Functional Study Of Cotton Chitinase Gene Ghchi6 On The Resistance To Aphid

Aphis gossypii is one of the important economic pests of cotton in the world,they feed on phloem sap of host plant with their sucking mouthparts.They do harm to host plant by depriving them of nutrient,spreading plant pathogenic virus and secreting honeydew caused coal pollution,and make serious impact on the yield and quality of cotton.Aphis gossypii,Because of the small size,strong fertility and short lifespan,is hard to control.In addition,Aphis gossypii prevention relies mainly on the use of chemical pesticides as for now,the misuse of pesticides has lead to the stronger resistance of Aphis gossypii to pesticides,as a result of which,the prevention and treatment of Aphis gossypii is more difficult.Thus,to foster efficient and insect-resistant cotton varieties has become the general breeding goal.Using the resistance to aphids of plant itself to control aphids is considered to be a highly efficient and environmentally friendly way to prevent and control aphids,and get the general attention of researchers.With the promotion of insect-resistant cotton,new problems began to emerge,17 aphids have become resistant to the plant?s anti-aphid genes.It is urgent to search for new plant aphid-resistant genes.Based on the perspective of cotton resistance to Aphis gossypii,we cloned red-chicken-feet-leaf cotton chitinase gene Ghchi6 by bioinformatics analysis and studied the function and mechanism of its resistance to aphids.The main results obtained are as follows:1.Cloning of Ghchi6 and sequence analysisGhchi6 gene was cloned from red-chicken-feet-leaf cotton,it consists of 975 bps,encoded a protein of 324 amino acids,and belongs to Glyco-hydro-19 subfamily,as the class I kind of chitin.Its molecular weight is 34.65 kDa and isoelectric point is 7.81.Through amino acid sequence comparison,cotton gene Ghchi6 and other plant chitinase genes are highly conservative,and is evolutionarily close relative to chitin of woody cotton.2.Expression analysis of Ghchi6 under Aphis gossypii attack or after mechanical injury inductionAfter Aphis gossypii attack,the expression level of Ghchi6 was suppressed in cotton leaves and stems.However,its expression level was up-regulated in the mechanical wounded cotton leaves,which suggested that Ghchi6 expression level was relative to the induction from cotton aphids or mechanical wounding.3.Analysis of resistance to green peach aphids of overexpressing Ghchi6 transgenic ArabidopsisBy using Agrobacterium-mediated transformation method,we got overexpressing Ghchi6 transgenic Arabidopsis plants.Three Lines(Line1,Line2 and Line4)were selected of them to analysis their resistance to green peach aphids.No matter the choice test or no-choice test,the number of aphid from transgenic Arabidopsis was less than that from the wild type.These results indicated that the overexpression of Ghchi6 can improve the aphid-resistance of transgenic Arabidopsis.4.Research on resistance machanisms to green peach aphids of overexpressing Ghchi6 transgenic ArabidopsisOverexpression of Ghchi6 in transgenic Arabidopsis can improve plant resistance to green peach aphids.In order to make clear the mechanism inside,we measured the content of soluble sugar,activities of four enzymes(Chitinase;Phenylalanine ammonia enzyme,PAL;Polyphenoloxidases or tyrosinases,PPO;Superoxide dismutase,SOD),and observed the leaf callus biosynthesis by using aniline blue staining method,and analyzed the expression level of defence response pathway ralated genes.The results show that the content of soluble sugar in transgenic Arabidopsis was defferent from that of wild type Arabidopsis.The activity of chitinase,PAL and PPO in leaves of transgenic Arabidopsis were higher than wild type,and the activity of SOD has also changed.What?s more,the callose content in transgenic Arabidopsis leaf was also more than that of wild type.These results suggested that the overexpression of Ghchi6 in Arabidopsis enhanced partial aphid-resistant indexes and improved Arabidopsis resistance to green peach aphids.In addition,in transgenic Arabidopsis,the expression level of SA(salicylic acid)and JA(jasmonic acid)defense pathway related genes were upregulated,suggusting that Ghchi6 might regulate transgenic Arabidopsis resistance to green peach aphids by SA and JA related pathway.