采用CTAB法提取经600mmol.L-1NaCl胁迫处理后的胡杨总RNA,利用SMART技术构建载体为λTriplEx2的盐胁迫条件下胡杨的表达型cDNA文库(SSL)。文库的滴度为1.2×106pfu.ml-1,重组率约为90.6%,扩增后的滴度为3×109pfu.ml-1,容量约为2.4×1011。插入片段长度均大于400bp,平均长度约为790bp。从文库中随机挑选32个阳性克隆进行3′端测序,并将测序结果通过NCBI(National Center for Biotechnology Information)数据库进行比较,发现有23个EST序列与已知序列有明显的同源性,而其余的与NCBI中的已知序列相似性较低(e值>10-4),可能是未知基因。其中序列SSL061、SSL179与脱水素序列有较高的序列一致性。同时合成引物,通过PCR扩增文库的方法,从中筛选获得存在于植物细胞膜上并在植物受盐胁迫时起重要作用的nhx基因,进一步验证了文库的质量。
A novel vacuolar Na+/H+ exchanger, CgNHX1, was cloned from a halophytic species Chenopodium glaucum by using reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique. Sequence alignment and phylogenetic analysis of 22 NHX genes from GenBank as well as the new CgNHX1 gene indicate that NHX genes shared a great degree of similarity, regardless of their glycophytic or halophytic origin. Expression of the CgNHX1 gene was induced by NaCl and peaked at 400 mmol/L NaCl. Overexpression of NHX1 genes in rice enhanced their tolerance to salt stress. However, there is no significant difference in salt tolerance among the transgenic rice plants overexpressing the NHX1 genes from either glycophytic or halophytic species. The Na+ content of both the wild type (WT) and transgenic plants increased when exposed to 50 and 100 mmol/L NaCl, and the Na+ concentration in transgenic plants was marginally higher than that of WT. Our data demonstrate that the overexpression of the NHX1 gene from either glycophytic or halophytic species resulted in the enhanced tolerance to salt stress at a similar level, suggesting that NHX gene per se might not be the reason accounting for the difference in salt tolerance between glycophytes and halophytes.
Jin-yao LIXiao-wei HELi XUJie ZHOUPing WUHui-xia SHOUFu-chun ZHANG
从植物组织中提取高质量的RNA是进行植物分子生物学研究的必要前提和关键。盐桦细胞中存在大量的胶质和多糖类物质,用Trizol试剂根本不能获得RNA。本研究采用CTAB-L iC l法,提出了一种适合盐桦叶片总RNA的简单快速提取方法。消除了蛋白质、DNA、多糖、多酚等污染。该方法提取的盐桦叶片总RNA,完整性好、纯度高,可用于RT-PCR等实验操作,此法无需特殊试剂和贵重仪器,实验结果稳定,重复性好,适合富含多糖和脂质的植物组织总RNA的提取。
