Expression Analysis And Functional Verification Of Three Cold Resistant Genes In Bemisia Tabaci(Hemiptera:Aleyrodidae)

Bemisia tabaci has strong temperature tolerance(heat resistance and cold resistance).However,there are significant differences in the distribution of invading species(MED cryptic species)and native species(Asia II 3 cryptic species)in China.Therefore,in this study,we used rapid-amplification of cDNA ends to clone the full length sequence of 3 cold resistant genes(Trichohyalin,Trehalase,Dopa decarboxylase),and qRT-PCR was used to detect the mRNA relative expression level of 3 important cold-resistance genes in the two B.tabaci cryptic species(MED and Asia II 3 cryptic species)under five gradient temperature stress,and RNAi technology was used to reduce gene expression level and verify gene function by observing its phenotypic changes.The purpose of the study is to explore the roles of cold-resistance genes underlying the successful invasion of B.tabaci MED cryptic species and widespread distribution in different climatic regions.The main findings and conclusions are as follows:1.The full length of Trehalase,Dopa decarboxylase gene was cloned from the two B.tabaci cryptic species,and the partial coding region of Trichohyalin gene was obtained.(1)The full length of Trehalase sequence in MED and Asia II 3 cryptic species is 3709 bp and 3026 bp,respectively,and the open reading frame is 1893 bp,encoding 630 amino acids,named as MEDBtTreh1 and Asia II 3BtTreh1.There is one different amino acid in the Trehalase amino acid sequence of the two B.tabaci cryptic species.(2)The full length of Dopa decarboxylase sequence in MED and Asia II 3cryptic species is 3655 bp and 3709 bp,respectively,and the open reading frame is1464 bp,encoding 487 amino acids,named as MEDBtDDC and AsiaII3 BtDDC,respectively.There are four different amino acid in the Dopa decarboxylase amino acid sequence of the two B.tabaci cryptic species.(3)The partial encoding region of Trichohyalin gene of MED cryptic species is 2037 bp,encoding 678 amino acids.2.The sequence analysis and phylogenetic analysis of Trehalase,Dopa decarboxylase,Trichohyalin gene were completed.The phylogenetic tree showed that the insect Trehalase and Dopa decarboxylase gene were relatively conservative inevolution,and Homologue insects could be clustered on the same evolutionary branch.The Trehalase and Dopa decarboxylas genes of the two B.tabaci cryptic species were highly similar.3.The expression pattern of Trehalase,Dopa decarboxylase,Trichohyalin gene has been detected under gradient temperature stress.Under short-term low and high temperature stress,general expression trend of Trehalase is falling,general expression trend of Dopa decarboxylase and Trichohyalin is on the rise.Both low temperature(0 ?,12 ?)and high temperature(35 ?,40 ?)could effectively induce gene regulation and did not cause death.The results showed that under high or low temperature stress,the three target genes in two B.tabaci cryptic species were overexpressed or low expressed,so it was suggested that the three genes could play a regulatory role under the stress of temperature adversity.4.The internal functional identification of Trehalase,Dopa decarboxylase and Trichohyalin gene were completed.Based on the results of internal functional verification by RNAi,it was found that the heat resistance and cold tolerance in the two B.tabaci cryptic species were significantly decreased after the reduction of the expression of the above three genes,but there was a certain difference between two B.tabaci cryptic species.The results showed that these three genes played a key role in the temperature tolerance of B.tabaci.Based on the above results,it is concluded that the three genes play a key role in the resistance to stress temperature in B.tabaci.Therefore,it is inferred that the abilitiy of cold resistance of B.tabaci can affect the establishment of population and the choice of settlement to some extent.It is expected to provide a new potential target for pest control and provide a theoretical basis for further study on the temperature adaptation mechanism of B.tabaci and the establishment of a sustainable control method for B.tabaci damage.