家蚕卵黄膜蛋白基因BmVMP23的3＇-UTR变异对其表达的影响  

Influence of 3′-UTR Mutation on Expression of Silkworm Vitelline Membrane Protein Gene BmVMP23

作　　者：陈安利[1,2] 夏定国[1,2] 裘智勇[1,2] 高鹏[1,2] 唐顺明[1,2] 赵巧玲[1,2] 

机构地区：[1]江苏科技大学,江苏镇江212003 [2]中国农业科学院蚕业研究所、农业部蚕桑遗传改良重点实验室,江苏镇江212018

出　　处：《蚕业科学》2013年第1期28-34,共7页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(No.31072083);2011年江苏省研究生科研创新计划(No.12508000439)

摘　　要：BmVMP23已被预测为一种编码家蚕卵黄膜蛋白的基因,该基因在家蚕"明"死卵突变体(l-em)中呈显著下调表达,并已证明其终止密码子后的序列发生了变异。为研究变异位点是否位于BmVMP23的3'-UTR区段及序列变异对该基因表达的影响,通过RACE扩增获得BmVMP23的全长cDNA序列,证实突变位点位于BmVMP23的3'-UTR区段。在此基础上,以突变体l-em及正常型的BmVMP23 3'-UTR序列为实验模型,构建以EGFP为报告基因的重组载体pMD18-T(A3-EGFP-SV40)、pMD18-T[A3-EGFP-3'UTR(WT)]和pMD18-T[A3-EGFP-3'UTR(l-em)],并将其分别转染BmN细胞观察EGFP的表达。结果显示,转染pMD18-T(A3-EGFP-SV40)和pMD18-T[A3-EGFP-3'UTR(WT)]质粒的细胞均能检测到绿色荧光,而转染pMD18-T[A3-EGFP-3'UTR(l-em)]质粒的细胞检测不到绿色荧光。上述结果证实BmVMP23的正常表达须具有完整的3'-UTR,由此认为"明"死卵突变体中BmVMP23基因表达量的降低是因其3'-UTR的变异引起的。It is predicted that BmVMP23 gene encodes a vitelline membrane protein of silkworm （ Bombyx mori）. Expression of this gene is significantly down-regulated in the lethal egg mutant of silkworm variety ＂ming＂ （l-em）. It has been demonstrated that its sequence behind the stop codon has mutated. To investigate whether the mutation site is located at 3＇-UTR of BmVMP23 and what influence of this sequence mutation has on gene expression, we obtained the full-length cDNA of BmVMP23 through RACE amplification and proved that the mutation site was located at the 3＇-UTR of BmVMP23. Based on these results, 3＇-UTR sequences of BmVMP23 in l-em mutant and normal type were used as experimental models to construct recombinant plasmids pMD18-T （ A3-EGFP-SV40）, pMD18-T [ A3-EGFP-3＇ UTR （WT） ] and pMD18-TEA3-EGFP-3＇UTR（ l-em） ] by using EGFP as the reporter gene, and the constructed plasmids were transfected into BmN cells for observation of EGFP expression. The results showed that green fluorescence could be observed in BmN cells transfected by plasmids pMD18-T （ A3-EGFP-SV40 ） and pMD18-T [ A3-EGFP-3＇ UTR （WT） ], but not detected in BmN cells transfected by plasmid pMD18-T[A3-EGFP-3＇UTR（l-em） ]. The above results confirmed that complete 3＇-UTR is needed for normal expression of BmVMP23. It is regarded that the decreased expression of BmVMP23 in l-em lethal egg mutant is due to mutation at 3＇-UTR.

关 键 词：家蚕 卵黄膜蛋白 BmVMP23基因 3′端非翻译区序列 基因表达 卵突变体 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]