饥饿胁迫下黑肩绿盲蝽转录组分析及S6K基因功能分析

Transcriptome analysis and functional analysis of S6K gene in Cyrtorhinus lividipennis under starvation stress

为扩大黑肩绿盲蝽Cyrtorhinus lividipennis的人工繁殖规模,利用RNA-seq技术对饥饿胁迫2 d的黑肩绿盲蝽雌成虫进行转录组测序分析,筛选参与生殖调控的相关信号通路,挖掘直接或者间接影响生殖的相关基因,采用实时荧光定量PCR(real-time fluorescence quantification PCR,qRTPCR)对筛选的相关基因进行验证,并通过试验分析沉默S6K基因和饥饿处理对黑肩绿盲蝽生殖的影响。结果显示,与取食褐飞虱Nilaparvata lugens卵(CK)的黑肩绿盲蝽相比,饥饿处理2 d的黑肩绿盲蝽雌成虫有11675个基因差异表达,其中有4264个基因表达量上调,7411个基因表达量下调。共筛选到7条与生殖调控相关的信号通路和6个与生殖调控相关的基因,除TSC2基因表达量上调外,其他S6K、INSR、Akt、HSP70-1、HSP70-2五个与生殖相关基因的表达量在7条生殖相关信号通路中均下调。qRT-PCR检测结果与转录组测序结果一致,说明转录组分析结果可靠。沉默S6K基因后,黑肩绿盲蝽雌成虫脂肪体和卵巢蛋白质含量、Vg基因表达量、雌成虫产卵量和Vg含量较对照显著降低。此外,饥饿处理2 d后黑肩绿盲蝽雌成虫产卵量也较对照显著减少。表明饥饿胁迫后黑肩绿盲蝽雌成虫的生殖相关通路可能受多个信号通路调控,S6K表达量下降显著影响黑肩绿盲蝽的生殖。

To expand the artificially fed population of Cyrtorhinus lividipennis,RNA-seq was used to sequence the transcriptome of female individuals of C.lividipennis after starvation stress for two days.The genes involved in fecundity regulation signaling pathway were screened,and the genes directly or indirectly influencing reproduction were identified.The screened genes were verified by real-time fluorescence quantification PCR(qRT-PCR),and the effects of silencing ribosomal protein small subunit 6 kinase(S6 K)gene and starvation stress treatment on the reproduction of C.lividipennis adult females were analyzed.The results showed that there were 11675 differentially expressed genes in response to starvation stress treatment for two days,among which 4264 genes were up-regulated and 7411 genes were down-regulated.Seven signaling pathways and six genes related to fecundity were identified,among which the expression levels of five genes(S6 K,INSR,Akt,HSP70-1 and HSP70-2)related to fecundity were significantly down-regulated in the seven signaling pathways,except TSC2 that was upregulated.The qRT-PCR results were consistent with those of RNA-seq.The protein contents in the fat body and ovary,vitellogenin(Vg)expression level,Vg abundance,the number of eggs laid under ds S6 K treatment were significantly decreased compared to untreated control in C.lividipennis female adults.In addition,the number of eggs laid by C.lividipennis female adults under starvation stress for two days was significantly decreased.These results suggested that the fecundity of C.lividipennis female adults was regulated by multiple signaling pathways under starvation stress.Silencing S6 K significantly affected the fecundity of C.lividipennis female adults.