Plant virus satellite DNA beta molecule, infectivity clone and method for preparing expression carrier
A DNA molecule and plant virus technology, applied in the field of genetic engineering, can solve the problems of low exogenous gene expression, laborious and time-consuming plant regeneration process, and affect the industrialization process, etc., to achieve fast proliferation, small genome, and low equipment requirements Effect
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Embodiment 2
[0028] The full-length DNA β of Chinese Saikui Yellow Vein Virus is 1348bp (GenBank accession number AJ421622), presumably encoding 8 ORFs in total, and contains a 115nt length between nucleotides 1249 and 15. All DNA β molecules are highly homologous and conserved Region (conserved region, CR) (SEQ ID NO.6), CR region contains the nonanucleotide sequence TAATATTAC shared with Saikui yellow vein virus DNA-A, it is speculated that this region is DNA-A coded replication protein recognition and interaction as a site. Saikui Huangvei virus DNAβ contains an A-rich region (SEQ ID NO.7) between nucleotides 767 and 971, and the nucleotide content is as high as 60.5% A. It is believed that the A-rich region is involved in the packaging process of DNAβ molecules associated with the size requirements. A 118aa-long C1 protein (SEQ ID NO.8) is encoded on the complementary strand of Saikui Yellowvein Virus DNAβ. After mutating the start codon ATG of C1ORF, the pathogenicity of the virus de...
Embodiment 3
[0031] The results showed that the complete DNAβ sequence of Saikui Yellow Vein Virus had the highest homology with Multan cotton leaf curl virus CLCuMVβ01 and CLCuMVβ02, which were 66.7% and 67%, respectively; followed by Rajasthan cotton leaf curl virus CLCuRVβ homology, which was 61.8%; The homology with the okra yellow vein mosaic virus BYVMVβ is 46%; the homology with the red thistle yellow vein virus AYVVβ, the Chinese tomato yellow leaf curl virus TYLCCNVY10β and the tobacco curly stem virus TCSVY2β are only 40.6% and 40.4%, respectively. % and 40.2%. It shows that the homology between DNAβ is very low. Example 3 Comparison of the nucleotide homology of the CR region of Saikui Huangvei virus DNAβ and other reported DNAβ molecules
[0032] With the race Kui yellow vein virus DNA β of the present invention and GenBank reported Sheng red thistle yellow vein virus (Ageratum yellow vein virus, AYVV β, AJ252072), okra yellow vein mosaic virus (Bhendi yellow vein mosaic virus...
Embodiment 4
[0036] Using the total plant DNA as a template, with SEQ ID NO.9 (5'-GGTCACTACGCTACGCAGCAGCC-3', without KpnI site) and SEQ ID NO.3 (5'- GGTACC TACCCTCCCAGGGGTACAC-3', containing the KpnI site) was amplified, and the PCR product was inserted into pGEM-T Easy Vector to obtain pGEM-T-β(2), which cloned another full-length genome copy. Digest pGEM-T-β(1) with KpnI to obtain a 1.3kb full-length genome fragment and insert it into the corresponding site of pGEM-T-β(2) to obtain pGEM-T-2β, and then use EcoRI at both ends of pGEM-TEasy Vector Two copies of DNAβ were excised at the site, inserted into the EcoRI site of the Agrobacterium vector pBINplus, and transformed into competent cells DH5α to obtain the DNAβ-infectious clone pBINplus-2β. Example 5 Construction of Saikui Yellow Vein Virus DNA-A Infectious Clones
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