Cloning And Functional Study Of The Telomere Reverse Transcriptase Gene Of Lipaphis Erysimi

The Lipaphis erysimi?Kaltenbach?is one of the agricultural insts endangering cruciferous vegetables,and it has a rapid propagation speed and is distributed all over the world.Telomerase reverse transcriptase?TERT?is a catalytic subunit of telomerase,which not only can induce telomerase activity to affect cell aging and apoptosis,but also has other biological functions besides telomerase dependent.In this study,we used gene amplification,RNAi technology,transcriptome sequencing and q RT-PCR to clone and obtain the CDs sequence of TERT gene of L.erysimi.We detected the dynamic changes of TERT gene expression under different treatments at m RNA level,and preliminarily explored the function of TERT gene in the growth and development of L.erysimi through RNAi technology.The main conclusions are as follows:1. According to the local transcriptome library,the related sequences of TERT gene of L.erysimi were screened,and the CDs sequences of TERT gene were cloned by gene amplification.The full-length sequence of the CDS region of the gene is 2658 bp,which can encode 885 amino acid residues.Its sondary structure contains 41 alpha-helixes and60 coiled coils.Five predicted tertiary structure models have been obtained.After uploading it to the NCBI,it was found that the gene has the highest homology with Diuraphis noxia,and has low homology with other species.2. The relative expression of TERT in different ages and tissues of aphid radish was analyzed by real-time PCR.The results showed that the relative expression of TERT gene in the adult stage of aphid radish was the highest,and there were significant differences with each age.The expression level of 1^st instar nymph was significantly lower than that of adults,but significantly higher than that of 2^nd,3^rd and 4^th instar nymphs.The expression level of 4^th instar nymph was the lowest,but there was no significant difference with that of 2^nd and 3^rd instar nymphs.Subsequently,the 4^th instar nymphs,which is basically mature and has the lowest expression level of TERT gene,was selected for disstion.We were able to simultaneously detect the expression of TERT gene in the head,chest and abdomen of 4^th instar nymphs with the highest expression in the abdomen.3. Two different sequences of TERT gene with different lengths were selected to explore the function of L.erysimi using RNAi technology.It was found that after 24 h of RNA interference,the relative expression of TERT gene in the treated group was down-regulated by 40.6%and 41.3%,respectively,which were significantly different from that in the control group,and there was no difference in downregulation levels between two ds RNA fragments with different interference lengths.After 48 h of RNA interference,the relative expression of TERT gene in the treatment group was significantly down-regulated by 73.9%and 57.0%,respectively,and the treatment group interfering with TERT gene fragment 2 was significantly different from the control group.After 72 h of RNA interference,the relative expression of TERT gene in the treated group was down-regulated by 69.0%and 48.2%respectively,which were significantly different from the control group,and there was no difference in downregulation levels between two ds RNA fragments with different interference lengths.The biological test results showed that interfering with the TERT gene of the L.erysimi could significantly increase the mortality rate,prolong the development period,and reduce the body length,fecundity and the adult longevity.4. The transcriptome of L.erysimi,which interfered with TERT and EGFP genes 72 hours later,was sequenced by Illumina sequencing platform.A total of 51,190,754 and461,296,156 cleaning reads were obtained,and 92,371 Unigene sequences were obtained by assembly,with an average length of 766 bp.GO annotation showed that molecular functional annotation accounted for the highest proportion,among which gene annotation related to ATP binding was the most,including 4,320 sequences accounting for 8.8%.KEGG annotation showed that human disease-related items were the most,and the proportion of gene annotation related to cancer pathway was the highest,including 644sequences accounting for 4.9%.KOG annotation showed that in addition to the unknown function,the largest proportion was signal transduction mechanism,accounting for 13%of all gene annotation.Then,the adjusted p-value?FDR?was selected as the key index of screening differential genes.A total of 545 differential genes were obtained through screening,of which 165 genes were up-regulated and 380 genes were down regulated.GO analysis showed that TERT gene inhibited the synthesis and metabolic processes of related pathways related to cells,organics,and advanced molecules after RNA interference.KEGG analysis showed that TERT gene induced down-regulation of related pathway genes such as ribosome synthesis,oxidative phosphorylation,and actin cytoskeleton regulation after RNA interference.