小麦亚硝酸还原酶基因及调控序列克隆、定位和表达分析

利用in silico及反向PCR技术, 从小麦中克隆了亚硝酸还原酶编码基因及其调控序列, 进一步利用原核诱导表达、半定量RT-PCR、AS-PCR及生物信息学手段对克隆的新基因进行鉴定及染色体定位分析。开放阅读框预测结合测序结果表明, 该基因gDNA长2 881 bp, 包含4个外显子和3个内含子, cDNA长1 830 bp, GenBank登录号分别为FJ555239和FJ527909, 预测编码产物大小约为65.7 kD, 与NCBI已公布的亚硝酸还原酶基因编码产物同源性达60%以上, 其中与其他单子叶谷类作物同源性达80%以上。IPCR技术延伸该基因5′端侧翼序列至-2 924 bp (以ATG起始计算), 经1 mmol L^-1 IPTG诱导后可表达大小约为70 kD的蛋白(含约3.8 kD的组氨酸标签)。RT-PCR结果显示, 30 mmol L^-1 KNO₃处理小麦幼苗1 h, 亚硝酸还原酶基因表达量最高。酶活性测定表明, 随着KNO₃处理时间延长亚硝酸还原酶活性增强。AS-PCR检测发现, 该基因在普通小麦6A及6B染色体上至少各存在1个拷贝。

Isolation, Chromosome Assignment, and Expression Assay of Nitrite Reductase Gene and Regulatory Sequence in Wheat

Nitrite reductase is wildly involved in N metabolism in plants and has shown excellent relevance to regeneration potential in the tissue culture of rice (Oryza sativa L.). In this study, in silico and inverse PCR techniques were employed to isolate nitrite reductase encoded gene and its regulatory sequences from wheat (Triticum aestivum L.). This gene was predicted to contain four exons and three introns. The gDNA and cDNA sequences were 2 881 bp and 1 830 bp in length, respectively, and they were both submitted to GenBank under the accession numbers FJ555239 and FJ527909. Its deduced encoding protein product was approximately 65.7 kD, sharing high (more than 60%) identity with other nitrite reductase genes deposited in the NCBI database, especially with those from other monocot cereal crops (more than 80%). The 5′ flanking region was isolated and extended to -2 924 bp (counting from the start code: ATG) through inverse PCR. After induced by 1 mmol L^-1 IPTG, a protein of ca. 70 kD was obtained in prokaryotic expression vector pET-28a, including a histidine tag of ca. 3.8 kD. The highest expression of gene in wheat seedlings was induced by 30 mmol L^-1 KNO₃ for 1 h. Measurement on nitrite reductase activity showed the enzyme activity increased following the treatment time extension at 5-d intervals under the above same condition. According to the result of AS-PCR analysis, at least one copy of the gene existed on chromosome 6A and 6B each in common wheat. The study provides a basis for functional determination of wheat nitrite reductase gene in subsequent research.