Cloning,Sequence Analysis and Prokaryotic Expression of the Coat Protein Gene of Tobacco Mosaic Virus from Pumpkin Plant

doi:10.7668/hbnxb.2010.05.001

Abstract

Abstract: Tobacco mosaic virus(TMV) is responsible for considerable economic losses in the growing of plant. There is no report that TMV infects pumpkin plant naturally. TMV was detected from one pumpkin(Cucurbita moschata) plant in Liaocheng,Shandong Province. The nucleotide sequences of coat protein (CP) gene of TMV isolate (TMV-liaocheng) was determined from RT-PCR derived cDNA clones.It was the first report that TMV naturally infected pumpkin. The obtained CP gene sequences were compared with sequences of TMV that are available in the GenBank. Results showed that the CP gene of TMV-liaocheng shared similarity of 86. 5%-9. 0% with those of other TMV isolates at nucleotide acid level,and identity of 93. 7%-99. 4% at amino acid level. The phylogenetic tree constructed with the complete nucleotide sequence of the CP genes showed that 32 TMV isolates were divided into 5 groups,and TMV-liaocheng,Nakron,Pathom,Fujian and 017 isolates belonged to group Ⅳ. There were probably recombination event during the evolution of TMV-liaocheng. It was the first report that TMV infected pumpkin. The CP gene of TMV-liaocheng was inserted to expression vector pET22b(+ ) and transferred into E. coli BL21(DE3) pLysS. SDS-PAGE showed that TMV-liaocheng CP gene was expressed as a 20 kDa fusion protein when induced with IPTG. The expressed fusion protein was purified through Ni2 +-NTA His. Bind column,and it could be used to immunize the mice for preparing the specific the antibody.

Key words: Pumpkin, Tobacco mosaic virus, CP gene, Sequence analysis, Prokaryotic expression

CLC Number:

LIU Jinliang, WANG Fengting, WEI Yi, PAN Hongyu, ZHANG Shihong. Cloning,Sequence Analysis and Prokaryotic Expression of the Coat Protein Gene of Tobacco Mosaic Virus from Pumpkin Plant[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2010, 25(5): 6-10. doi: 10.7668/hbnxb.2010.05.001.

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