Dissertation > Agricultural Sciences > Plant Protection > Pest and Disease Control > Horticultural Crops Pest and Disease Control > Fruit tree pests and diseases > Pome pests and diseases > Pear pests and diseases

Cloning and Construction of Prokaryotic Expression Vector of Coat Protein Gene of Apple Stem Pitting Virus in Korla Pear and Its Expression in Escherichia Coli

Author LiWenHui
Tutor NiuJianXin
School Shihezi University
Course Pomology
Keywords Apple stem pitting virus Xinjiang Korla Pear Coat protein Clone The prokaryotic expression
CLC S436.612
Type Master's thesis
Year 2010
Downloads 23
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China's commercial orchard fruit tree cultivars carrying the virus is widespread. Fruit tree acreage expanding cultivation time, the virus becoming more damaging, so researchers, the fruit production operators to increase the emphasis on virus. Apple stem pitting virus (apple stem pitting virus, ASPV) Foveaviris (Martelli GP, 1998), on behalf of species, a wide host range and distribution of the virus can infect a variety of fruit trees. Pear stem pitting disease early, pears yellow disease, the pear red mottled disease, Pear vein yellow disease, pear stone pox disease, Apple stem pitting disease pathogens classified as different virus species (Ya-Qin Wu et al, 2000), but Jelkmann confirmed the above several diseases caused by ASPV. ASPV differences in their different host and geographical distribution, has been isolated from multiple isolates. Korla Pear, Apple stem pitting virus coat protein isolates are cloning, sequencing, sequence analysis, and induction of the isolates prokaryotic expression of the functional areas of Apple stem pitting virus. 1. Acquisition by RT-PCR analysis to determine the biennial branches of the pear with Apple stem pitting virus (Apple stem pitting virus, ASPV) as test material, amplified ORF5 coat protein design and synthesis of primers the EU095327 sequences in the GenBank The nucleic acid sequence fragment. Target fragment into the cloning vector, the resulting fragment was confirmed by restriction enzyme digestion, sequencing purposes fragment nucleotides homologous sequences with MHczAp, ASPV isolate24, ASPV isolate38 isolates were 81% -83% amino acid homology rates were 70% -71%. Digested connection purpose cloning vector fragment, purification of the objective fragment, transferred to the expression vector pET-3a. Positive clones were sequenced, and confirmed the correct frame of the expression of the gene sequence. The connection destination gene fragment expression vector is transferred to E. coli BL21, after IPTG induction were obtained at different times in the expression, and the expression of the protein product size and the expected value of the molecular weight, and can be specifically recognized by the antibody. In addition, the gene expression of the BL21 timeliness characteristics ,1-3h, higher expression levels of recombinant protein expression in the hours after began to decline. Forecast ASPV CP protein relative molecular mass of 43.7kDa, the relative molecular mass is consistent with the experimental detection of recombinant proteins. This article Cloning and prokaryotic expression technology used in the study of Korla Pear Apple stem pitting virus, Apple stem pitting virus vector construction and expression, Apple stem pitting virus isolates in Xinjiang, to be compared with isolates at home and abroad, provide the basis for future preparation of antiserum

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