家蚕多聚泛素基因的电子克隆、序列分析及表达谱预测

In silico Cloning, Sequence Analysis and Electronic Expressed Pattern of Polyubiquitin Gene in the Silkworm, Bombyx mori (Lepidoptera: Bombycidae)

具选择性蛋白质降解功能的泛素在昆虫生长发育过程中起着重要的调控作用。本研究采用电子克隆的方法钓取家蚕基因组中多聚泛素基因序列,命名为Bm-polyUB(GenBank登录号:AADK01019318),并进行序列分析和电子表达谱预测。序列分析表明,该编码区长3426bp,编码1141个氨基酸残基的15聚体,预测分子量和等电点分别为127.97kD和7.33,二级结构中α-螺旋、延伸带、β-转角和无规则卷曲各占17.09%、32.78%、14.37%和35.76%,亚细胞定位于细胞质、细胞核和线粒体各占43.5%、34.8%和21.7%,无前导肽、信号肽和跨膜区;多重序列比对显示,15个泛素单体基因序列间同源性和遗传距离分别介于89.0%～100.0%和0.000～0.120,其中Bm-UB3与Bm-UB10、Bm-UB5与Bm-UB7及Bm-UB10与Bm-UB14的序列相同;遗传多样性分析可见,共检出33个多态性位点,共生成12个单倍型,单倍型多样性(Hd=0.962)、平均核苷酸差异数(K=7.495)、核苷酸多样性(Pi=0.03287)、密码子有效值(ENC=41.623<61)、偏爱指标(CBI=0.496>0)和χ2检验计算(χ2=0.713)显示泛素单体间遗传多样性较丰富且呈现较强的密码子偏爱性;电子表达谱预测结果表明Bm-polyUB基因在家蚕中高表达的组织和发育阶段所占比例显著高于低表达。本文的研究结果可为进一步开展该基因进化机制、表达特性和生理功能等方面的实验研究提供基础资料。

Ubiquitin that has the function of selective protein degradation may play important roles in the regulation of insect growth and development.In this research,the coding sequence of polyubiquitin gene from Bombyx mori genome named Bm-polyUB （GenBank accession No.： AADK01019318） was found by in silico cloning method,then its sequence analysis and electronic expression pattern predicate were carried out.The sequence analysis showed that the length of the coding sequence was 3 426 bp,encoding 1 141 amino acids with 15 repeats of ubiquitin coding unit,the calculated molecular weight of 127.97 kD and theoretical isoeletric point of 7.33.The predicated secondary structure composition for Bm-polyUB had about 17.09% alpha helixes,32.78% extended strands,14.37% beta turns and 35.76% random coils in further study.Subcellular localization analysis demonstrated that the gene of cytoplasm,cell nucleus and mitochondrial occupied about 43.5% ,34.8% and 21.7% respectively,however,leader peptide,signal peptide and transmembrane domain had not been found.On the other hand multiple sequence alignment indicated that the homology and genetic distances among 15 ubiquitin monomers were 89.0%~100.0% and 0.000~0.120,respectively.Bm-UB3~Bm-UB10,Bm-UB5~Bm-UB7 and Bm-UB10~Bm-UB14 had exactly the same sequence.During the genetic diversity analysis,the total 33 polymorphic sites were detected from 15 sequences and 12 hapolotypes were sorted.Abundant genetic diversity and strong codon usage bias were found by the haplotype diversity （Hd=0.962）,the average number of nucleotide difference （K=7.495）,the nucleotide diversity （Pi=0.032 87）,the effective number of codons （ENC=41.62361）,the codon bias index （CBI=0.4960） and the scaled Chi-square （χ2=0.713）.Electronic expression pattern indicated that the proportions of higher expression of Bm-polyUB gene in different tissues and developmental stages of B.mori were significantly higher than those of lower expression.The results of this investigation might provide some elements data for further studies on the evolutionary mechanism,expressed characteristics and physiological functions and so forth of Bm-polyUB.