The Transcriptome Sequencing And Diapause Related Gene-cloning Research Of Bactrocera Minax

Insects can avoid damage from harsh environmental conditions to their growth and developmentthrough diapause, thus ensuring the continuation of the population. The diapause could be classified intoegg diapause, larval diapause, pupal diapause and imaginal diapause, according to the developmental stageswhen the insects enter diapause. The Chinese citrus fruit flies belong to the class of pupal diapause insects,they overwinter in the form of pupae and then enter the eclosion and oviposit in the young fruit, the larvaehazards in fruit. Besides hormone, there are many factors in affecting insectdiapause, and stress genes arealso involved, such as heat shock protein gene showed different expression patterns in the form of diapauseoverwintering insects, suggesting that it may play a role in differet degrees of resistance to lowtemperature environment in diapause insect.In order to study the regulatory factors involved in the pupal diapause of the large citrus fruit flies, thepaper first sequenced the transcriptome of the flies at pre-diapause (larvae) stage, diapause maintenancephase (60days’ old pupae) and post-diapause (second-grade pupa) state, and bioinformatics analysis to findthe genes related to diapause such as the diapause hormone gene and prothoracicotropic hormone gene.Then we cloned the cDNA complete sequence of3heat shock genes (hsp): hsp23, hsp70and hsp90, andPheylogenetic tree analyzed the amino acid sequence encoding the corresponding protein and theirexpression in the mRNA at different development periods. Finally, RNAi technology was used to verify thefunctions of the three genes so as to reveal the molecular mechanism of the regulation of Chinese citrusfruit flies, which can be served as a theoretical foundation for breaking the diapause and conducting indoormass rearing. Also, the mechanism can provide a premise for carrying out the irradiation infertility andgenetic technology to control the damage caused by the flies. The main findings are as follows: 1. After the transcriptome sequencing and the pretreatment on obtained reads, we got CleanRead of threesamples with their size167,509,796respectively. After using SOAPdenovo software to stitch all thesereads samples, the transcript length mainly located in the section of200bp-1500bp, with the totalnumber over20,000. Through RPKM calculation, we found that different genes in different diapauseperiods have different amounts of expression. Using Bowtie to compare transcripts and DESeq tocompare differences two by two, we discoverd that the differently expressing genes between DP and DMwere335, DR and DP554, and DM and DR177. In addition, Unigenes of the measured samples are24,741, and most of them have widespread existence in other species, especially among diptera insects.2. A810bp,2269bp and1730bp fragment, the complete cDNA sequence of hsp23, hsp70and hsp90including both the ORF and3’end of the hsp23, hsp70and hsp90were respectively cloned using thehomologous amplification and RACE method. The sequences encode170,637and363amino acidsseparately. Comparing the amino acids the sequences encode with those of the known species using theNCBI-BLAST, we find that the hsp23, hsp70and hsp90of the flies share a homology of32%~81%、72%~96%and76%~98%respectively with the ones of the other species, indicating that hsp70andhsp90are strongly conservative.3. Ten genes α-TUB、18s rRNA、28s rRNA、β-actin、GAPDH、RPL32、β-TUB、ELF1-β、G6PDH andEF1-α were chosen as the endogenous reference genes and their middle fragments were cloned from theprimers. The expression of the10genes in different instars (eggs, larvae, and30,90,160day-old pupae,female adults and male adults) were detected through the semi-quantification PCR. The results showedthat the α-TUB、RPL32and EF1-α were stably expressed at different development stages of the flies,whereas the other7’s expression existed significant differences at different development periods. 4. Using the Quantitative PCR to detect the expression of the three genes in the flies’ mRNA atpre-diapause(larvae) stage, diapause maintenance phase (60days’ old pupae) and post-diapause(secondgrade pupae) stage, we found that hsp23, hsp70and hsp90all maintained a certain amount of expressionin the process of the whole diapause, but the amount of hsp23gene expression is high relatively, therelative expression quantity of hsp70and hsp90is low, and the hsp90gene is showing the level ofexpression is high relatively in pre-diapause and post-diapause stage, suggesting that hsp23plays themain role in the process of the Chinese citrus fruit fly overwintering, and hsp90gene may be involved inregulating the growth and developmentof the insect;5. In order to establish the appropriate system of RNA interference, we injected with the quality of dsRNAin the Chinese citrus fruit fly larvae was2μg and3μg respectively, and then we achieved the larvae in0d、1d、2d、3d and5d respectively. Then through the quantitative PCR detection, we found that whenthe concentration of injection of dsRNA is2μg/μL, the target gene of insect achieves the best result intime and effect of silence.