Cloning，Sequence Analysis and Prokaryotic Expression of Three Spliceosomes of Abd‑A from Silk Gland of Bombyx mori

doi:10.15933/j.cnki.1004-3268.2022.04.016

Abstract

Abstract: In order to explore the splicing form and sequence characteristics of transcription factor Abdominal‑A （Abd‑A） from silk gland of Bombyx mori，clarify the relationship with the Abd‑A sequences of other insects，the cDNA of silk gland tissue was used as the template to amplify Abd‑A by PCR.The sequence characteristics of Abd‑A and the evolutionary relationship of Abd‑A among different insects were analyzed by bioinformatics methods.The Abd‑A was recombinantly expressed in vitro by prokaryotic expression system，and its protein expression level was detected by SDS‑PAGE and Western blot.The results showed that three spliceosomes of Abd‑A were cloned from the silk gland of B.mori，with
coding regions of 1 059，1 044，1 032 bp，named BmAbd-A L（A59T），BmAbd-A S，BmAbd-A 3，respectively.Multiple sequence alignment of Abd‑A in different insects indicated that their amino acid sequences differed greatly，but all had the DNA‑binding domain（homeobox domain） and the transcriptional activation domain（Abd‑A domain）.The homeobox domain was completely conserved，while the carboxyl terminal sequence of Abd‑A domain was quite different. The three‑dimensional structure prediction indicated that the proteins encoded by three spliceosomes of Abd‑A had four α‑helix and random coil structures，among which the random coil structures varied greatly.Phylogenetic analysis showed that the amino acid sequences encoded by Abd‑A of 35 different insects were mainly divided into four orders，which were Diptera，Lepidoptera，Coleoptera and Hymenoptera，respectively.The Abd‑Asequences of B.mori were closer to Galleria mellonella（Lepidoptera：Pyralidae） in evolutionary relationship，followed by Ostrinia furnacalis.The results of SDS‑PAGE and Western blot showed that the fusion proteins of BmAbd‑A L（A59T），BmAbd‑A S，BmAbd‑A 3 were mainly expressed in soluble form in Escherichia coli，and their theoretical molecular weights were 52.99，52.35 and 51.91 ku，respectively，which were consistent with the expectation.In conclusion，three spliceosomes of Abd‑A were successfully cloned from the silk gland of B.mori.Their sequences and three‑dimensional structures were different，and their molecular evolution process was consistent with the phylogenetic evolution of species.Soluble Abd‑A recombinant proteins were obtained by prokaryotic expression system.

Key words: Bombyx mori, Silk gland, Abd?A, Spliceosome, Prokaryotic expression

摘要： 为探究转录因子Abdominal-A（Abd-A）在家蚕丝腺中的剪接形式及其序列特征，明确与其他昆虫Abd-A的亲缘关系，以家蚕丝腺组织cDNA为模板，对转录因子Abd-A进行PCR扩增，利用生物信息学方法，对Abd-A的序列特征以及不同昆虫间Abd-A的进化关系进行分析；并通过原核表达系统对Abd-A进行体外重组表达，利用SDS-PAGE和Western blot检测其蛋白质表达水平。结果显示，从家蚕丝腺中克隆到Abd-A的3种剪接体，其编码区长度分别为1 059、1 044、1 032 bp，分别命名为BmAbd-A L（A59T）、BmAbd-A S、BmAbd-A 3。不同昆虫Abd-A的多序列比对结果表明，它们的氨基酸序列差异较大，但都具有DNA结合结构域Homeobox domain和转录激活结构域Abd-A domain，其中Homeobox domain序列完全保守，而Abd-A domain的羧基端序列存在较大差异。三维结构预测结果表明，Abd-A的3种剪接体编码蛋白质中均有4个α螺旋结构和无规则卷曲结构，其中无规则卷曲结构差异较大。系统发育分析结果表明，35种不同昆虫的Abd-A编码的氨基酸序列主要分为4个目，分别为双翅目、鳞翅目、鞘翅目、膜翅目，家蚕的Abd-A氨基酸序列与大蜡螟（鳞翅目：螟蛾科）在进化关系上更近，其次为玉米螟。SDS-PAGE及Western blot分析表明，BmAbd-A L（A59T）、BmAbd-A S、BmAbd-A 3的融合蛋白主要以可溶形式在大肠杆菌中表达，其理论分子质量分别为52.99、52.35、51.91 ku，与预期一致。综上，从家蚕丝腺中成功克隆到Abd-A的3种剪接体，它们的序列及三维结构间具有差异性，其分子进化过程与物种系统进化一致，并利用原核表达系统获得可溶性的Abd-A重组蛋白。

关键词: 家蚕, 丝腺, Abd-A, 剪接体, 原核表达

CLC Number:

S881.2

LU Qingjun, LUO Zhuxing, LIU Huawei, WEI Meng, WANG Yuan, ZHANG Zhaofeng, LI Youshan. Cloning，Sequence Analysis and Prokaryotic Expression of Three Spliceosomes of Abd‑A from Silk Gland of Bombyx mori[J]. Journal of Henan Agricultural Sciences, 2022, 51(4): 138-150.

路庆君, 罗竹星, 刘华伟, 魏梦, 王圆, 张照锋, 李游山. 家蚕丝腺中Abd-A 3 种剪接体的克隆、序列分析及原核表达[J]. 河南农业科学, 2022, 51(4): 138-150.

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