甘蔗CDK基因的cDNA全长克隆与表达分析

Cloning and Expression Analysis of CDK Gene in Sugarcane

植物细胞周期依赖性蛋白激酶(cyclin-dependent kinases,CDKs)是一类丝氨酸/苏氨酸蛋白激酶,与周期蛋白(Cyclins)协同作用,是重要的细胞周期性调控因子。本研究从甘蔗受花叶病毒侵染的转录组数据库中获得一条与高粱(Sorghum bicolor)CDK基因(Gen Bank登录号为XP_002466536.1)高度同源的Unigene序列,通过RT-PCR扩增获得长度为1799 bp的甘蔗Sc CDK基因(Gen Bank登录号为KR258796)。序列分析显示Sc CDK基因含一个完整的开放阅读框(65~1603 bp),编码512个氨基酸,且具有CDK典型保守结构域,如ATP结合位点、磷酸结合位点及活化环A-loop。此外,生物信息学预测显示该基因编码的蛋白定位于细胞核,为可溶性蛋白,不存在信号肽,二级结构元件多为无规则卷曲,主要参与蛋白翻译。实时荧光定量PCR分析表明,Sc CDK基因的表达具有组织特异性,其在蔗芽上的表达量最高,其次是在蔗肉、蔗根、叶鞘和蔗皮中;在PEG、Na Cl和ABA的胁迫诱导过程中,Sc CDK均表现上调作用,且受ABA胁迫后表达量最高,约为对照组的1.9倍。推测该基因的表达与甘蔗抗干旱和抗渗透胁迫有关,同时受ABA的诱导,参与细胞周期分裂。

Plant cyclin-dependent kinases （CDKs） are a specific class of serine/threonine protein kinases, which synergize with cyclins and play an important role in the process of cell regulation. In this study, a unigene that was highly homologous to the Sorghum biocolor CDK sequence （GenBank accession No. XP_002466536.1） was isolated from the sugarcane transcriptome in response to Sorghum mosaic virus （SrMV） infection. This gene was validated by RT-PCR and named as ScCDK （GenBank accession No. KR258796）. ScCDK has a full-length cDNA of 1799 bp and contained a complete open reading frame （65-1603 bp） encoding 512 amino acids. The ScCDK protein contains the typical conservative CDK domains, such as ATP binding site, polypeptide substrate binding site and activation loop. Bioinformatics prediction indicated that ScCDK is soluble protein located in cell nucleus, very likely involved in intermediary metabolism, and has no signal peptide but with random coil according to its secondary structure, qRT-PCR assay revealed that ScCDK gene had tissue-specific expression in sugarcane, with the highest expression level in bud, followed by stem pith, root, leaf sheath and epidermis. The gene expression level was up-regulated by PEG, NaC1, and ABA, particularly ABA treatment resulted in 1.9-fold higher expression of ScCDK compared to the control. These resuits suggest that ScCDK participates in the response of drought and osmotic stresses and the cell cycle division in sugarcane and can be induced by ABA.