Cloning And Function Identification Of Synthetase Gene Of Glycosphingolipid Of Bt Receptor In Diamondback Month, Plutella Xylostella

The diamondback month (DBM), Plutella xylostella, is one of the most destructive pests of cruciferous vegetables. It causes enormous losses to vegetables every year. Moreover, it is also one of the pests which can develop most serious resistance to insecticides, and has developed some degree of resistance to 60 kinds of insecticides. Bacillus thuringiensis (Bt) has been widely used against DBM as a biological insecticide, and DBM has found to be the first insect to develop resistance to Bt in the open field.In this research, techniques of bioassay, biological observation and molecular clonig were used to study DBM resistance mechanism to Bt toxin. We selected the resistance strains, analyzed fitness cost, cloned synthetase gene sequences of GSLs, and compared relative expression of synthetase gene of GSLs by real-time qPCR. The results will provide fundermental informations for further understanding of Bt resistance mechanism of P. xylostella. The main results and conclusion were as follows:1. Based on continuously selecting DBM by Cry1Ac and Btk toxins, respectively, CrylAc resistance (DBM1Ac-R) and Btk resistance strains (DBM.Bt-R) were obtained. The resistance leve of DBM1Ac-R and DBM.Bt-R strains were 797.8 and 3.9-fold compared with DBM1Ac-S strain treating by CrylAc.2. The biological characteristics of DBM1Ac-S, DBMlAc-R and DBM.Bt-R strains were observed and compared by constructing life tables. The results showed that the intrinsic rates of increase(r), gross reproductive rates(GRR), net reproductive rates(R0), mean generation time(T) and the relative fitness(Rf) of DBMlAc-R and DBM.Bt-R strains were lower than those of DBM1Ac-S strain. On the other hand, the fecundity, female/male ratio, daily fecundity, oviposition time, survival rate of female and life expectancy showed a decreasing trend both in DBM1Ac-R and DBM.Bt-R strains when compared with DBM1Ac-S strain.3. Using RT-PCR, the full-length of bre-3, bre-4 and bre-5 from DBM 1 Ac-S and DBMlAc-R strains were cloned. The open reading frame (ORF) length of bre-3, bre-4 and bre-5 are 1383bp,1230bp and 1041bp, and encoding 460,409 and 346 amino acids, respectively. There were 3,4 and 7 amino acids differences sites in bre-3, bre-4. and bre-5 between DBM1Ac-S and DBM1Ac-R respectively. All of the differences sites are not in the catalytic conservative domains of each gene family. 4. The relative expression levels of synthetase gene of GSLs bre-3 and bre-5 of DBMlAc-S and DBM1Ac-R strains were determined using real-time qPCR, from 2nd-instar to mature larvae, pupa and midgut of 4th-instar larvae. The results showed that both bre-3 and bre-5 mRNA were found to express in different development stages and midgut; The relative expression of bre-3 mRNA in DBM1Ac-S strain was significant higher than DBM1Ac-R, exhibiting 2.36,5.54 and 2.68 times in 3th-instar, 4th-instar and mature larvae respectively; The relative expression of bre-5 mRNA in DBM1Ac-S strain were 1.19,3.13,1.78,1.75 and 1.65 times respectively compared with DBMlAc-R from 2th-instar to mature larvae. The results suggested that there may be association between the lower relative expression of bre-3 and bre-5 mRNA in DBM1Ac-R strain from 3th to mature larvae and the long-term CrylAc toxin selection to DBM1Ac-R. In the same stage of DBM1Ac-S and DBM1Ac-R strains, the mRNA level expression of bre-5 was higher than bre-3.