摘要
【目的】卵黄原蛋白受体(vitellogenin receptor,VgR)属于低密度脂蛋白受体,通过介导内吞作用为发育中的卵母细胞摄取卵黄原蛋白,为胚胎发育提供营养物质,在昆虫生殖过程中发挥关键作用。为研究黑尾叶蝉Nephotettix cincticeps VgR(NcVgR)基因的生理功能及其在生殖中的作用,本研究克隆并解析了NcVgR基因的序列,并对其时空表达进行了研究。【方法】根据黑尾叶蝉转录组数据信息,利用RT-PCR克隆了NcVgR基因,并进行了生物信息学分析;利用实时荧光定量PCR研究了不同发育时期、成虫不同组织NcVgR的表达水平。【结果】NcVgR c DNA序列全长6 676 bp,开放阅读框长度5 568 bp,编码1 855个氨基酸,预测编码蛋白的分子量为206 k D,N端前17个氨基酸为信号肽。序列分析显示,NcVgR具有低密度脂蛋白家族的5个经典保守域,即:配体结合域(ligand-binding domain,LBD)、表皮生长因子前体同源域(EGF-precursor homology domain,EGFP)、O-糖链结构域(O-linked sugar domain,OLSD)、跨膜域(transmembrane domain,TMD)和胞质尾域(cytoplasmic domain)。系统发育分析表明,NcVgR与褐飞虱N.lugens VgR亲缘关系最近。实时荧光定量PCR结果显示,NcVgR转录起始时间为5龄若虫,羽化后转录水平逐渐上升,至羽化后8 d达到峰值,随后下降。有意思的是,随着黑尾叶蝉产卵,NcVgR转录水平再次上升,至羽化后16 d达到最高水平。组织定位结果显示,NcVgR在黑尾叶蝉雌成虫卵巢中特异性高表达,而在雌成虫脂肪体和肠道中微量表达,在雌成虫脑及雄成虫中均未检测到表达。【结论】NcVgR在黑尾叶蝉雌成虫卵巢中特异性表达,并且不同发育时期具有不同的表达量,这为研究黑尾叶蝉的生殖调控机理提供了分子信息。
【Aim】Vitellogenin receptor（VgR）,which belongs to low-density lipoprotein receptor（ LDLR） superfamily,mediates vitellogenin /yolk protein absorption by developing oocytes for embryonic development and plays a critical role in insect reproduction. In order to define the physiological functions of VgR and its roles in reproduction of the green rice leafhopper,Nephotettix cincticeps,we cloned and characterized the VgR gene（ NcVgR）,and monitored its expression profiles. 【Methods】NcVgR was cloned using RT-PCR according to previous transcriptomic data of N. cincticeps and analyzed by bioinformatictools,and the mRNA levels of NcVgR in different developmental stages and adult tissues were detected via real-time PCR. 【Results】The complete c DNA of NcVgR is 6 676 bp in length with a 5 568 bp of open reading frame,encoding a 206 k D protein with 1 855 amino acids including a putative 17-aminoacid signal peptide at the N-terminus. NcVgR has 5 typical conserved domains of LDLR family,including ligand-binding domain（ LBD）,EGF-precursor homology domain（ EGFP）,O-linked sugar domain（ OLSD）,transmembrane domain（ TMD） and cytoplasmic domain. Phylogenetic tree analysis indicated that NcVgR was most closely related to N. lugens VgR. Real-time PCR results showed that the expression of NcVgR started at the 5th instar nymphal stage,increased rapidly after adult eclosion,reached the peak at 8 d post adult eclosion,and decreased subsequently. Interestingly,the transcriptional level of NcVgR increased again and reached the peak at 16 d post adult eclosion when N. cincticeps female adults started to lay eggs. The mRNA level of NcVgR was very specially high in ovary,but only trace level was measured in fat body and gut of female adults. NcVgR was not expressed in both brains of female and male adults. 【Conclusion】We cloned the NcVgR gene,and demonstrated that it is specifically expressed in ovaries of female adults and displays a higher expression after adult eclosion. Our results provide the molecular information for further studying the regulation mechanisms of reproduction of female N.cincticeps.
出处
《昆虫学报》
CAS
CSCD
北大核心
2016年第9期925-937,共13页
Acta Entomologica Sinica
基金
国家重点基础研究发展规划(“973”计划)项目(2014CB138404)
关键词
黑尾叶蝉
卵黄原蛋白受体
基因克隆
序列分析
表达模式
Nephotettix cincticeps
vitellogenin receptor（VgR）
gene cloning
sequence analysis
expression profile
作者简介
陈利鹏，男，1991年11月生，湖南永州人，硕士研究生，研究方向为昆虫生理生化，E-mail：clpzju@163．com
通讯作者Correspondingauthor，E—mail：chu@zju．edu．cn
