Expression And Function Analysis Of A Laccase-1Gene From Bemisia Tabaci Cryptic Species Middle East-Asia Minor1(Hemiptera:Aleyrodidae) And Trialeurodes Vaporariorum (Hemiptera:Aleyrodidae)

Bemisia tabaci (Gennadius) Middle East-Asia Minor1(MEAM1) cryptic species (Hemiptera, Aleyrodidae)(previously known as "B biotype") and Trialeurodes vaporariorum (Westwood)(Hemiptera, Aleyrodidae) are invasive alien pests. Morphologically similar, they often blended occur alternately in different seasons and had caused huge economic losses to agricultural production, and their alternative physiological and molecular mechanisms of competition in the field is not clear yet.Both the B. tabaci and T. vaporariorum are phloem-feeding insects. The plant defense responses induced by phloem-feeding insects often have a negative impact on subsequent feeding insects, including extending the developmental duration, reducing the number of progeny, increasing mortality. The various response to different whiteflies accelerated replacement between MEAM1and Trialeurodes vaporariorum, and saliva secreted by the whiteflies played an important role in inducting plant defense reaction. The saliva of piercing-sucking insects containsmany kinds of enzymes, these enzymes play an important role in the physiology of insects, which could help them to puncture plant tissue, digest plant juice, detoxificate and overcome the defensive response of the plants. Moreover, these enzymes are related to the transmission of plant viruses and induced plant pathological response.Polyphenol oxidase is the predominant enzyme in saliva of piercing-sucking insects. It can interfere with the normal oxidation-reduction reaction in host plants of insects and make food source more acceptable for itself. Insect laccase-1(Lac-1) belongs to the polyphenol oxidase family, and it has been verified that it is involved in the iron homeostasis, immune defense and detoxification. But the function of Lac-1in B. tabaci is not clear. In this study, molecular cloning, expression and function analysis of Lac-1gene from B. tabaci MEAM1and T. vaporariorum were examined.The main research contents of this paper are as follows:1. The enzyme activity of laccase in saliva of B. tabaci MEAM1and T. vaporariorum was analysized.B. tabaci MEAM1and T. vaporariorum were fed on tomato leaves for24h, in order to detect the enzyme activity, saliva of these two species of whitefly was extracted by artificial method. The result showed that the enzyme activity of laccase1in T. vaporariorum was higher than that in B. tabaci MEAM1.2. Cloning and sequence analysis of MEAM1and Trialeurodes vaporariorum Lac-1genes.The Lac-1of B. tabaci MEAM1and T. vaporariorum were cloned using bioinformatics methods, and named as BtLac-1and TvLac-1respectively. The nucleotide sequence length of BtLac-1fragment is1452bp, encoding483amino acids. The length of TvLac-1fragment is641bp, encoding213amino acids, and the identity of two nucleotide sequences is99.8%.3. The Lac-1gene expression of B. tabaci MEAM1and T. vaporariorum in different ages and different tissues were analysized.The expression levels of Lac-1in different developmental stages and different tissues were analysized by RT-RCR. The results showed that, Lac-1in B. tabaci ME AMI and T. vaporariorum were expressed in cephalothorax, midgut and abdomen, and the highest expression level was detected in midgut; the Lac-1gene were expressed in each developmental stage, and the relative expression level of adult was higher than that in nymph; the total relative expression level of B. tabaci MEAM1was higher than T. vaporariorum; but in midgut, the relative expression level of T. vaporariorum was higher than B. tabaci, and in both insects, the expression level in midgut were higher than that in cephalothorax and abdomen.4. The function research of Lac-1of B. tabaci MEAM1and T. vaporariorum.The RNAi effect was explored by rearing B. tabaci adding dsRNA to the artificial diet.. With the feeding time extending on tomato leaves, a decreasing of the gene silencing effection was observed, and the Lac-1even appeared overexpression5days after feeding. The expression level of Lac-1had some correlation with insect lifespan, the higher of the Lac-1expression level, the higher insect survival rate will be observed; however, no significantly impact on the number of offsprings was detected.The physiological responses of B. tabaci MEAM1and T. vaporariorum feeding on plants sprayed with salicylic acid were explored. Salicylic acid induced plant defense response, compared with the control, after insects feeding plant which sprayed salicylic acid, the relative expression level of Lac-1was decreased, and expression level of Lac-1in B. tabaci MEAM1decreased more than that in the T. vaporariorum.With the feeding time on salicylic acid sprayed plant increasing, the survival rate of B. tabaci MEAM1decreased, and the differences was significant, the survival rate of T. vaporariorum also decreased, but the difference was not significant; the offsprings number of B. tabaci MEAM1reduced significantly, T. vaporariorum also reduced but the difference was not significant. The results indicated that the plant resistance mechanism induced by SA had impact on expression of Lac-1, and B. tabaci MEAM1was more sensitive to this mechanism than T. vaporariorum.