朝鲜碱茅抗坏血酸过氧化物酶的基因克隆和表达

本研究从朝鲜碱茅叶cDNA文库分离得到序列长1125bp，开放读码框ORF区876bp，编码291个氨基酸的抗坏血酸过氧化物酶基因（PutAPx）全长cDNA。推测的编码蛋白质的相对分子质量和等电点分别为32kD和7．71。同源性比较发现，PutAPx基因与禾本科5个物种（水稻、大麦、小麦、黑麦草和玉米）在氨基酸水平上具有高度（89．7％，94．3％，94．2％，50．7％和79．6％）的同源性。系统发育树显示朝鲜碱茅与大麦、小麦遗传距离最近，跨膜结构与亚细胞定位表明为过氧化物体APX。将该基因构建到酵母表达载体pYES2-PutAPx，并导入酵母IVSCI菌株后在半乳糖的诱导下分析具有抗氧化性。本研究成功克隆了抗坏血酸过氧化物酶基因（PutAPx）编码区，并做了初步分析，为进一步研究逆境诱导的氧化胁迫的作用机理研究奠定了基础。

Cloning of an Ascorbate Peroxidase Gene from Puccinellia tenui.flora and its Expression Analysis

The full-length gene of an ascorbate peroxidase （PutA Px） was isolated from Puccinellia tenuiflora Ohwi eDNA library. The gene is 1 125 bp in length and it has an ORF of 876 bp, which encoded a protein of 291 amino-acid with an estimated molecular weight 32 kD and an isoelectric point of 7.71. Blasting at NCBI, we found that PutA Px showed high similarity （89.7%, 94.3%, 94.2%, 50.7% and 79.6%） to 5 different gramineae species （Oryza sativa L., Hordeum vulgare L., Triticum aestivum, Lolium perenne L., and Zea mays ）. The result ofphylogenetic tree showed that PutA Px has the elosdest genetic distance with Hordeum vulgate L. and Tritieum aestivum. Transgenie yeast （InVSC 1）, expressing PutA Px gene, under the inducement with β-galactose showed higher stress resistance in oxidation stress than control. In this study, we succeeded cloned an ascorbate peroxidase （PutAPx） and studied in primary levle, which established the foundation for the future study in the mechanism of oxidative stress mechanism of the role of the foundation establish.