水稻OsAMTR310基因的克隆与在干旱胁迫下的功能分析

Cloning of OsAMTR310 and Functional Analysis under Drought Stress in Rice

为了探究水稻转氨酶基因Os AMTR310的功能以及明确其作用机制,利用多种分子生物学手段对Os AMTR310基因进行了功能分析。实时定量PCR的分析结果表明,Os AMTR310在干旱条件下相对基因表达水平是正常条件下的3.0倍;根据NCBI上获得的Os AMTR310基因序列设计引物,扩增其全长CDS序列,并利用NCBI对蛋白序列的功能结构域进行预测;将Os AMTR310的CDS序列连入p CAMBIA1302中构建超表达载体,并转入受体材料,通过PCR及蛋白质免疫印迹分析来确定Os AMTR310阳性转基因植株,进而分析Os AMTR310转基因植株与野生型植株在干旱胁迫下的差异来验证Os AMTR310基因的功能。结果表明,Os AMTR310 c DNA全长1 873 bp,CDS全长1 533 bp,编码510个氨基酸。Os AMTR310蛋白具有3个保守的结构域,属于乙酰鸟氨酸氨基转移酶家族。在正常条件下,Os AMTR310转基因植株与野生型相比没有明显的差别;然而,在干旱条件下,Os AMTR310转基因植株的存活率是野生型的1.5~2.0倍。通过测定叶片游离脯氨酸含量发现,Os AMTR310转基因植株游离的脯氨酸含量在正常条件下与野生型相比并无显著差异,而Os AMTR310转基因植株的游离脯氨酸含量在干旱条件下是野生型植株的1.5~2.0倍,说明水稻转氨酶基因Os AMTR310赋予了水稻更高的耐旱性。

In order to analyze the function and molecular mechanism of OsAMTR310 in rice,multiple molecular biological methods were performed. Real-time quantitative PCR analysis showed that the relative gene expression level of OsAMTR310 under drought conditions was three times of that under normal conditions. The primers were designed according to the gene sequence on NCBI. The full-length CDS sequence was amplified and the conserved domain of the protein sequence was predicted by NCBI. The CDS sequence of OsAMTR310 banding to the overexpression vector was constructed in pCAMBIA1302 and was transduced into the receptor rice material. OsAMTR310 positive transgenic plants were identified by PCR and Western Blot analysis,and the difference between OsAMTR310 transgenic plants and wild plants under drought stress were analyzed to verify the function of OsAMTR310 gene. Results indicated that the full length of OsAMTR310 cDNA was 1 873 bp and CDS was 1 533 bp in length which encoded 510 amino acids. The OsAMTR310 protein had three conserved domains,belonging to acetyl ornithine aminotransferase family;Under normal condition,there was no obvious difference between OsAMTR310- overexpression transgenic plants and wild-type;however,survival rate of OsAMTR310- overexpression transgenic plants was 1.5- 2.0 times compared with wild-type under drought stress. By investigating the free proline content in leaves,it was no significantly difference in OsAMTR310 transgenic plants compared with wild type under normal conditions and it was found that OsAMTR310- overexpression transgenic plants were 1.5-2.0 times of free proline content under drought conditions than wild type,suggesting that OsAMTR310 confers higher drought tolerance in rice.