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根据已报道的其他植物H+-PPase基因的保守序列设计一对简并性引物,以马蔺幼根总RNA为模板,采用RT-PCR方法克隆出马蔺H+-PPase基因片段并克隆到p UCm-T载体,命名为Il VP。阳性克隆经PCR鉴定后进行测序,序列分析结果表明:该基因片段长度为893 bp,编码297个氨基酸,所得到的序列与Gen Bank中注册的高等植物液泡膜H+转运无机焦磷酸酶(H+-PPase)核苷酸碱基排列顺序的同源性均在70%以上、氨基酸序列的同源性达79%以上。这为马蔺Il VP全长基因的克隆及其耐盐分子机理的研究奠定基础。
A pair of degenerate primers was designed according to the reported conserved sequences of other plant H + -PPase genes. The total RNA of Iris seedling root was used as a template, and the H-PPase gene fragment was cloned by RT-PCR and cloned into pUCm-T vector , Named Il VP. The positive clones were identified by PCR and sequenced. The results of sequence analysis showed that the fragment was 893 bp in length and encoded 297 amino acids. The obtained sequence was identical to the H + translocated inorganic pyrophosphatase (H + PPase nucleotide sequences were more than 70% homology, amino acid sequence homology of more than 79%. This laid the foundation for the cloning of Iris full-length gene and its salt-tolerant molecular mechanism.