Cloning,Expression And Function Analysis Of Pax6 Gene Segmental Fragment In Mythimna Separata

Mythimna separata,belonging to Insecta,Lepidoptera,Noctuidae,is a kind of typical seasonal migratory pest,which is widely distributed in many countries.Mythimna separata seriously endanger the corn,wheat and rice,which are three main crops around the world.Because of the characteristics of migratory,gluttonous,gregarious and omnivorous,it is the most important agriculture pest of China.In China,there have been outbreaked in other places except Tibet and Xinjiang.The compound eye is the most important visual receptor for insects,especially for fast flying insects.Development of insect compound eye is regulated by a variety of genes,and Pax6 is one of the most important gene.It mainly controls the development of insect optic disc,optic vesicle,retina and cornea.Pax6 is highly conserved,from drosophilas to vertebrates.In vertebrates,Pax6 is the key to start the cell differentiation of lens fiber.In addition,Pax6 gene also associated with the differentiation of neurons.In cell proliferation and differentiation,the self-renewal of stem cells and the inhibition of cell cycle are controlled by Pax6,besides adjusting of the development of olfactory neurons in the nervous system.In this study,we mainly cloned the Pax6 gene segmental fragment sequence of Mythimna separata by means of molecular biology,compared with the homology species,analyzed the expression pattern of Pax6 of Mythimna separata in differenf days,researched the function of Pax6 by RNA interference,the main research results are as follows:We extracted the total RNA of head,synthesised the first strand cDNA,then designed specific primers,cloned a Pax6 segment sequence of 699 bp of Mythimna separata by PCR.The structure of Pax6 segmental fragments sequence was analyzed,and the amino acid sequence was predicted.We also compared with other homology species by bioinformatics software,the results showed that the Pax6 gene is highly conservative.In order to investigating the expression pattern of Pax6 in Mythimna separata,we used real-time fluorescence quantitative method to check its expression from larval 6 age to thefirst day of adult,the analysis shows that Pax6 has expressed persistently,and the expression increased from larval 6 age to the second day of pupa,then declined in third day of pupa,finally reached maximum in the first day of adult.The method of RNA interference was used to verify the function of Pax6 gene.Results showed that the dsRNA interference had a certain effect,and the expression of the target gene was significantly increased,and we can concluded preliminarily that the Pax6 gene played a role of negative regulation.After the RNA interference,the phenotypic observation showed that there was no significant change in compound eyes of all adults with normal growth.However,its mouthpart can not develop properly,which lead to the death of the moth.In addition,the wing of the moth folded severely and affected its flight ability.It is concluded that Pax6 gene may encode an organ forming factor,which was related to the development of multiple organs.