Identification And Functional Verification Of Genes Which Regulated The Biosynthesis Of Lac In Kerria Chinensis(Hemiptera:Tachardiidae)

Lac insect is a resource insect with important economic value,its female secrete lac through the gland,which is a pure natural biolipid.It is widely used in military industry,medicine,chemical industry and other industries because of its excellent properties such as strong adhesion and good insulation.Since its important economic value,it is of great significance to understand the mechanism of lac synthesis at the molecular level for increasing the lac secretion of lac insects and establishing a sustainable and efficient lac production system.In this paper,Kerria chinensis was taken as the object of study.The amount of individual secretion and rate of lac secretion in different periods were measured to reveal the lac secretion characteristics and dynamic of K.chinensis.By analyzing the transcriptomes of different stages of K.chinensis,key genes of lac synthesis signaling pathway and differential gene expression analysis,candidate genes closely related to lac secretion were screened out.And the functional verification of major genes for lac synthesis using gene silencing?RNAi?Technology.The main results are as follows:1.The dynamic rules of lac secretion for K.chinensisThe amount and rate of lac secretion of female at different stages of K.chinensis were measured.The results showed that,in larval phase the lac secretion was less,the secretion in adult stage was large,and the highest individual secretion rate was in the mid-late stage of adults,it can reach 4.82×10^-1±0.0005 mg/d.In the whole life cycle of K.chinensis,the amount of individual lac secretion was about 24.9±0.0037 mg.2.Analysis of transcriptomes in different stages of K.chinensisAccording to the lac secretion dynamics of K.chinensis,samples from larval stage?L?,early adult stage?A1?and middle-late adult stage?A2?were selected for transcriptome sequencing analysis.A total of 205 581 transcripts were obtained,of which 183 899 unigenes were annotated.The results of differential genes expression analysis showed that a total of 6435 differential expressed genes were obtained in three stages.Further analysis found that compared larval stage with lac less secretion with adult stage with large secretion,there were202 genes up-regulated and 1 187 genes down-regulated.Through the correlation of gene expression level with lac secretion rate and speculation of gene function,28 candidate genes that may be involved in lac synthesis were preliminarily screened.It includes 19 genes involved in fatty acid synthesis;4 genes involved in terpenoids biosynthesis and 5 genes involved in UDP-driven glycosylation.Real-time fluorescent quantitative PCR assay detection of 14 candidate genes related to terpenoids and fatty acid synthesis and metabolism,The results showed that the trend of expression level of these candidate genes was mostly consistent with that of gene expression level in transcriptome data.The results of q RT-PCR confirmed the accuracy of transcriptome data and analyzed the expression pattern of key genes in lac synthesis.3.The functional verification of FAD gene by RNAiIn this study,a recombinant vector containing FAD gene of K.chinensis FAD-L4440 was successfully constructed,transfered to HT115 competent cells,cultured to bacterial solution OD₆₀₀=0.4,IPTG was used as inducer to express dsRNA corresponding to target gene,and FAD-L4440-HT115 was obtained.Finally,transfection of dsRNA into K.chinensis by coating-drying method.The results showed that the expression of FAD gene decreased significantly after interference.Compared with L4440-HT115 bacterium solution control group,the treatment of low concentration?6.2 ng/ml?for 12 h and medium concentration for 24 h had significant difference,which decreased by 90.90%and 86.52%respectively.There were significant differences in individual lac secretion among different treatment groups compared with CK group,and low concentration treatment had the most significant effect on individual lac secretion,indicating that FAD gene had a regulatory effect on lac secretion.In this study,28 major genes closely related to lac synthesis were screened by transcriptome sequencing,which accumulated genetic data for the subsequent analysis of the molecular mechanism of lac synthesis.The RNA interference vector of K.chinensis was constructed,and a RNAi system for bacterial expression of dsRNA was introduced in gene function verification,which provided a technical reference for insects that not apply to inject or feed methods in the process of RNAi transfection.