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当前位置: 首页 > 文章 > 琥珀蚕内参基因的克隆及表达稳定性检测 蚕业科学 2018 (3) 390-397
Position: Home > Articles > Cloning and Expression Stability Analysis of Antheraea assama Internal Reference Genes Science of Sericulture 2018 (3) 390-397

琥珀蚕内参基因的克隆及表达稳定性检测

作  者：
陈安利;钟健;刘增虎;李琼艳;廖鹏飞;杨伟克;董占鹏
单  位：
云南省农业科学院蚕桑蜜蜂研究所
关键词：
琥珀蚕;内参基因;全长cDNA序列;表达稳定性
摘  要：
琥珀蚕(Antheraea assama)是一种重要的野蚕种质资源。为了开展琥珀蚕功能基因的研究,选择合适的内参基因非常必要。通过c DNA末端快速扩增技术获得琥珀蚕功能基因研究的3个常用内参基因β-actin、GAPDH和18S rRNA的c DNA全长序列,采用实时荧光定量PCR(qRT-PCR)分析3个基因在蚕体不同组织中的转录水平,并利用标准化分析软件ge Norm和Norm Finder分析其表达的稳定性。结果显示,3个内参基因的表达稳定性依次为β-actin、GAPDH、18S rRNA。就组织表达而言,β-actin在幼虫的中肠、脂肪体、马氏管、气孔、丝腺和卵巢以及蛹的输卵管中表达相对稳定;就发育时期的表达而言,β-actin在蛾期的表达也相对稳定。此外,GAPDH在幼虫头部、表皮和血液中的表达相对稳定;18S rRNA在幼虫精巢中的表达相对稳定。对琥珀蚕功能基因表达进行qRT-PCR所需内参基因数量的分析表明,选用2个内参基因即可满足试验需求。
译  名：
Cloning and Expression Stability Analysis of Antheraea assama Internal Reference Genes
作  者：
Chen Anli;Zhong Jian;Liu Zenghu;Li Qiongyan;Liao Pengfei;Yang Weike;Dong Zhanpeng;The Sericultural and Apicultural Research Institute,Yunnan Academy of Agricultural Sciences;
关键词：
Antheraea assama;;Internal reference gene;;Full-length cDNA sequence;;Expression stability
摘  要：
Antheraea assama is an important germplasm resource of wild silkworm. It is necessary to choose suitable internal reference gene for gene function research in Antheraea assama. In this research,we obtained full-length c DNA sequences of reference genes β-actin,GAPDH and 18 S rRNA by cloning with rapid amplification of c DNA ends. Transcriptional levels of these three reference genes in different tissues were investigated by qRT-PCR,and expression stability of them were analyzed using two kinds of standardized analyzing software: ge Norm and Norm Finder. The results showed that the expression stability of three reference genes was in the order of β-actin,GAPDH and 18 S rRNA. In view of expression tissue,expression of β-actin is relatively stable in midgut,fat body,Malpighian tubule,stoma,silk gland,ovary of larva,and oviduct of pupa. In view of expression phase,expression of β-actin is relatively stable at adult stage. Expression of GAPDH is relatively stable in head,epidermis and blood of larva. Expression of 18 S rRNA is relatively stable in spermary of larva. Relative quantification of functional genes in Antheraea assama by qRT-PCR demonstrated that two internal reference genes are sufficient to meet the requirement in experiment.

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