A VIGS silencing vector and silencing system of Lycoris radiata LcCLA gene and its construction method and application

Abstract

The invention belongs to the field of biotechnology, and specifically relates to a VIGS silencing vector and silencing system of a Chinese Lycoris radiata LcCLA gene, and a construction method and application thereof. The invention constructs a VIGS silencing vector and a silencing system of a Chinese Lycoris radiata LcCLA gene, introduces the silencing system into Chinese Lycoris radiata leaves by needle injection, induces silencing of endogenous LcCLA of Chinese Lycoris radiata, effectively reduces the expression level of the Chinese Lycoris radiata LcCLA gene, obtains a virus-induced gene silencing plant, can verify the function of the Chinese Lycoris radiata LcCLA gene, and has application value in the functional gene research of Chinese Lycoris radiata.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a Lycoris radiata LcCLA gene VIGS silencing vector, a silencing system, and a construction method and application thereof. Background Art

[0002] Chinese Lycoris (Lycoris chinensis Traub) is an important ornamental plant of the genus Lycoris. It has ornamental characteristics such as bright colors, beautiful flower shapes, and tall and upright flower stems. It is not demanding on the environment and has strong adaptability. It is a good choice for landscaping. Molecular breeding is the trend and common method of flower breeding research in the future. It can create excellent conditions for improving ornamental traits, improving flowering period, and cultivating new germplasm. However, the genetic transformation system of Lycoris plants is not yet mature, making it difficult to study gene functions.

[0003] Virus-induced gene silencing (VIGS) is a technique based on post-transcriptional gene silencing (PTGS) that exploits the natural defense mechanisms employed by plants to prevent invading viruses. Virus-infected plants induce double-stranded RNA-mediated PTGS, which degrades viral RNA. Recombinant viruses carrying partial sequences of host target genes are used to infect and spread throughout the plant. Viral gene transcripts are recognized and degraded by endogenous PTGS along with plant target genes, resulting in reduced expression of the target gene. Compared with other transgenic technologies, VIGS technology can avoid plant transformation and has the advantages of shorter cycles, low costs, and simple operations. It has been applied to a variety of plants for gene function verification, but this system has not yet been established and applied in Lycoris radiata in China. Summary of the invention

[0004] The purpose of the present invention is to provide a VIGS silencing vector and a silencing system of the LcCLA gene of Lycoris radiata, and a construction method and application thereof, so as to verify the gene function of Lycoris radiata.

[0005] The invention provides a VIGS silencing vector of Lycoris radiata LcCLA gene. The silencing vector comprises a specific fragment of the Lycoris radiata LcCLA gene. The nucleotide sequence of the specific fragment is shown as SEQ ID NO.1.

[0006] The present invention provides a method for constructing the Chinese Lycoris radiata LcCLA gene VIGS silencing vector, comprising the following steps:

[0007] The specific fragment was connected to pTRV2 to construct the silencing vector pTRV2-LcCLA.

[0008] Preferably, the specific fragment is obtained by PCR amplification; the primer pair used for PCR amplification includes an upstream primer CLA1-XbaI-F and a downstream primer CLA1-KpnI-R; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.5.

[0009] The present invention also provides a VIGS silencing system of LcCLA gene of Chinese Lycoris radiata, which comprises: a mixed solution of Agrobacterium bacterial solution containing pTRV1 and Agrobacterium bacterial solution containing pTRV2-LcCLA; the volume ratio of the Agrobacterium bacterial solution containing pTRV1 and the Agrobacterium bacterial solution containing pTRV2-LcCLA is 1:1.

[0010] The present invention also provides application of the silencing vector or the silencing system in identifying the function of the LcCLA gene of Lycoris radiata.

[0011] The present invention also provides a method for identifying the function of the LcCLA gene of Lycoris radiata, comprising the following steps:

[0012] The leaves of Lycoris radiata were infected with the silencing system and then cultured to observe the phenotypic changes of the leaves.

[0013] Preferably, the infection method includes: injecting the silencing system into the leaf vein duct at the tip of the leaf by needle injection.

