两个与中华拟同形溞生殖相关基因的筛选及其RNA干扰研究

SCREENING AND RNA INTERFERENCE STUDY OF TWO GENES RELATEDTO REPRODUCTION IN DAPHNIA SINENSIS

为了筛选与中华拟同形溞(Daphnia sinensis)生殖相关的基因,选择4个生长阶段的中华拟同形溞(雌性幼溞JF、成熟前1龄雌溞BM、第1成龄雌溞MA和第4成龄雌溞RF)进行转录组测序。在BM vs.JF、MA vs.JF、RF vs.JF、RF vs.MA、MA vs.BM、RF vs.BM等6个组合中,选择每个组合的前30个上调基因进行筛选,获得了163个差异表达基因。这些差异表达基因以鱼类信息素培养的第4成龄雌溞为样本进行RT-qPCR检测,共筛选出19个与生殖相关的候选基因,选择其中2个基因(Cluster-13168.32243,Dsussp和Cluster-13168.22885,Dsmapk)进行RNAi实验。在RNAi后,Dsussp和Dsmapk基因的表达量在含有其dsRNA的大肠杆菌(Escherichia coli)浓度组均显著小于对照组(Enhanced Green Fluorescent Protein,EGFP),且与EGFP组相比,Dsmapk和Dsussp基因的表达量在2%和5%大肠杆菌浓度组分别下降了28.1%和32.3%、41.1%和55.6%。在RNAi后,中华拟同形溞在Dsussp和Dsmapk组的怀卵数及产幼溞数与对照组相比均减少,且5%大肠杆菌浓度组均小于2%大肠杆菌浓度组。随着龄数的增加,RNAi对中华拟同形溞怀卵数和产幼溞数的抑制影响均加强。因此,Dsussp和Dsmapk基因被证实在中华拟同形溞的生殖发育中起重要作用。

Reproductive biology of cladocera has been a research focus among ecologists,however,the molecular mechanism on the reproduction of cladocera remains unclear.In this paper,the genes related to reproduction of Daphnia sinensis were screened.D.sinensis at four growth stages(JF:Juvenile female,BM:the 1st instar female before maturity,MA:the 1st adult instar female,and RF:the 4th adult instar female)were selected for transcriptome sequencing.6 combinations including BM vs.JF,MA vs.JF,RF vs.JF,RF vs.MA,MA vs.BM,RF vs.BM,were analyzed,and the top 30 upregulated genes from each combination were selected.This resulted in a total of 163 differentially expressed genes.To validate these genes,RT qPCR was performed on the fourth adult females of D.sinensis cultured with fish kairomones,leading to the identification of 19 candidate genes related to reproduction.Among these candidates,two genes(Cluster-13168.32243,Dsussp and Cluster-13168.22885,Dsmapk)were selected for functional analysis.Subsequently,RNAi was conducted,resulting in significantly lower expression levels of Dsussp and Dsmapk genes in E.coli containing their dsRNA compared to the control group(Enhanced Green Fluorescent Protein,EGFP).Compared with the EGFP group,the expression levels of Dsmapk and Dsussp genes decreased by 28.1%and 32.3%in the 2%E.coli concentration,and by 41.1%and 55.6%in the 5%E.coli concentration,respectively.Compared with the control group,the number of eggs and offspring of D.sinensis decreased in the Dsmapk and Dsussp groups after RNAi,particularly in the 5%E.coli concentration compared to the 2%E.coli concentration.The inhibitory effects on the number of eggs and offspring of D.sinensis were strengthened after RNAi.Therefore,the Dsmapk and Dsussp genes have been confirmed to play an important role in the reproductive development of D.sinensis.