色氨酸阻遏结合蛋白(tryptophan repressor-binding protein,WrbA)是一种重要的抗氧化酶类,参与了多种逆境反应。笔者通过RACE-PCR的方法克隆得到青杄WrbA基因的cDNA全长,共1 045 bp,编码区651 bp,编码203个氨基酸。利用生物信息学工具对其进行理化性质、二级结构和三级结构的分析,该蛋白理论分子质量21.80 ku,pI值6.43,N端11—15位氨基酸和C端112—165位氨基酸是FMN结合位点。荧光定量PCR和半定量RT-PCR发现青杄PwWrbA在各组织中都有表达,但在针叶中表达量最高。同时,PwWrbA在NaCl和ABA胁迫处理后表达量升高,H2O2也会影响其表达的改变,H2O2处理6 h后表达量上调。因此,PwWrbA是一个新的WrbA基因,推测其可能在青杄逆境胁迫应答中发挥作用。
Abstract
The tryptophan(W)repressor-binding protein(WrbA)is an important antioxidant enzyme which has been shown to play roles in a variety of adversities. In this study,PwWrbA cDNA was cloned by RACE-PCR method. Bioinformatics analysis showed that the full length of PwWrbA cDNA was 1 045 bp, including the ORF 651 bp, encoding 203 amino acids. The theoretical molecular weight was 21.80 ku and value of pI was 6.43.The ScanProsite online software showed that the N terminal 11-15 aa and the C terminal 112-165 aa were supposed to be the FMN binding sites. RT-qPCR and semi-quantitative RT-PCR analysis showed PwWrbA expressed in various tissues, while the highest expression level was in pollen. Meanwhile, the expression of PwWrbA increased gradually after stress treatment with NaCl and ABA.In contrast to NaCl and ABA treatment, the expression of PwWrbA decreased in early stage and increased at 6 h after H2O2 treatment. Our results suggested that PwWrbA was a new WrbA gene and played roles during different abiotic stresses.
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基金
收稿日期:2013-09-04 修回日期:2013-12-12
基金项目:国家自然科学基金面上项目(31270663); 国家转基因生物新品种培育科技重大专项(2013ZX08009-003-002)
第一作者:张通,硕士生。*通信作者:张凌云,副教授,博士。E-mail: lyzhang73@sohu.com。
引文格式:张通,周燕妮,李长江,等. 青杄PwWrbA基因克隆及表达分析[J]. 南京林业大学学报:自然科学版,2014,38(4):34-38.
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