Plant DNA virus satellite silent carrier and method for constructing and using same

[0026] Example 1 Construction of TbCSV DNA1 virus satellite silencing vector with a single multiple cloning site

[0027] Total DNA was extracted from TbCSV-infected tobacco plants [Murray MG et al., 1987, EMBO J, 6:3901-3907], since invasive cloning of geminiviruses requires a full-length genome with 1.3-2.0 positive repeats ( contains the common region sequence of the two geminiviruses). Using TbCSV tobacco plant total DNA as a template, specific primers designed according to the gene sequence of SEQID NO. -3' introduction of KpnI restriction site) PCR amplification of the total DNA of TbCSV tobacco plants, while designing another pair of specific primers according to the gene sequence of SEQ ID NO.1, DNA1S (5'-GTCGACGAGTATAAATACGTTAATTTTGC-3' to introduce SalI enzyme cleavage site) and DNA1X (5'-TCTAGAGTATTTAGTCCAAATACTCGTCGC-3'introduction of XbaI restriction site) as specific primers for PCR amplification of TbCSV tobacco plant total DNA PCR reaction system: 50μl reaction s

[0028] Example 2 Cloning of plant transgenic or endogenous gene cDNA fragments

[0029] Extraction of 16C plants (transgenic Nicotiana benthamiana, literature see Voinnet and Baulcombe, 1997, Nature, 389:553), N. benthamiana, tomato and petunia total RNA (Johansen, LK, and Carrington, JC, 2001, Plant Physiol , 126: 930-938), using mRNA in total RNA as a template and specific primers for each gene to perform two-step RT-PCR amplification of target gene cDNA fragments. The specific method is as follows: using ReverseTranscription System kit (Promega Company) to synthesize the cDNA of the first strand. RNA 5μl in DEPC-treated Eppendorf tube, Olige dT primer 1μl, Rnase free H 2 O 5μl, water bath at 70°C for 10min; add 5×cDNA buffer 4μl, 0.1mol / L DTT 2μl, 10mM dNTP 1μl, Rnase inhibitor 1μl, water bath at 42°C for 2min, add 1μl M-MLV at 42°C for 50min, then inactivate at 70°C for 15min Live reverse transcriptase, which synthesizes the first strand of cDNA for the synthetic transgen

[0030] Example 3 Construction of DNA1 Recombinant Virus Satellite Silencing Vector Containing Plant Transgene and Plant Target Gene Fragment

[0031] 170bp GFP cDNA fragment GFP170 (SEQ ID NO.2) amplified from 16C plant mRNA and verified by sequencing, 351bp N. benthamiana Su cDNA amplified from N. benthamiana and tomato mRNA and verified by sequencing Fragment NbSu351 (SEQ ID NO.3) and tomato Su cDNA fragment ToSu351 (SEQ ID NO.4), amplified from N. benthamiana mRNA and verified by sequencing 350bp PCNA cDNA fragment PCNA350 (SEQ ID NO.5) and The 351bp CHS cDNA fragment CHS351 (SEQ ID NO. 6) amplified from the petunia mRNA and verified by sequencing was inserted into the viral satellite silencing vector pBINPLUS-2mDNA1 after double digestion with XbaI and BamHI, and constructed to induce 16C plants , recombinant viral satellite silencing vectors of pBINPLUS-2mDNA1-GFP170, pBINPLUS-2mDNA1-NbSu351, pBINPLUS-2mDNA1-ToSu351, pBINPLUS-2mDNA1-PCNA350 and pBINPLUS-2mDNA1-CHS351 gene si