[0014] Preferably, before using the silencing system to infect the leaves of Lycoris radiata, the method further includes dark culturing the silencing system, and using the dark-cultured silencing system to infect the leaves of Lycoris radiata; the temperature of the dark culturing is 24-26° C., and the time is 3-5 hours.

[0015] Preferably, after the infection, the method of culturing Lycoris radiata includes dark culturing at 17-19° C. for 2 days and then normal culturing.

[0016] Preferably, the Agrobacterium comprises Agrobacterium GV3101.

[0017] The present invention provides a VIGS silencing vector for the LcCLA gene of Lycoris radiata, wherein the silencing vector comprises a specific fragment of the LcCLA gene of Lycoris radiata, and the nucleotide sequence of the specific fragment is shown in SEQ ID NO. 1. The present invention obtains the specific fragment of the LcCLA gene shown in SEQ ID NO. 1 by sequencing the transcriptome of Lycoris radiata bulb, and constructs a VIGS silencing vector for the LcCLA gene of Lycoris radiata for the first time, which can effectively reduce the expression level of the LcCLA gene in Lycoris radiata.

[0018] The invention transforms a pTRV2 virus silencing expression vector plasmid containing a specific fragment of an LcCLA gene into an Agrobacterium, mixes an Agrobacterium bacterial solution containing pTRV1 and an Agrobacterium bacterial solution containing pTRV2-LcCLA to obtain a silencing system (i.e., an Agrobacterium bacterial solution mixture), and infects Chinese Lycoris leaves with the silencing system in a needle injection manner to induce silencing of endogenous LcCLA in Chinese Lycoris, effectively reduce the expression level of the Chinese Lycoris LcCLA gene, obtain the virus-induced gene silencing trait, can verify the function of the Chinese Lycoris LcCLA gene, and has application value in the research of functional genes in Chinese Lycoris. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below.

[0020] Figure 1 The method is the leaf tip needle injection method of Example 1 and the leaf penetration method of Comparative Example 1;

[0021] Figure 2 The leaf albino phenotypic trait that appears after the silencing vector infects the Chinese Lycoris radiata leaves in Example 1;

[0022] Figure 3 This is the cDNA fragment detection diagram after LcCLA gene silencing in Lycoris radiata;

[0023] Figure 4 Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of LcCLA in Lycoris radiata. DETAILED DESCRIPTION

[0024] The invention provides a VIGS silencing vector of Lycoris radiata LcCLA gene. The silencing vector comprises a specific fragment of the Lycoris radiata LcCLA gene. The nucleotide sequence of the specific fragment is shown in SEQ ID NO.1, specifically: 5'--3'.

[0025] The present invention obtains the LcCLA gene specific fragment (i.e., CDS sequence) by sequencing the Chinese Lycoris radiata bulb transcriptome, and its specific nucleotide sequence is shown in SEQ ID NO. 1. The LcCLA gene specific fragment is used as the target fragment to construct the LcCLA gene VIGS silencing vector, which can verify the function of the Chinese Lycoris radiata LcCLA gene.

[0026] The present invention also provides a method for constructing the VIGS silencing system of the Chinese Lycoris radiata LcCLA gene, comprising the following steps:

[0027] The specific fragment was connected to pTRV2 to construct the silencing vector pTRV2-LcCLA.

[0028] In the present invention, the specific fragment is preferably obtained by PCR amplification, and the primer pair used for the PCR amplification preferably includes an upstream primer CLA1-XbaI-F and a downstream primer CLA1-KpnI-R. The nucleotide sequence of the upstream primer CLA1-XbaI-F of the present invention is preferably as shown in SEQ ID NO.4 (5'-CTAGTCTAGATTGACGGGCAGGAGGGAC-3'), and the nucleotide sequence of the downstream primer is preferably as shown in SEQ ID NO.5 (5'-CGGGGTACCGCGGAGGAAGCAGTTTAGGC-3'). The present invention does not specifically limit the source of the template used in the PCR amplification. The specific fragment can be obtained by synthesis, or the cDNA obtained by reverse transcription of the RNA of Chinese Lycoris radiata can be used as a template. In the embodiments of the present invention, cDNA is preferably used as a template, but it cannot be identified as the entire protection scope of the present invention.

