酵母和人剪接因子Prp5在家蚕中部丝腺的表达  

Expression of Yeast and Human Splicing Factor Prp5 in Middle Silk Gland of Silkworm,Bombyx mori

作　　者：李文萃 黄俊逸[1] 徐永镇 孙霞[3,4] Li Wencui;Huang Junyi;Xu Yongzhen;Sun Xia(College of Life Science,Shanghai University,Shanghai 200000,China;Institute of Plant Physiology and Ecology,Shanghai Institute for Biological Sciences,Chinese Academy of Sciences,Shanghai 200032,China;College of Biotech-nology,Jiangsu University of Science and Technology,Zhenjiang Jiangsu 212018,China;Sericultural Research Institu-te,Chinese Academy of Agricultural Sciences,Zhenjiang Jiangsu 212018,China)

机构地区：[1]上海大学生命科学学院,上海200000 [2]中国科学院上海生命科学研究院植物生理生态学研究所,上海200032 [3]江苏科技大学生物技术学院,江苏镇江212018 [4]中国农业科学院蚕业研究所,江苏镇江212018

出　　处：《蚕业科学》2018年第4期530-536,共7页ACTA SERICOLOGICA SINICA

基　　金：中国科学院重点部署项目(No.KJZD-EW-L12)

摘　　要：剪接因子Prp5是一种三磷酸腺苷（ATP）水解酶/RNA解旋酶,在内含子的识别和预剪接体的组装过程中具有重要的调控作用。分别克隆了酵母（Saccharomyces cerevisiae）与人（Homo sapiens）Prp5蛋白的核心编码序列Sc Prp5-M和h Prp5-M,然后在序列上游分别添加8×His标签序列,再与转基因质粒p Bac-Seri1-IE1ds Red（含seri1基因启动子和信号肽序列）进行重组,得到p Bac-Sc Prp5-M和p Bac-h Prp5-M转基因质粒。利用家蚕转基因技术将转基因质粒分别注射到蚕卵中,通过串联红色荧光标记筛选阳性转基因家蚕,在基因组水平检测到有Sc Prp5-M和h Prp5-M插入的家蚕个体,进一步的RNA和蛋白质检测也证明2种外源蛋白在家蚕中部丝腺中特异性表达,并且在5龄第4天和第5天的表达量较高。此外,在家蚕中部丝腺表达的2种重组蛋白均能随丝蛋白分泌到体外存在于茧层中,经裂解和纯化后,每1 g鲜茧茧层可分别获得6.25μg重组Sc Prp5-M蛋白和12.5μg重组h Prp5-M蛋白。利用家蚕丝腺生物反应器表达外源Prp5蛋白,为进一步研究Prp5蛋白的功能奠定了一定的实验基础。Splicing factor Prp5 is an ATPase/RNA helicase,which plays an important role in the identification of introns and assembly of pre-spliceosome. The core coding sequences of yeast（ Saccharomyces cerevisiae） and human（ Homo sapiens） Prp5 protein,Scprp5-M and hprp5-M,were cloned. After adding 8×His tag sequences at the upstream,they were recombined with the transgenic plasmid p Bac-Seri1-IE1 ds Red（ containing the seri1 promoter and signaling peptide sequences） to yield two transgenic plasmids p Bac-Sc Prp5-M and p Bac-h Prp5-M,which were injected into silkworm eggs respectively by transgenic technology. Positive transgenic silkworms were screened by observing the red fluorescence under microscopy. The successful transgenic silkworms with Scprp5-M or h Prp5-M sequence insertion were detected at the genomic level. Further RNA and protein tests showed that the two heterologous proteins were specifically expressed in middle silk gland with relatively high expression levels in day 4 and day 5 larvae of the 5 th instar.Besides,the two heterologous proteins expressed in middle silk gland can be secreted and stored in cocoons.After lysis and purification,6. 25 μg Sc Prp5-M and 12. 5μg h Prp5-M can be obtained from 1 g fresh cocoon shellsrespectively. Expression of Prp5 protein in silk gland bioreactor provides a good experimental basis for further functional study of Prp5 protein.

关 键 词：丝腺生物反应器 中部丝腺 转基因家蚕 外源蛋白 剪接因子 Prp5 

分 类 号：Q786[生物学—分子生物学] S881.2[农业科学—特种经济动物饲养]