The Expression Characteristics Of Chitinase Genes And Function Of The Key Genes In Acyrthosiphon Pisum

The pea aphid（Acyrthosiphon pisum Harris）,the major agricultural pests around the world,is an effective vector of a variety of plant viruses,which is serious threat to the leguminous plants.The control of A.pisum mainly depends on chemical insecticides for a long time,but the aphid has evolved resistance to a variety of chemicals.Therefore,it is particularly important to find new targets for killing peas.Insect chitinase play a significant regulatory role in degrading chitin in the exoskeletal and gut linings of insects.Targeting chitin degraded by insect chitinases may be an effective approach in integrated pest management programs because they are harmless for the plants and vertebrates,which do not have chitin in their tissues.The ability of chitinases to digest chitin in the peritrophic matrix or exoskeleton raises the possibility to use them as an insect pest control strategy.Therefore,the identification and excavation more functional genes of chitinases for the developmental stages of pest is of great guiding importance for the design of environment-friendly specific new insecticides.Based on the transcriptome and genome of A.pisum,analyze expression patterns of the chitinase gene of A.pisum and systematically studied the physiological functions of the identified key chitinase genes.The main results are listed as follows:1.Transcriptome analysis between different developmental stages in A.pisumBased on 15 samples from five developmental stages,including 1^st instar nymph,2^nd instar nymph,3^rd instars nymph,4^th instars nymph and newly emerged adult,the transcriptome database of A.pisum between different developmental stages was obtained by second-generation sequencing.A total of 10087 differentially expressed genes（DEGs）between different developmental stages were obtained in A.pisum.There were 1734,3123,3052 and 2178 DEGs obtained during 1^st vs.2^nd,1^st vs.3^rd,1^st vs.4^th,1^st vs.adult respectively.By systematically comparing the differentially expressed genes,it was found that two Cht genes were differentially expressed during ages.qRT-PCR was used to detect the expression of differential genes,which confirmed the reliability of transcriptional sequencing data.2.Identification and bioinformatics analysis of chitinase genes of A.pisumFive chitinase family genes were identified by comparing the genome and transcriptome of A.pisum.The number of introns and exons of different chitinase genes varies greatly.All the 5 chitinase contained chitinase catalytic domains,ApCht7 and ApCht10 had 2 and 4 catalytic domains respectively,while ApCht3,Ap IDGF and Ap ENGase have only one catalytic domain.Moreover,multiple sequence alignment analysis showed that the DWEYP in the conserved domain II of ApCht10-1,ApCht10-2 and Ap IDGF was replaced by other amino acids,and Ap ENGase conservative domain II is incomplete,which suggesting that it catalyze loss of chitinase activity.Phylogenetic analysis showed that the five chitinase were clustered in five groups,namely Group II（ApCht10）,Group III（ApCht7）,Group V（Ap IDGF）,Group X（ApCht3）and ENGase（Ap ENGase）,respectively.3.Expression pattern analysis of chitinase genes in A.pisumThe expression patterns of chitinase genes in different developmental stages and abiotic stresses were analyzed by qRT-PCR technique.Developmental expression analysis revealed that the five A.pisum chitinase genes were expressed at whole developmental stages with different relative expression patterns.ApCht10 was highly expressed in the 1^stand 2^ndinstar nymphs,and ApCht7 was significantly highly expressed in the 4^th instar nymphs.Compared with other chitinase genes,the relative expression level of ApCht3 increased by a great margin in adult stage.Analysis of temperature stress of A.pisum chitinase genes suggested that low temperature（10℃）significantly reduced the relative expression levels of ApCht3,ApCht10,Ap IDGF and Ap ENGase,while the expression levels of ApCht7 significantly increased.After 36 h of high temperature（30℃）,the expression of ApCht3 and ApCht10 