[0029] The present invention has no strict requirements on the connection method, and adopts conventional gene recombination technology, for example, adding an XbaI restriction site to the 5' end of the upstream primer CLA1-XbaI-F of the primer pair, and adding a KpnI restriction site to the 5' end of the downstream primer CLA1-KpnI-R of the primer pair, and inserting the specific fragment obtained by PCR amplification into pTRV2 by enzyme ligation. The present invention has no strict requirements on the source of the vector pTRV2, and it can be purchased conventionally. The vector pTRV2 used in the specific implementation process of the present invention was purchased from https: / / www.addgene.org / 148969 / .

[0030] The present invention also provides a Chinese Lycoris LcCLA gene VIGS silencing system, comprising: a mixed solution of an Agrobacterium bacterial solution containing pTRV1 and an Agrobacterium bacterial solution containing pTRV2-LcCLA; the volume ratio of the Agrobacterium bacterial solution containing pTRV1 to the Agrobacterium bacterial solution containing pTRV2-LcCLA is 1:1.

[0031] The present invention mixes an Agrobacterium solution containing pTRV1 and an Agrobacterium solution containing pTRV2-LcCLA to obtain an infection solution, which is the silencing system. The present invention can improve the infection effect by limiting the volume ratio of the Agrobacterium solution containing pTRV1 and the Agrobacterium solution containing pTRV2-LcCLA to 1:1.

[0032] In the present invention, the preparation method of the Agrobacterium liquid containing pTRV1 and the Agrobacterium liquid containing pTRV2-LcCLA is not strictly required, and pTRV1 and pTRV2-LcCLA can be respectively transferred into Agrobacterium by conventional methods, such as electroporation or heat shock method, and then cultured. The Agrobacterium of the present invention is preferably Agrobacterium GV3101.

[0033] The present invention also provides the use of the silencing vector or silencing system in identifying the function of the LcCLA gene in Lycoris radiata. The silencing vector or silencing system of the present invention can effectively reduce the expression level of the LcCLA gene in Lycoris radiata, obtain virus-induced gene silencing plants, and verify the function of the LcCLA gene in Lycoris radiata.

[0034] The present invention also provides a method for identifying the function of the LcCLA gene of Lycoris radiata, comprising the following steps:

[0035] The leaves of Lycoris radiata were infected with the silencing system and then cultured to observe the phenotypic changes of the leaves.

[0036] Before the silencing system is used to infect the leaves of Lycoris radiata, the silencing system is preferably dark-cultured, and the leaves of Lycoris radiata are infected by the dark-cultured silencing system. The temperature of the dark culture in the present invention is preferably 25°C; the time of the dark culture is preferably 3 to 5 hours, and more preferably 4 hours. The present invention does not specifically limit the dark culture method, and a conventional culture method can be used, such as culture on a shaker at 100 rpm. The dark culture of the silencing system in the present invention can induce the infection ability of Agrobacterium and improve the infection efficiency of the silencing system.

[0037] After the dark culture is completed, the present invention uses the silencing system after dark culture to infect the leaves of Chinese Lycoris radiata. The infection method of the present invention is preferably needle injection, specifically, the infection solution after dark culture is injected into the leaf tip vein duct by needle injection, and the needle part is pinched with fingers to slowly push the bacterial solution until the entire leaf is infiltrated. The injection volume of the silencing system after dark culture of the present invention is preferably 1-2mL; the infection time of the silencing system after dark culture is preferably 15-20s, and more preferably 16-18s. The present invention adopts a needle direct injection method for the special leaf structure of Lycoris radiata plants, and directly infiltrates the bacterial solution along the parallel veins to completely infiltrate the entire leaf, effectively avoiding the problem that the leaf surface infiltration method is difficult to penetrate the wax on the leaf surface. In a relatively short period of time, infection can be achieved using a small amount of silencing system, and it is used for subsequent verification of the LcCLA gene function of Chinese Lycoris radiata.

