疏叶骆驼刺耐盐基因AsNHX的3′-RACE克隆及序列分析

Clone and Sequence Analysis of 3′-RACE of Na+/H+Antiporter Gene As NHX1 of Alhagi sparsifolia

通过Trizol法提取骆驼刺叶片总RNA的方法,反转录合成c DNA,PCR扩增得到一条600-800bp的DNA条带,与报道的Asp NHX1基因的核心序列基本相符,经3′-RACE扩增的DNA片段并连接到载体上进行测序.测序结果显示c DNA长度为1484bp,编码470个氨基酸,与猪毛菜Sa NHX1(Salsola affinis)、梭梭Ha NHX1(Haloxylon ammodendron)、盐爪爪Kf NHX1(Kalidium foliatum)的核苷酸序列同源性高达96%、89%、89%,氨基酸序列同源性达到97%、93%和90%.生物信息学分析显示As NHX蛋白的分子式为C4578H7674N1484O1907S291,蛋白的分子量为123347.5,理论等电点p I为5.01,不稳定参数为46.41,是一个富含疏水性氨基酸的不太稳定蛋白,理论上有35个苏氨酸的磷酸化位点,属于NHX1家族.说明己经在骆驼刺叶片中成功的克隆到Na+/H+逆向转运蛋白基因c DNA序列的部分片段,为下一步克隆骆驼刺叶片Na+/H+逆向转运蛋白基因的c DNA全序列提供了可能.

A lhagi sparsifolia is a very good biological resistance material with the characteristics of cold resistance, drought resistance, salt resistance and wind characteristics. In order to study on the core sequence of the A lhagi sparsifolia＇s NHX1 gene, Extracted the total RNA from the Alhagi sparsifolia＇s leaves used Trizol kit method, synthesis the eDNA, and amplified by PCR, gained a DNA belt about 600bp to 800bp, which conforms to the core sequence of the NHX1 gene that had published.Amplified and sequenced the core As-NHX by 3＇-RACE. The sequence result shows the length of the As-NHX is 1484bp,encode 470 amino acid. The nucleotide sequence ho- mology is 96%, 89%and 89% ,The amino acid sequence homology is 97%, 93% and 90% by compared the se- quence with NHX 1 between SaNHX 1 （Salsola affinis）, HaNHX 1 （ Haloxylon ammodendron ）and Kf NHX 1 （ Kalidi- urn foliatum）reported in NCBI. The result analyses with bioinformatics shows the formula of As-NHX protein is C4578H7674N 148401907S291, Mr is 123347.5, the isoelectric point （pI）is 5.01,it is a kind of unstable protein with rich hydrophobic amino acids, 35 Threonine phosphorylation sites. We had cloned the fragment of NHX from Alhagi sparsifolia leaf and lay a foundation for the molecular biology research works of the resistance aspects on A lhagi sparsifolia.