Isolation, molecular cloning and characterization of a cold-responsive gene, AmDUF1517, from Ammopiptanthus mongolicus
Isolation, molecular cloning and characterization of a cold-responsive gene, AmDUF1517, from Ammopiptanthus mongolicus
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沙冬青冷响应基因 AmDUF1517 的分离、分子克隆和鉴定
DOI：
10.1007/s11240-014-0433-4
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发表时间：
2014-05-01
影响因子：
3
通讯作者：
Cheng, Hongmei
中科院分区：
文献类型：
--
作者：
Gu, Lijiang;Cheng, Hongmei
温度是影响植物生长发育的重要因素之一。采用基因扩增片段长度多态性方法，从沙冬青中筛选出344个温度相关转录衍生片段(TDF)。在这项研究中，我们通过半定量RT-PCR证实了其中15个TDF在低温或高温下表达上调。通过快速扩增、聚合酶链式反应和基因组步行等方法，克隆并鉴定了AmDUF1517的全长cDNA和启动子序列。AmDUF1517基因的906bp开放阅读框编码一个301个氨基酸残基的蛋白质。相应的基因组DNA序列包含两个外显子和一个内含子。生物信息学分析表明，AmDUF1517与其同源物(AtDUF1517)之间存在一个预测的叶绿体转运肽的切割位点、一个DUF1517结构域、两个跨膜区和两个可能的总甲基化位点。我们进一步证明了GFP标记的AmDUF1517确实针对拟南芥原生质体中的叶绿体。AmDUF1517基因在叶片中的转录水平在低温胁迫下显著增加。此外，乙烯、水杨酸、赤霉酸或氯化钠处理诱导了AmDUF1517的转录。拟南芥中DUF1517突变表现出对冷胁迫的敏感性增强，同时伴随着电解质外渗率、丙二醛含量的增加和可溶性糖、脯氨酸含量的下降。有趣的是，AmDUF1517在拟南芥突变体中的异源表达显著挽救了它们的冷敏感表型。综上所述，我们的数据提示了AmDUF1517和AtDUF1517在调节冷应激耐受性方面的潜在作用。
Temperature is one of important factors that influence plant growth and development. Using cDNA-amplified fragment length polymorphism approach, we previously screened 344 temperature-related transcript-derived fragments (TDFs) from Ammopiptanthus mongolicus. In this study, we confirmed that 15 of these TDFs were upregulated in response to low- or high-temperature by using semi-quantitative RT-PCR. Based on the rapid amplification of cDNA ends, PCR and genome walking approaches, full-length cDNA and promoter sequence of AmDUF1517 was cloned and identified. The 906 bp open reading frame of the AmDUF1517 gene encoded for a protein of 301 amino acids residues. The corresponding genomic DNA sequence contains two exons and one intron. Bioinformatic analysis showed that a predicted cleavage site for chloroplast transit peptide, a DUF1517 domain, two transmembrane domains and two putative sumoylation sites were conserved between AmDUF1517 and its homolog from Arabidopsis thaliana (AtDUF1517). We further showed that GFP-tagged AmDUF1517 was indeed targeted to the chloroplast in Arabidopsis protoplast. The transcript levels of AmDUF1517 were increased specifically in leaves in response to cold stress. In addition, treatment of ethylene, salicylic acid, gibberellic acid or NaCl induced the transcription of AmDUF1517. Mutation of DUF1517 in Arabidopsis exhibited enhanced sensitivity to cold stress, which was coupled with increased electrolyte leakage, malondialdehyde content and decreased contents of soluble sugar, proline. Interestingly, heterologous expression of AmDUF1517 in Arabidopsisatduf1517 mutants significantly rescued their cold-sensitive phenotypes. Altogether, our data suggest the potential roles of both AmDUF1517 and AtDUF1517 in the regulation of cold stress tolerance.