[0038] After the infection is completed, the present invention cultivates the infected leaves of Lycoris radiata. The cultivation of the present invention preferably includes first culturing at 17-19°C, more preferably 18°C ​​in the dark for 2 days and then normal cultivation. The normal cultivation of the present invention is specifically: alternating light and dark cultivation, specifically, culturing for 16 hours in light and then 8 hours in dark; the light intensity of the light cultivation is 4000lx, the temperature of the light cultivation is 24-26°C; the temperature of the dark cultivation is 21-23°C. During the normal cultivation of the present invention, the relative humidity of the air is maintained at 70%-80%.

[0039] After normal cultivation for 8 to 13 days (i.e., infection for 10 to 15 days), the present invention observes the phenotypic changes of the leaves and / or samples and measures the expression of the LcCLA gene in the leaves to achieve the identification of the function of the Chinese Lycoris LcCLA gene. The specific identification method is: when the leaves lose their green color, turn white or light yellow, it indicates that the Chinese Lycoris LcCLA gene functions.

[0040] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] The molecular biology experimental techniques used in the following examples include RNA extraction and reverse transcription, PCR amplification and other experiments, and are usually performed according to conventional methods unless otherwise specified.

[0042] Example 1

[0043] (1) Cloning of the specific fragment of LcCLA gene

[0044] According to the transcriptome of Lycoris radiata, sequence assembly, annotation and alignment analysis were performed, and it was found that LcCLA had a complete ORF (full length 1980 bp). Total RNA from Lycoris radiata leaves was extracted using an RNA extraction kit (purchased from Beijing Huayueyang Biotechnology Co., Ltd., item number 0416-50gk), and reverse transcribed using oligo (dT) 18 (purchased from Thermo Fisher, item number SO131). Full-length primers CLA1-F (recorded as SEQ ID NO. 2): 5'-TTGACGGGCAGGAGGGAC-3' and CLA1-R (recorded as SEQ ID NO. 3): 5'-GCGGAGGAAGCAGTTTAGGC-3' were designed according to the transcriptome of Lycoris radiata. PCR amplification was performed to obtain LcCLA specific bands, and agarose gel electrophoresis, gel excision purification, cloning and sequencing were performed to obtain the final sequence.

[0045] (2) Vector construction

[0046] According to the final sequence of step (1), primers CLA1-XbaI-F and CLA1-KpnI-R were designed for amplifying the specific fragment on the CDS domain of the LcCLA gene. An XbaI restriction site was added to the 5' end of the upstream primer, and a KpnI restriction site was added to the 5' end of the downstream primer to PCR amplify the specific fragment of the LcCLA gene.

[0047] Total RNA from Lycoris radiata leaves was extracted using an RNA extraction kit (purchased from Beijing Huayueyang Biotechnology Co., Ltd., catalog number 0416-50gk), and reverse transcribed using oligo(dT)18 (purchased from Thermo Fisher, catalog number SO131) to obtain a cDNA template.

[0048] The PCR reaction system (20 μL) was as follows: 1 μL of cDNA template, 1 μL of upstream and downstream primers (10 μM), 0.4 μL of dNTPs (10 mM), 4 μL of 5×Phusion HF buffer, 0.2 μL of Phusion DNA polymerase, and double distilled water was added to 20 μL. The amplification conditions were as follows: 98°C pre-denaturation for 30 s; 98°C denaturation for 5 s, 60°C annealing for 20 s, 72°C extension for 10 s, 35 cycles; 72°C final extension for 7 min, and a total of 852 bp of LcCLA specific sequence (SEQ ID NO.1) was obtained, which was stored at 4°C.

[0049] After PCR gel excision and purification, the A tail was added, and the T vector (purchased from TaKaRa, item number D101A) was connected and transformed into E. coli. The positive clones were picked and shaken and sequenced at General Bio. The clones whose sequencing results were consistent with the known sequence were selected, shaken and the plasmid was extracted, named pMD19-LcCLA. Then, the pMD19-LcCLA and pTRV2 vector plasmids were double-digested with XbaI / KpnI, and the enzyme digestion products were recovered respectively, and the positive clones were connected, transformed and screened; thus, the VIGS recombinant vector pTRV2-LcCLA was obtained.

[0050] (3) Preparation of infection solution and infection method

[0051] ① Preparation of infection buffer: 100 mL of infiltration solution contains 1 mL of MgCl2 solution (1 mol / L), 1 mL of MES solution (1 mol / L), and 200 μL of acetosyringone solution (1 mol / L). Adjust the pH to 5.6 and make up to volume with sterile distilled water. It needs to be prepared and used immediately.

