摘要
以本实验室构建的大鲵皮肤cDNA文库为材料,通过文库质量检测、随机测序、ESTs拼接、COG软件进行基因注释等对文库进行了分析,并从中分离获得了大鲵胞质动力蛋白轻链2(dynein light chain,LC8-type 2,Dynll 2)基因的cDNA序列,对Dynll 2基因进行了生物信息学分析及组织表达研究。结果表明,本实验室构建的大鲵cDNA文库初级库容为1.50×106 cfu,文库重组率为94.8%,插入片段平均长度约为1 kb。400个随机克隆测序共获得343个有效ESTs,其中214条ESTs序列E-值<10-6,经COG软件归为9大类,其中与分泌蛋白、细胞代谢、细胞骨架以及信号转导相关的基因表达量最高,分别为31.3%、14.0%、13.0%和12.6%。表明大鲵皮肤分泌活动强烈,代谢旺盛,这和大鲵皮肤发挥免疫、呼吸以及渗透压平衡等生理功能相一致。获得的大鲵Dynll 2基因全长为682 bp,其中5'-UTR为57 bp,3'-UTR为313 bp,生物信息学分析表明,该基因编码104个氨基酸,分子量为12.03 ku,等电点pI值为7.05。Dynll 2蛋白在88～104处有由里向外和由外向里的2个跨膜螺旋区,且二级结构以α—螺旋为主;在69～87处有典型的"ZnF-C2H2"结构模体;经比较,其三级结构与鼠Dynll 2蛋白相似。Dynll 2蛋白的进化分析表明,大鲵与无尾两栖类相比,更接近陆生动物。荧光定量实验表明,大鲵Dynll 2基因在肌肉中高表达,在其他组织中低度表达。
Dyneins are a group of evolutionarily highly conservative molecular proteins,which can be divided into two groups：cytoplasmic dyneins and axonemal dyneins.Cytoplasmic dynein probably moves along the microtubule essential for transporting cargo in eukaryotes.It is also probably involved in the movement of chromosomes and positioning the mitotic spindles for cell division.Dynein light chain 2,cytoplasmic,is a protein that in humans is encoded by the Dynll 2 gene.To clone dynein light chain,LC8-type 2（Dynll 2）gene from A.davidianus,and to analyze the characteristics of the functional gene by bioinformatics analysis,the structure of Dynll 2 gene was isolated from skin cDNA library.A cDNA library of the skin tissue was constructed by using the isolated mRNA as the template during reverse transcription.The skin cDNA library of A.davidianus were detected and sequenced by picking clones randomly.It was confirmed that the titer of the skin cDNA library of A.davidianus was 1.50×106 cfu,the recombination rate was 94.8%,and the PCR results showed that the average size of inserts segment was about 1 000 bp.A total of 343 ESTs from the library were sequenced and made alignment with sequences in GenBank database.Moreover,at least 214 clones（E-value10-6）derived from these identified clones were categorized into nine categories.Immune-related genes accounted for 31.3% of the largest distribution,metabolism genes（14.0%）,cytoskeleton genes（13.0%）and signaling pathway-related genes（12.6%） took up a larger gene distribution though.It revealed that skin of A.davidianus made intense activities in the form of secretions and took part in metabolism reactions,according to their physiology function in the area of immunity,breathing,and osmotic pressure equilibrium.In order to investigate the contribution of Dynll 2 to motor protein in A.davidianus,molecular cloning,analyzing cDNA sequence and expression analysis of Dynll 2 gene from A.davidianus were performed.The relative mRNA expression levels of Dynll 2 gene were investigated in A.davidianus using real-time RT-PCR.The Dynll 2 cDNA contained a 682 nt of continuous complete open reading frame encoding a polypeptide of 104 amino acids with a calculated molecular mass of 12.03 ku.Analytic results of TMHMM,Phyre,Protscale and SMART showed that Dynll 2 localized to membrane.Dynll 2 contained conserved ＂ZnF-C2H2＂ motif of Dynein family.Dynll 2 shared high amino acid sequence identity with other organisms and the tertiary structure of the Dynll 2 of A.davidianus was highly homologous to the one of Rattus norvegicus.Then phylogenesis analysis of Dynll 2 was obtained with other 26 animals,and it showed Dynll 2 of A.davidianus was highly homologous to terrestrial animals or freshwater fishes.Moreover,we were able to express Dynll 2 mRNA in 6 tissues of A.davidianus,followed by bowel,muscle,liver,lung,skin and stomach.The data of qRT-PCR,analyzed by 2-ΔΔCt method,indicated the plentiful presence of Dynll 2 mRNA in muscle rather than other 5 tissues.
出处
《水产学报》
CAS
CSCD
北大核心
2011年第6期801-808,共8页
Journal of Fisheries of China
基金
陕西省自然科学基金项目(2003C124)
西北农林科技大学重点项目(01140513)
