摘要
从柽柳(Tamarix hispida)根中的cDNA库中分离出一个与盐相关的类锌指基因(ThZFL)。ThZFL基因全长514bp,开放读码框自103位的ATG起,止于421位的TAA,为318bp,共编码105个氨基酸,推测其分子量为11.25kD,等电点为9.97。对其进行生物学信息分析,这个基因与动物锌指蛋白有部分相似性。
Northern blot分析表明盐和渗透剂处理都提高了ThZFL在柽柳根部的表达,也能被ABA诱导。亚细胞定位表明ThZFL蛋白是定位在细胞壁中的蛋白。
将ThZFL基因克隆到酵母表达载体pYES2中,转化酿酒酵母(Saccharomyces cerevisiae)中,空载体pYES2转酵母为对照。分别用NaCl和甘露醇胁迫处理转ThZFL与对照酵母,表明ThZFL基因对盐和渗透有抵抗能力。
为进一步研究ThZFL的功能,用农杆菌介导法将ThZFL基因转入烟草中,以GUS基因作为对照,得到了转基因烟草。PCR、Southern杂交和Northern杂交检测表明ThZFL和GUS基因分别整合到烟草基因组中,并能够表达。
选取3个转ThZFL基因株系的种子及叶片进行了非生物胁迫实验,转GUS基因及非转基因烟草作为对照。分析逆境胁迫条件下非转基因、转GUS基因植株和转ThZFL基因植株种子发芽率、相对生长量及丙二醛(MDA)含量。结果显示,胁迫条件下,转ThZFL基因的发芽率、相对生长量明显高于对照,丙二醛含量低于对照。这些结果表明ThZFL的过表达提高了转基因植株的抗盐和耐渗透能力。
This study served to isolate a Zinc-finger-like gene(ThZFL) from the Tamarix hispida roots cDNA library in response to salt treatments. The length of the ThZFL gene was 514bp. The gene open reading frame(ORF) was 381bp in length that was from 103 of ATG to 421 TAA. The ThZFL gene encodes a predicated polypeptide of 105 amino acid with a molecular weight off 11.25kDa and a pI of 9.97. Analyzing the gene by bioinformatics indicated the ThZFL gene has low sequence homology to animal Zinc-finger genes.
Expression of ThZFL in Tamarix in response to abiotic stress was studied by Northern blot. The results showed that ThZFL was response to treatments of salt and osmotic in T.hispida roots and ThZFL was induced by ABA treatment. Subcellular localization ananlysis showed that the ThZFL protein is localized in the cell wall.
The ThZFL gene was inserted into pYES2 and transformed into yeast cells(Saccharomyces cervisiae). Yeast cells transformed and pYES2 empty vector were stressed by different abiotic stresses. The results demonstrated that ThZFL exhibits a wide range of abiotic stresses.
To further characterize the function of ThZFL in plant, we transformed ThZFL gene into tobacco by using Agrobacterium mediated transformation.Furthermore, the GUS gene was transform into tobacco as the control.The transgenic lines were examined by PCR、Northern hybridization and Southern hybridization, confirming that ThZFL gene and GUS gene have been integrated int the genome of tobacco, the foreign ThZFL gene and GUS gene were successfully expressed in all the transgenic lines.
Based on the above results, tree transgenic lines were selected for investigating the tolerance of abiotic stresses, the GUS gene transgenic line and WT line as contol. Germination percentage of the seeds,relative growth rate and activity of MDA were measured and analysised under different abiotic stresses. The results showed that under abiotic stresses, germination percentage of seeds and relative growth rate in all transgenic lines were higher than that of the control. The MDA content was lower than that of the control. Theses results showed that overexpression of ThZFL in transgenic tobacco increased tolerance to salt and osmotic.
引文
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