[0052] ② Preparation of bacterial solution: The obtained recombinant vector pTRV2-LcCLA plasmid, pTRV2 empty vector plasmid and pTRV1 (purchased from https: / / www.addgene.org / 148968 / ) plasmid were transformed into Agrobacterium GV3101 by freeze-thaw method. The pTRV1 plasmid, pTRV2 plasmid and pTRV2-LcCLA plasmid transformed with Agrobacterium GV3101 were streaked and cultured on LB (Kan, 50μg / mL; Rif, 25μg / mL) agar plates. After culturing at 28℃ for 48h, the freshly cultured Agrobacterium GV3101 transformant single colony containing pTRV1 plasmid, pTRV2 plasmid and pTRV2-LcCLA plasmid was picked and added to 3mL YEB liquid medium (Kan, 50μg / mL; Rif, 25μg / mL), and cultured in a shaker at 28℃ and 170rpm / min for 16h. Take 1 mL of cultured bacterial solution and add it to 100 mL of YEB liquid medium (Kan, 50 μg / mL; Rif, 25 μg / mL), culture at 28°C, 170 rpm / min for 20-24 h. Detect the OD value of the bacterial solution. 600 When the OD is about 1.0, take 40 mL and centrifuge at 4000 rpm for 10 min to collect the bacterial cells. Add an appropriate volume of infection buffer and resuspend to a final concentration of 2.0 (OD 600 ).

[0053] ③ Preparation of infection solution: Mix the Agrobacterium GV3101 bacterial solution carrying the pTRV1 vector in step ② with the bacterial solution carrying pTRV2 and the bacterial solution carrying the pTRV2-LcCLA plasmid at a volume ratio of 1:1 to prepare two mixed bacterial solutions. Place the mixed resuspension in a 25°C shaker at 100 rpm / min, incubate in the dark for 4 hours, and use it to infect Lycoris radiata leaves.

[0054] ④ Infection with Agrobacterium tumefaciens: Figure 1 As shown in (a), the leaf needle injection method is used to infect the leaves of Lycoris radiata. The steps are as follows: first absorb the infection solution cultured in step ③, insert the needle of the syringe into the leaf tip of the leaf vein duct, and pinch the needle with your fingers to slowly push the bacterial solution in until the entire leaf is infiltrated. (Gloves and syringes need to be changed when injecting two different bacterial solutions).

[0055] Culture after injection: Culture in the dark at 18°C ​​for 2 days after infection, and then culture in alternating light and dark conditions at a relative humidity of 70%-80%. The intensity of light culture is 4000lx and the temperature is 25°C; the temperature of dark culture is 22°C, and the light-dark ratio is 16h / 8h.

[0056] Comparative Example 1

[0057] Same as Example 1, except that in step (3), when infecting with Agrobacterium solution, Figure 1 The leaf infiltration method shown in (b) was used for infiltration.

[0058] Test Example 1

[0059] Comparison of infection efficiency of Agrobacterium tumefaciens liquid

[0060] In Example 1, the needle injection method only requires 1-2 mL of bacterial solution to infiltrate a whole leaf, and it takes 15-20 seconds. In Comparative Example 1, the leaf infiltration method belongs to the traditional infiltration injection method. Because the surface of the leaves of Lycoris plants is waxy, it is difficult to inject the bacterial solution into the entire leaf during infiltration. In order to achieve the effect of infiltrating the entire leaf, multiple wounds need to be made. Therefore, the bacterial solution is easily lost during the infiltration process, and multiple wounds are not conducive to the normal growth and development of the plant, affecting the experimental results. The leaf infiltration method requires at least 5 mL of bacterial solution to infiltrate a whole leaf, and it takes 1-2 minutes.

[0061] The needle injection method of the present invention is easier to infiltrate the entire leaf, has higher efficiency, is easier to operate, and can save more bacterial liquid.

[0062] Test example 2.

