa tattctgtgt atgttaaaca tctgcatttc 2760
aaattcataa cgaatgtgtc gatttgctgc aacaggttgt acatgatgtt gacctgcaga 2820
agctgcctgt aagatttgct atggatagag ctggccttgt tggtgcagat ggtccaacac 2880
attgtggggc tttcgatgtc actttcatgg catgcctccc caacatggtc gtaatggctc 2940
cttctgatga ggctgagctt tttcatatgg ttgccacagc tgcagccata gatgaccgtc 3000
ctagctgttt tcgttacccg agaggaaatg ggatcggagt tcagttgccg ccagggaaca 3060
aaggtgttcc tcttgaggtg actttcgatc aaatatgtgc atattcctat tcgtaaagaa 3120
acaaagacta aatgaaacat accaacaact taatccaaaa tgcgtacaat gtccttacag 3180
atcggcaaag gtagggtact gatagaagga gaaagagtgg cactattagg ttatggatct 3240
gcagttcaaa gctgcttagc agccgcctcg ttattggaat cctacggctt acagctgact 3300
gttgcggacg cacgattttg taaaccattg gatcactccc tcatccggga gctggcaaaa 3360
tcacatgaag ttctgatcac agtcgaagaa ggatcgatcg gtggcttcgg ttctcatgtg 3420
gcacagttcc tagctcttga tggtcttctt gatggcaaag taaaggtatg cgaatccaaa 3480
atcgcagact tgaatgtcca taattggaag ctgatataac cgtagaacaa tttgtttctt 3540
tgcattaata tttttcccca atttgcagtg gaggccagtg gttcttcccg atcgatacat 3600
cgaccatggc tcacccgccg accagttggc cgaagctggt ctgacaccat cgcacattgc 3660
agcaacagtg ttcaatgtgc ttggacaaaa aagagaggct cttgggatca tgtcgtcgag 3720
aaactgaaga aattcttcag ctaaactttc attttatgta caaaaatttt catttcaaat 3780
gaatggtagt tagtttaaac atatatatgt atatttatga tcaaaattta tatctaaaca 3840
atgatacagt tttaccataa atatttacag ttttcactct tctaacagat gtaaaatact 3900
ggcagaaaga tctttgaggc ctttaatttc aaaattgagc aaataagtcc atttcagaga 3960
aaatagaacc agtccagtcc tgtcattttt gaaagcaaac aatc 4004
<210> 2
<211> 55
<212> DNA
<213> Artificial sequence
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ggggacaagt ttgtacaaaa aagcaggctt cattatgtct tcaatgctcc tagag 55
<210> 3
<211> 54
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<213> Artificial sequence
<400> 3
ggggaccact ttgtacaaga aagctgggtc acaacattat tcctttcacc tttc 54
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<213> Artificial sequence
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attatgtctt caatgctcct agagacaaga tattatggga tgttggacat cagtcttacc 60
ctcataaaat cttgactggg agaagagata agatgcatac catgaggcaa actaatggat 120
tggccggatt cacgaaacga gctgagagcg aatatgattg ctttgggact ggtcacagtt 180
caaccacaat ctctgctggc ttgggaatgg ctgtgggaag ggatctgaaa ggtgaaagga 240
ataatgttgt 250
<210> 5
<211> 25
<212> DNA
<213> Artificial sequence
<400> 5
tcgcgttaac gctagcatgg atctc 25
<210> 6
<211> 24
<212> DNA
<213> Artificial sequence
<400> 6
gtaacatcag agattttgag acac 24
<210> 7
<211> 26
<212> DNA
<213> Artificial sequence
<400> 7
tgctcctaga gacaagatat tatggg 26
<210> 8
<211> 26
<212> DNA
<213> Artificial sequence
<400> 8
caacattatt cctttcacct ttcaga 26
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence
<400> 9
ttgcagaccg tatgagcaag 20
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence
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atcctccgat ccagacactg 20
Claims (7)
1. Kenaf 1-deoxyxylulose-5-phosphate synthase geneHcCLA1The method is characterized in that: the 1-deoxyxylulose-5-phosphate synthase geneHcCLA1The nucleotide sequence of the full-length gene is shown in SEQ ID NO. 1.
2. A method for silencing the kenaf 1-deoxyxylulose-5-phosphate synthase gene according to claim 1HcCLA1Is/are as followsHcCLA1A gene-specific fragment characterized by: the above-mentionedHcCLA1The nucleotide sequence of the gene specificity fragment is shown as SEQ ID NO. 4.
3. The kenaf 1-deoxyxylulose-5-phosphate synthase gene according to claim 1HcCLA1The VIGS viral vector of (1), wherein: the technology of using gateway recombinant cloning is toHcCLA1The gene specific fragment is connected to a VIGS virus framework vector pTRV2 to construct and obtain a recombinant virus vector pTRV2-HcCLA1(ii) a The above-mentionedHcCLA1The nucleotide sequence of the gene specificity fragment is shown as SEQ ID NO. 4.
4. The kenaf 1-deoxyxylulose-5-phosphate synthase gene according to claim 1HcCLA1The construction method of the VIGS silencing system comprises the following steps:
(1) freezing and thawing method for recombinant viral vector pTRV2-HcCLA1Transformation into agrobacterium GV 3101;
the recombinant viral vector pTRV2-HcCLA1Is prepared by using gateway recombinant cloning technologyHcCLA1The gene specific fragment is connected to a VIGS virus framework vector pTRV2 to construct and obtain a recombinant virus vector pTRV2-HcCLA1(ii) a The above-mentionedHcCLA1The nucleotide sequence of the gene specificity fragment is shown as SEQ ID NO. 4;
(2) mixing the transformed positive agrobacterium liquid and pTRV1 liquid according to the liquid volume ratio of 1:1 to prepare mixed liquid;
(3) soaking seeds in the mixed bacterial liquid obtained in the step (2) for 24 hours at 24 ℃;
(4) dropping the soaked seeds in the soil thoroughly watered with clear water, growing at 24 deg.C/20 deg.C for 14 h/10 h in light/dark cycle, observing the phenotype of true leaves of kenaf, and detecting the leavesHcCLA1The expression condition of (1) is to construct the kenaf 1-deoxyxylulose-5-phosphate synthase geneHcCLA1The silencing system of (1).
5. The method of claim 2HcCLA1Silencing 1-deoxyxylulose-5-phosphate synthase gene of kenaf by gene specific fragmentHcCLA1The use of (1); the kenaf 1-deoxyxylulose-5-phosphate synthase geneHcCLA1The nucleotide sequence of (A) is shown in SEQ ID NO. 1.
6. The kenaf 1-deoxyxylulose-5-phosphate synthase gene according to claim 3HcCLA1The VIGS viral vector is used for silencing 1-deoxyxylulose-5-phosphate synthase gene of kenafHcCLA1The use of (1); the kenaf 1-deoxyxylulose-5-phosphate synthase geneHcCLA1The nucleotide sequence of (A) is shown in SEQ ID NO. 1.
7. The method according to claim 4, wherein the silencing of kenaf 1-deoxyxylulose-5-phosphate synthase gene is performedHcCLA1The use of (1); the kenaf 1-deoxyxylulose-5-phosphate synthase geneHcCLA1The nucleotide sequence of (A) is shown in SEQ ID NO. 1.
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