家蚕eIF2α全长cDNA克隆及生物信息学分析

Cloning and Bioinformational Analysis of Full-Length cDNA Sequence of Bombyx mori Eukaryotic Translation Initiation Factor 2 Alpha subunit

目的克隆家蚕eIF2α全长cDNA,分析家蚕抗BmDNV-Z品系、感性品系eIF2α的点突变。方法利用荧光差异显示技术,在易感浓核病毒中国镇江株(BmDNV-Z)的家蚕品系华八中获得感性特异条带G13-1b650,通过电子延伸,设计特异引物验证。结果克隆了家蚕eIF2α(BmeIF2α)全长cDNA。其全长为1100bp(GenBank登录号:DQ073458),ORF框为999bp,编码332个氨基酸。BmeIF2α蛋白与草地贪夜蛾eIF2α蛋白同源性高达94.3%。具有保守的磷酸化位点S(51)R(52)R(52)R(54)R(56)K(60)R(63)。BmeIF2α有推定的3个蛋白激酶C磷酸化、5个酪氨酸激酶磷酸化、9个酪蛋白激酶Ⅱ磷酸化、3个依赖cAMP与cGMP蛋白激酶磷酸化位点,2个酪氨酸硫酸盐化位点,1个糖基化位点。结论家蚕易感品系的eIF2α在113～127位的“HVAELLHYETSEQSE”为酪氨酸硫酸盐化位点,而完全抗性品系eIF2α发生C378→T突变,导致Ser126→Leu,缺少了113～127位的酪氨酸硫酸盐化位点。

[Objective] Clone full cDNA sequence of Bombyx mori elF2α, analyse cite mutation between susceptible and resistant strain infected by Bombyx mori densovirus-Z （BmDNV-Z）. [Method] Using fluorescent differential display （FDD） technique, a special band named G13-1b650was obtained in highly susceptible strain HUABA infected by Bombyx mori densovirus-Z （BmDNV-Z）. Then electrically extended and further confirmed by using specific primers, a full cDNA sequence of Bombyx mori elF2α （BmelF2α） gene was cloned. [Result] The full length of the obtained cDNA sequence was 1 100 bp （GenBank accession number： DQ073458）. Its open reading frame （ORF） includes 999 bp and encodes 332 amino acid residues. The deduced amino acid sequence showed 94.3% identity to elF2α of Spodopterafrugiperda. It contains one putative conservative phosphorylation site （S （51） R （52） R （52） R （54） R （56） K （60） R （63））, three phosphorylation sites of Protein kinase C, five phosphorylation sites of Tyrosinase, nine phosphorylation sites of Tyrosinase II, three phosphorylation sites of protein kinase depended on cAMP or cGMP, two tyrosine sulphation sites and one glycosylation site. [Conclusion] BmelF2α in highly susceptible strain contains one tyrosine sulphation site by analyzing sequences from 113-127, H V A E L L H Y E T S E Q S E. While in resistant strains, a C^372 to T mutation which leads＇ Serine126 mutaton to Leucine results in the absence of the tyrosine sulphation site in the sequences from 113-127.