二化螟鞘磷脂合酶相关基因CsSMSr的克隆、真核表达及表达谱分析

CLONING,EUKARYOTIC EXPRESSION AND EXPRESSION PATTERN OF SPHINGOMYELIN SYNTHASE-RELATED GENE(CsSMSr) FROM Chilo Suppressalis(WALKER)

鞘磷脂合酶(SMS)基因家族在鞘脂质代谢中起着关键作用,可以通过调节神经酰胺和甘油二酯的平衡调控细胞的生存和凋亡。本研究利用RT-PCR和RACE技术克隆得到1个二化螟鞘磷脂合酶相关基因的全长cDNA,命名为CsSMSr(Genbank登录号:JQ664739)。CsSMSr开放阅读框1587bp,可编码528个氨基酸;序列分析表明,CsSMSr基因编码的蛋白N端具有保守的SAM结构域,含6个跨膜区域,并且有4个SMS家族特有的保守区域D1～D4,保守区D3和D4上有活性中心包括3个氨基酸残基(催化三联体):His302,His345,Asp349。CsSMSr在粉纹夜蛾Tn细胞内成功表达,Western blot检测表达产物分子量约60kDa。应用实时荧光定量PCR方法分析该基因在二化螟不同发育阶段和不同组织的表达,结果表明,CsSMSr随着二化螟的生长发育表达水平逐渐升高,在成虫时期达到最大;CsSMSr的表达具有组织特异性,二化螟雌成虫和雄幼虫生殖器官中的CsSMSr表达量都显著高于其他器官,在二化螟雄成虫精巢中表达量也较高,但与中肠和马氏管差异不大。CsSMSr的表达有时间特异性和组织特异性,推测该基因在二化螟的发育和生殖中有重要作用。本研究成功克隆并表达了二化螟鞘磷脂合酶相关基因,为进一步对该基因进行功能鉴定奠定了基础。

Sphingomyelin synthase（SMS） plays an important role in regulating the metabolism of sphingolipids,and activation of SMS promotes cell survival and suppresses apoptosis either by decreasing the pro-apoptotic ceramide or by increasing the levels of mitogenic factor DAG.The full-length cDNA encoding SMSr gene（CsSMSr,genebank ID： JQ664739） was obtained by using RT-PCR and rapid amplification of cDNA ends（RACE） strategies.The full length of SMSr gene contains a 1587bp open reading frame（ORF） encoding a 528 amino acid residues.Amino acid sequence analysis revealed that CsSMSr contains a N-terminal sterile alpha motif（SAM） domain and six transmembranes,it also shares four highly conserved sequences motifs,designated D1~D4.Among them,Motifs D3 and D4 include the His302,His345,Asp349 which form a catalytic triad.CsSMSr gene was successfully eukaryotically expressed in Tn cell,the recombinant protein molecular weight was 60kDa.RT-PCR was carried out to analyze the temporal and spatial gene expression patterns,the results showed that the expression level of CsSMSr was much higher in the adult stage than in the pre-adult stages,and highly expressed in the reproductive organs of male larvae and female adults,furthermore the expression level of CsSMSr was much higher in testis,but not significant different from midgut and Malpighian tubules in male adults,suggesting that CsSMSr gene may play a key role in organ maturity and reproductive function.All these results laid a basis for further research on the physiological and population ecological functions of CsSMSr gene.