[0063] Detection of silencing efficiency of LcCLA in Lycoris radiata after infection

[0064] (1) Example 1 After 10-15 days of infection, the phenotypic changes of the injected Lycoris radiata leaves were observed. The results are as follows Figure 2 As shown, Figure 2 From left to right (ac) are the leaves of Lycoris radiata infected with pTRV1+pTRV2-LcCLA in Example 1, the leaves of Lycoris radiata infected with pTRV1+pTRV2 in Example 1, and the leaves of Lycoris radiata that are not infected and grow normally. Figure 2 It can be seen that after being infected with pTRV1+pTRV2-LcCLA, the leaves of Lycoris radiata lost their green color and turned white, indicating that the needle injection method of the present invention can silence the LcCLA gene of Lycoris radiata and exert its function.

[0065] (2) When the leaf phenotype of Lycoris radiata was white, the leaves were taken and the total RNA of the leaves without injection, injected with pTRV1+pTRV2, and pTRV1+pTRV2-LcCLA was extracted using the Huayueyang Rapid Universal Plant RNA Extraction Kit, with three replicates per group. Then, 1 μg of total RNA from each sample was used as a template to remove genomic DNA and reverse transcribe the first-chain cDNA using the Prime Script TM RT reagent Kit with gDNAEraser (Perfect Real Time) Kit (Takara), which was then stored at -20°C for later use.

[0066] The injection efficiency was detected by PCR. Specifically, the primers used were: LcCLA1-RNAi-RT-F (denoted as SEQ ID NO.6): 5'-GTTGCTCATTTCTTGGCACTCA-3' and LcCLA1-RNAi-RT-R (denoted as SEQ ID NO.7): 5'-CAGCACCAACGGTCTCCACT-3'. The detection results are shown in Figure 3 , where 1-3 are uninjected leaf samples, 4-6 are pTRV1+pTRV2-injected leaf samples, and 7-9 are pTRV1+pTRV2-LcCLA-injected leaf samples. Figure 3 It can be seen that no band is seen in the 1-3 uninjected leaf holes, and a band is obvious at about 250 bp in the 4-9 injected leaf holes, indicating that the needle injection method of the present invention achieves infection of Lycoris radiata leaves and the injection is successful;

[0067] Gene expression was detected by qRT-PCR, with EXP as the internal reference gene, and primer sequences were EXP1-RT-F (denoted as SEQ ID NO.8): 5'-TTGATGTTGACAAGGTAAGGTGC-3' and EXP1-RT-R (denoted as SEQ ID NO.9): 5'-AGGCAGGAAATCTCCAAAGC-3'. The detection results are shown in Figure 4 , where CK is the control group, which is the leaves infected with the empty vector pTRV2, and the relative expression of LcCLA gene is 1.00; LcCLA is the leaves infected with the recombinant viral vector pTRV2-LcCLA in Example 1, and the relative expression of LcCLA gene is 0.37. Compared with the control CK, the expression of LcCLA gene in the leaves of Lycoris radiata infected with the recombinant viral vector Agrobacterium carrying pTRV2-LcCLA is significantly reduced, indicating that the silencing system of the present invention is successfully constructed.

[0068] The Chinese Lycoris radiata LcCLA gene VIGS silencing system provided by the present invention can induce silencing of endogenous LcCLA in Chinese Lycoris radiata, effectively reduce the expression level of the Chinese Lycoris radiata LcCLA gene, and obtain the virus-induced gene silencing trait.

