家蚕C型凝集素16基因(BmCTL 16)的电子克隆及序列特征和表达谱  

In Silico Cloning,Sequence Features and Expression Profile Analysis of Bombyx mori C Type Lectin 16 Gene

作　　者：王立鹏[1] 王华[1] 朱文娟[1] 何月[1] 纪海鹏[1] 王海英[1] 杜贝贝[1] 刘影霞[1] 徐世清[1] 司马杨虎[1] 

机构地区：[1]苏州大学基础医学与生物科学学院,江苏苏州215123

出　　处：《蚕业科学》2015年第2期254-262,共9页ACTA SERICOLOGICA SINICA

基　　金：现代农业产业技术体系建设专项(No.CARS-22)

摘　　要：C型凝集素是在动物先天免疫中发挥重要作用的模式识别受体。从家蚕杂交一代超亲表达的基因中筛选出一个C型凝集素16基因（Bm CTL 16）,通过电子克隆、序列拼接获得了总长度为1 105 bp的序列,其5＇端和3＇端分别为63 bp、385 bp,预测ORF为657 bp,在3＇端终止密码子下游314 bp位置为加尾信号胞质多聚腺苷化特异因子（CPSF）结合位点。预测Bm CTL16编码218个氨基酸,组成蛋白质的氨基酸有20种,数量最多的是亮氨酸（有19个）,蛋白质理论等电点（p I）6.65,分子质量25.124 4 k D。Scan Prosite结构域预测Bm CTL 16存在与其它鳞翅目昆虫CTL一样保守的糖蛋白识别位点CRD。系统进化分析表明Bm CTL 16与烟芽夜蛾、小菜蛾、刺舌蝇、埃及伊蚊的同源CTL序列相似度均高达99%。对电子拼接序列进行RT-PCR验证,扩增片段的测序结果与电子克隆的Bm CTL 16序列完全一致。qRT-PCR分析Bm CTL 16在5龄第1-2天幼虫中肠组织中的表达量较高,之后呈现下降趋势,推测可能是家蚕5龄初期旺盛的生命活动提高了蚕体免疫功能。C type lectin is a pattern recognition receptor which plays important roles in the innate immune system of animals. From the hybrid F1 offspring transgressive gene expression library of silkworm,we obtained a C type lectin 16 gene（ Bm CTL 16）. By in silico cloning and sequence splicing,the full-length of Bm CTL 16 with 1 105 bp was obtained,of which 5＇ and 3＇ end length was 63 bp and 385 bp respectively. It was predicted that its ORF is 657 bp long and a binding site of polyadenylation signal CPSF（ cleavage and polyadenylation specificity factor） is located at 314 bp downstream of the stop codon. Bm CTL16 encodes 218 amino acids with theoretical isoelectric point of 6. 65 and molecular mass of 25. 124 4 k D. It contains all 20 kinds of amino acids,among which the number of leucine（ 19 in total） is the highest. Scan Prosite prediction indicated that Bm CTL 16 has conserved glycoprotein recognition site（ CRD）,being consistent with CTL of other lepidopteran insects. Phylogenetic analysis showed that the sequence similarity of Bm CTL 16 and CTL protein of Heliothis virescens,Plutella xylostella,Glossina morsitans morsitans and Aedes aegypti was as highas 99%. RT-PCR verification revealed that the sequencing result of amplified fragment was consistent with the electronically spliced sequence. qRT-PCR analysis indicated that the expression level of Bm CTL 16 was high in midgut of day 1 to day 2 larvae of the 5th instar and then showed a downward trend on the followingdays,which suggest that the strong vitality at the beginning of the 5th instar may have greatly improved the immune function of silkworm.

关 键 词：家蚕 先天免疫 C型凝集素16基因 电子克隆 序列分析 基因表达 

分 类 号：S881.2[农业科学—特种经济动物饲养]