significantly decreased.Imidacloprid down-regulated the expression of these five chitinase genes.Besides,the expression of A.pisum chitinase genes was also induced and regulated by20E,and the positive and negative regulation was related to the treatment time of 20E.20E-10min enhanced and ApCht10 expression in A.pisum,but reduced ApCht7expression.After 20E-30 min treatment,the expression of ApCht10 reached the highest at 48 h,and then decreased significantly.It can be seen that Apcht10 is closely related to 20E regulation.4.Functional study of ApCht7 and ApCht10 during the development of A.pisumThe expression levels of ApCht7 and ApCht10 decreased significantly,weight loss and mortality increased of A.pisum after 2^nd instar nymph treated by dsRNA treatment24 h and 48 h,and the cumulative mortality after 96 h treated by RNAi was 22.78%and 26.11%respectively,which significantly higher than the control group.By observing the lethal phenotype and death of aphids,it was found that after RNAi ApCht7 and ApCht10,there were difficulties in molting,deformity of body and aphid tube,shrinkage and curling of body wall.Among them,the proportion of death due to difficulty in molting was 13.83%and 10.67%,respectively,which indicated that ApCht7 and ApCht10 were related to the molting of A.pisum.In addition,compared with the control,RNAi ApCht7 and ApCht10 in adult aphid significantly reduced the fecundity.Furthermore,evaluated the effect of RNAi on the offspring aphid,it was found that the expression of ApCht7 and ApCht10 decreased significantly,the silencing efficiency was 25.45%-30.94%and 33.27%-33.62%respectively,and the average body weight of the offspring aphid was also significantly lower than control treatment.This shows that RNAi effect can be transferred from female to offspring and then affect the offspring growth.5.Regulation of RNAi ApCht7 and ApCht10 on chitin metabolism in A.pisumRNAi ApCht7 and ApCht10 can affect the expression levels of genes related to chitin metabolic pathway in A.pisum.The expression levels of ApCDA1、ApCDA3 and ApCDA4 were decreased by RNAi ApCht7 and ApCht10 for 24 h and 48 h,and the expression levels of ApCDA2 increased after 48 h.In addition,RNAi ApCht7 and ApCht10 also regulates genes in its chitin synthesis pathway.Among them,the expression levels of Ap HK,Ap GFAT,Ap PGM and ApCHS were significantly up-regulated after interference with ApCht7 and ApCht10.The expression levels of Ap TRE and Ap GNPAN were up-regulated first and then down-regulated,while Ap G6PI and Ap UAP were down-regulated first and then up-regulated.These results suggest that RNAi ApCht7 and ApCht10 in A.pisum can regulate chitin metabolism related gene and then regulate chitin metabolism.6.Regulation of RNAi ApCht7 and ApCht10 on energy metabolism in A.pisumThe protein content of A.pisum after RNAi ApCht7 and ApCht10 was 1.10mg/m L and 1.09 mg/m L,respectively,which was significantly lower than control.Compared with the control,the total fat content increased significantly,and the content of carbohydrate（soluble sugar,glycogen and trehalose）decreased significantly after RNAi ApCht7 and ApCht10 treatment.In addition,RNAi ApCht7 and ApCht10 resulted in significant down-regulation of the relative expression levels of Ap TRE,Ap TPS and Ap GP,and significantly up-regulated the relative expression levels of Ap AKH and Ap AKHR.This shows that inhibition of key chitinase genes ApCht7 and ApCht10 of A.pisum can significantly affected energy metabolism and broke the dynamic balance of energy substances,thus affecting the normal growth of A.pisum.In conclusion,this dissertation enriched the bioinformatics database of A.pisum,identified 5 chitinase genes of A.pisum and clarified their expression patterns in different developmental stages,and abiotic stresses;In addition,the functions of ApCht7 and ApCht10 in the growth and development and the regulatory mechanism of RNAi ApCht7 and ApCht10 on chitin metabolism and energy metabolism of A.pisum were explored.The results provide a theoretical basis for the development and utilization of novel insect pest inhibitors targeting chitinase gene.