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坛紫菜丝氨酸羟甲基转移酶基因的克隆及表达特征
林颖辉, 王文磊, 徐 燕, 纪德华, 陈昌生, 谢潮添
集美大学水产学院 厦门 361021
摘要:
丝氨酸羟甲基转移酶(SHMT)在植物应答逆境胁迫中发挥着重要作用。本研究以坛紫菜(Pyropia haitanensis)为研究材料,采用普通PCR技术克隆得到2条坛紫菜的SHMT全长基因序列,分别命名为PhSHMT-1(GenBank收录号:MF687405)和PhSHMT-2(GenBank收录号:MF687406)。其中,PhSHMT-1序列全长1710 bp,包含一个1491 bp的开放阅读框,所编码的多肽包含497个氨基酸,分子量为121.443 kDa,等电点为4.93;PhSHMT-2序列全长1957 bp,包含一个1395 bp的开放阅读框,所编码的多肽包含465个氨基酸,分子量为113.969 kDa,等电点为4.95。多序列比对和系统进化树分析结果确认PhSHMT-1和PhSHMT-2基因属于SHMT基因家族。qRT-PCR定量分析结果表明,高温胁迫条件下,2条PhSHMT基因的表达模式基本一致,均表现为先上调后下调再上调的趋势,这说明SHMT基因可能在坛紫菜应答高温胁迫过程中发挥作用。
关键词:  坛紫菜  高温胁迫  丝氨酸羟甲基转移酶  活性氧  基因克隆
DOI:
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基金项目:
Cloning and Expression Analysis of Serine Hydroxyl Methyltransferase (SHMT) Genes from Pyropia haitanensis
LIN Yinghui, WANG Wenlei, XU Yan, JI Dehua, CHEN Changsheng, XIE Chaotian
College of Fisheries, Jimei University, Xiamen 361021
Abstract:
Serine hydroxyl methyltransferase (SHMT) plays an important role in plant response to stress. Under adversity stress, the SHMT gene not only promotes the Calvin cycle to participate in the anti-stress reaction, but also participates in the synthesis of glycine, and then synthesis of glutathione, scavenging reactive oxygen species. Pyropia haitanensis, naturally grows in the coastal intertidal zone, is an important economic macroalgae in Fujian, Zhejiang and Guangdong provinces. Because of the special living environment, P. haitanensis has the unique ability to resist adversity. With global warming, the temperature of seawater increases, which causes damage to seedlings of P. haitanensis and leads to great economic losses in the aquaculture industry. As for the physiological and molecular response of P. haitanensis to high temperature and dehydration stress conditions, our previous studies found SHMT plays an important role in stress resistance. In this study, based on unigene sequences which were obtained from whole transcriptome sequencing of P. haitanensis, two full-length PhSHMT genes were obtained by ordinary PCR, and named PhSHMT-1 (GenBank accession No: MF687405) and PhSHMT-2 (GenBank accession No: MF687406). The full-length cDNA of the PhSHMT-1 gene comprised 1710 nucleotides and contained an open reading frame (ORF) of 1491 bp, encoding a protein of 497 amino acid residues, with the predicted molecular weight of 121.443 kDa and theoretical isoelectric point of 4.93; and the full-length cDNA of the PhSHMT-2 gene comprised 1957 nucleotides and contained an ORF of 1395 bp, encoding a protein of 465 amino acid residues with the predicted molecular weight of 113.969 kDa and theoretical isoelectric point of 4.95. Multiple sequence alignment and phylogenetic analysis indicated that the PhSHMT-1 and PhSHMT-2 belong to the plant SHMT enzyme family. The expressions of the two PhSHMT genes, as measured by real-time fluorescence quantitative PCR (qRT-PCR), were consistent under high temperature stress. During high-temperature stress, the expression levels of the two PhSHMT genes were significantly upregulated first and then decreased, but as the high-temperature stress continued, the expression levels were upregulated again. Results suggested that the gene of PhSHMT enzyme plays an important role in responses of P. haitanensis to high-temperature stress.
Key words:  Pyropia haitanensis  High temperature stress  Serine hydroxyl methyltransferase  Active oxygen  Gene cloning