黄瓜硝酸还原酶基因(CsNR)序列分析及正反义表达载体的构建

Sequence Analysis and Construction of Sense and Antisense Expressing Vector of Cucumber Nitrate Reductase(CsNR)

为进一步研究CsNR的作用机制,从黄瓜幼叶中克隆获得硝酸还原酶基因编码区序列(CDS)全长和片段(CsNR;EC1.6.6.1),并运用生物学软件对该基因进行序列分析。其中CDS全长2 748 bp,编码915个氨基酸;CDS片段360 bp。CDS全长和p ROKⅡ经Kpn I单酶切和连接,构建了CsNR正义表达载体;CDS片段和p ROKⅡ经Kpn I和Sac I双酶切,构建了CsNR反义表达载体。通过与其他高等植物NR蛋白进行同源性比对分析,结果发现,CsNR基因的氨基酸序列与甜瓜、烟草、拟南芥、油菜NR基因编码的氨基酸序列高度同源,其中与甜瓜NR蛋白之间同源性最高,为98.25%。

To further study the mechanism of CsNR, a fragment and full length CDS of cucumber nitrate reductase（CsNR; EC1.6.6.1） was isolated from cucumber（Cucumis sativus L.） young leaves, respectively. Meanwhile, the gene sequence was analyzed by the biology software. The fragment and full-length CDS was 2 952 and 360 bp, respectively. The full-length CDS and p ROKII were digested to construct sense expressing vector. The fragment CDS and p ROKII were digested to construct sense expressing vector. The homology alignment analysis indicated that the protein encoded by CsNR was in high similarity with NR-encoding protein in Cucumis melo, Nicotiana tabacum, Arabidopsis thaliana and Brassica campestris. The homology with amino acid sequences of CsNR compared with Cucumis melo was the highest by 98.25%.