Molecular Cloning And Bacterial Expression Of Heat Shock Proteins And Their Relationship With Stress Tolerance In Bemisia Tabaci(B Biotype)

Bemisia tabaci (Gennadius)(Homoptera:Aleyrodidae)(B biotype) is one of the most devastating agricultural pests in the world.It has caused severe damage to vegetables,ornamental crops.and other economic crops since its invasion to China.ln the past decades, Some researches have indicated its successful invasion is closely related to its competition with the local insects and then substituting for them. The development of B. tabaci (B biotype) resistance to insecticide also played the important roles in its invasion. In this paper, Hsps, which is closely associated with B. tabaci (B biotype) tolerance to stress, was studied to understand the difference in the stress-tolerance between different B. tabaci biotypes, between B biotype and its close sib-whiteflies in the greenhouse, and between the resistant type and the sensitive one. .Comparison of whiteflies undergoing various stress conditions gives an new insight in the relationship between the stress-tolerance and the successful invasion of B.tabaci.The ecological and evolutionary roles of Hsps were discussed.The main results are as follows:(1) A successful cDNA library from mRNA of heat-shocked B. Tabaci was constructed after packaging.The cDNAs molecule weight range from 400-5,500bp was identified with agar electrophoresis.The recombination rate were 98.9%,measured by plate with IPTG and x-Gal.Ten recombinat were picked out randomly and were self-cycled into plasmid.The plasimid were digested by two enzymes,the inserted fragments range from 400bp-2 , 500bp were identified with agar electrophoresis,also.(2) Eleven complete or partial cDNA sequences (GenBank Accession number are DQ093378> DQ093379, DQ093380, DQ093381, DQ093382, DQ093383, DQ093384, DQ093385, DQ093386 and DQ093387)were cloned by RT-PCR or screened from cDNA library. Two full-lenth cDNAs of the eleven display the classical amino acid(aa) stretches representing the Hap70 and Hsp90 signature.They potentially encode a 644aa and a 721aa proteins,with calculated molecular mass of 70 kDa and 83 kDa similar to the Drosophila homologous proteins. Both of them displayed a polyA tail at their 3'end, confirming that we had obtained the sequences in their integrity at this end.The start and stop codons were located respectively at positions 40 andl939 in the case of hsp70, and at positions 135 and 2295 in the case of hsp90. The displayed ORFs were thus 1904 and 2163 bp long, respectively.(3) Bacterial expression vectors of Hsp70 and Hsp90 were constructed and successfully expressed in E.coli.The proteins recombinated with HIS tag were purified by IDA resin.Polyclonal antibodies against Hsp70 and Hsp90 were prepared to identify the protein of stressed whiteflies.by Western blot. Within the three hours after stressed in 43℃ for 30min,Hsp70 were accumulated with times.No visible changes were showed in the case of Hsp90.(4) A perfect method has been developed to measure mRNA expression of Hsps genes within whiteflies through real-time RT-PCR.The mRNA of Hsps expressed after stressed were compared between B and China-ZHJ-1 biotype whiteflies. Analyzation of the results showed that B biotype was more tolerant than China-ZHJ-1 biotype when they underwent cold stress.and two whiteflies showed the simililar tolerance when undergoing heat stress. With respect to thermal tolerance.B biotype whitefly had...