[0069] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention. Sequence Listing <110> Institute of Botany, Chinese Academy of Sciences, Jiangsu Province <120> A VIGS silencing vector and silencing system of Lycoris radiata LcCLA gene and its construction method and application <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 852 <212> DNA <213> Artificial Sequence <400> 1 ttgacgggca ggagggacaa aatgcccaca atgagacaaa caaacggatt agctgggttt 60 accaaacgct ccgagagcga gcatgattgc ttcggtactg gccacagctc aaccagcatc 120 tcagcagcac taggaatggc agttggaaga gacttgaagg gaggaaagaa caatgtgatt 180 gcagtgatag gagatggagc catgacagca ggccaagctt atgagcaat gataatgca 240 gggtatttgg attctgatat gattgtata ttaatgata ataagcaggt ttctctgccg 300 actgcaaatc ttgatgggcc ataccacct gttggagctt taagcagtgc tcttagtaaa 360 ttgcagtcta gcaggcctct tagagaactc agggaggtgg aaagggagt aaaaaacag 420 cttggtgggg ggatgcacga attagcagca aaagttgatg atatgctcg aggaatgatc 480 agtggatcgg gttccacctt gttcaccgag cttggcttt actacatagg tccagttgat 540 ggccacaaca tgacgacct tgtcacaatt ctcaagaag tgagagcac aaaaacaact 600 ggtccagttc taatccatgt cataacagag aaaggaag gatatcccta tgcagaaaga 660 gctgctgata agtaccatgg tgtggtgaag tttgacccag caacgggaaa acagttcaaa 720 accagttctc agactcagtc ctataccaat tattttgctg aggctttgat agccgaagct 780 gaagtagaca aggacattgt tgcaattcat gctgcaatgg ggggaggac tggcctaac 840 tgcttcctcc gc 852 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 ttgacgggca ggagggac 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 gcggaggaag cagtttaggc 20 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <400> 4 ctagtctaga ttgacgggca ggagggac 28 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <400> 5 cggggtaccg cggaggaagc agtttaggc 29 <210> 6 <211> twenty two <212> DNA <213> Artificial Sequence <400> 6 gttgctcatt tcttggcact ca 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 cagcaccaac ggtctccact 20 <210> 8 <211> twenty three <212> DNA <213> Artificial Sequence <400> 8 ttgatgttga caaggtaagg tgc 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 aggcaggaaa tctccaaagc 20

Claims

1. A Chinese Lycoris (Lycoris chinensis Traub) LcC The gene VIGS silencing vector is characterized by: The silencing vector includes pTRV2 and Lycoris radiata LcC A specific fragment of a gene, the nucleotide sequence of the specific fragment is shown as SEQ ID NO.

1.

2. Chinese Lycoris radiata according to claim 1 LcC The method for constructing a gene VIGS silencing vector is characterized in that: The following steps are involved: The specific fragment of claim 1 was connected to pTRV2 to construct the silencing vector pTRV2- LcC .

3. The construction method according to claim 2, characterized in that: The specific fragment is obtained by PCR amplification; the primer pair used for PCR amplification includes an upstream primer CLA1-XbaI-F and a downstream primer CLA1-KpnI-R; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

5.

4. A Chinese Lycoris LcC The gene VIGS silencing system is characterized by: The silencing system includes: an Agrobacterium solution containing pTRV1 and an Agrobacterium solution containing pTRV2- LcC The Agrobacterium liquid containing pTRV1 and the Agrobacterium liquid containing pTRV2- LcC The volume ratio of Agrobacterium bacterial solution is 1:1; Chinese Lycoris LcC The CDS sequence of the gene is shown in SEQ ID NO.

1.

5. The silencing vector according to claim 1 or the silencing vector obtained by the construction method according to claims 2-3 or the silencing system according to claim 4 is used to identify Lycoris radiata LcC Applications in gene function; Chinese Lycoris LcC The CDS sequence of the gene is shown in SEQ ID NO.

1.

6. A method for identifying Lycoris radiata LcC A method for gene function, characterized in that The steps include: After infecting the leaves of Lycoris radiata with the silencing system described in claim 4, the leaves are continuously cultured and the phenotypic changes of the leaves are observed; when the leaves lose green color, turn white or light yellow, it indicates that the Lycoris radiata LcC Genes function.

7. The method according to claim 6, characterized in that The infection method includes: injecting the silencing system into the leaf vein duct at the tip of the leaf by needle injection.

8. The method according to claim 6, characterized in that Before using the silencing system to infect the leaves of Lycoris radiata, the method further comprises dark culturing the silencing system, and infecting the leaves of Lycoris radiata with the silencing system after dark culturing; the temperature of the dark culturing is 24-26° C., and the time is 3-5 hours.

9. The method according to claim 6, characterized in that After the infection, the method of culturing Chinese Lycoris radiata includes dark culturing at 17-19° C. for 2 days and then normal culturing.

10. The method according to claim 6, characterized in that The Agrobacterium includes Agrobacterium GV3101.

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