| Light receptor CRY can sense the blue light and near ultraviolet light optical signal,in the biological clock and biological rhythm adjustment process plays an important role.Circadian rhythm is the most common biological rhythm of the insect community.It has been found in the study of Drosophila that CRY deficiency may cause serious changes in Drosophila growth and reproductive rhythm.The reproductive behavior of insects is directly regulated by the reproductive-related genes,but whether CRY is still unknown by regulating the expression of these reproductive-related genes and thus affecting the reproductive behavior of insects.Insect CRY gene is divided into CRY1 with photoreceptor and CRY2,and CRY1 plays a leading role in regulation of behavioral rhythm.Based on this,two CRY1 and CRY2 photoreceptor genes were found in the genus Spodoptera litura by transcriptome analysis.The full-length mRNA sequences of these two genes were obtained by RACE technique and sequencing.The two genes were analyzed by fluorescence quantitative analysis.And the expression of CRY1 was further analyzed by RNAi,and the influence of CRY1 gene silencing on the expression of reproductive gene was further analyzed.The results were as follows:(1)The expression of CRY1 gene was analyzed by RNAi.:The CRY1 full-length mRNA sequence has 3686 bases,of which 1647 bases of open reading frames encode a protein with 549 amino acid residues.The CRY2 full-length mRNA sequence has 3366 bases,of which 2376 bases open reading frames encode a protein with 791 amino acid residues.The homology of CRY1 and CRY2 was similar to that of Lepidoptera insects(>85%),but had lower homology with the corresponding proteins of Diptera,Hymenoptera and Coleoptera(<40%).Real-time quantitative analysis showed that the expression levels of CRY1 and CRY2 were very low in the larval stage,and the expression level of CRY1 and CRY2 in the pupal stage was increasing.The expression of CRY1 and CRY2 was the highest in adult stage.The changes of CRY1 and CRY2 expression were similar in larval stage and pupal stage,and the trend of adult and female male and female were opposite:the expression levels of CRY1 and CRY2 were decreased first and then decreased.The expression of CRY1 and CRY2 For the first rise after the drop.This study also found that mating can cause CRY1 and CRY2 expression down for the first time.In this study,RNAi(through dsRNA synthesis and injection)successfully interfered with CRY1 gene expression,interference efficiency of 60%or more.Further studies on CRY2 and PBAN(pheromone synthesis regulatory genes),SPR(post-mating reproductive behavior regulation gene)and Fligtin(migration and reproductive behavior-related genes)were studied under CRY1 RNAi conditions in the daytime and night conditions,The expression of the female and male tissues.The results showed that the down-regulation of CRY1 resulted in significant down-regulation of CRY2,PBAN,SPR and Fligtin genes(P<0.05)in most cases,and showed no significant trend in other cases(P>0.05).In this study,CRY1 and CRY2 full-length mRNA sequences were obtained for the first time,and the CRY gene library of Lepidoptera was improved.And the expression changes of CRY1 and CRY2 in different life cycle and different mating conditions were studied in detail.The expression of CRY1 and other genes related to reproductive genes were analyzed for the first time,suggesting that CRY1 gene may regulate the reproductive rhythm of insects by affecting the expression of reproductive direct gene.The study provides insight into the biological clock and the biological rhythm mechanism,which helps to achieve a breakthrough in the field of research. |
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