Cloning of an H+-PPase gene from Thellungiella halophila and its heterologous expression to improve tobacco salt tolerance
Cloning of an H+-PPase gene from Thellungiella halophila and its heterologous expression to improve tobacco salt tolerance
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DOI：
10.1093/jxb/erl090
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发表时间：
2006-09-01
影响因子：
6.9
通讯作者：
Zhang, Juren
中科院分区：
文献类型：
--
作者：
Gao, Feng;Gao, Qiang;Zhang, Juren
从盐藻(Thellungiella Halophila)中克隆了一个与基本生化和生理机制有关的H+焦磷酸酶(PPase)基因，命名为TSVP。该翻译产物在生物信息学方面与拟南芥和水稻等其他物种的H+-PPase具有相似的特征。TSVP在酵母突变体ena1中的异源表达抑制了Na+过敏性，证实了TSVP作为H+-PPase的功能。在300 mM盐胁迫下，转TSVP基因烟草的干重比野生型烟草高60%，叶肉原生质体在盐激胁迫下的存活率也较高。TSVP和AVP1是另一种来自拟南芥的H+-PPase，分别在酵母突变体ena1和烟草中异源表达。TSVP或AVP1酵母转化子与转基因烟草的耐盐性提高到几乎相同的水平。对H+-PPase水解酶活性最高的转基因烟草品系TL3和TL5进行了进一步研究。在盐胁迫条件下，转基因烟草比野生型植株积累的溶质多25%，Na+多20%~32%。盐胁迫条件下，转基因烟草品系TL3和TL5虽然积累了较多的Na+，但丙二醛含量和细胞膜损伤均小于野生型。据推测，液泡中Na+的区域化减少了它对植物细胞的毒害作用。这一结果支持了过表达H+-PPase导致Na+在液泡中而不是在细胞质中积累的假说，从而避免了过量Na+对植物细胞的毒害。
An H+-pyrophosphatase (PPase) gene named TsVP involved in basic biochemical and physiological mechanisms was cloned from Thellungiella halophila. The deduced translation product has similar characteristics to H+-PPases from other species, such as Arabidopsis and rice, in terms of bioinformation. The heterologous expression of TsVP in the yeast mutant ena1 suppressed Na+ hypersensitivity and demonstrated the function of TsVP as an H+-PPase. Transgenic tobacco overexpressing TsVP had 60% greater dry weight than wild-type tobacco at 300 mM NaCl and higher viability of mesophyll protoplasts under salt shock stress conditions. TsVP and AVP1, another H+-PPase from Arabidopsis, were heterologously expressed separately in both the yeast mutant ena1 and tobacco. The salt tolerance of TsVP or AVP1 yeast transformants and transgenic tobacco were improved to almost the same level. The TsVP transgenic tobacco lines TL3 and TL5 with the highest H+-PPase hydrolytic activity were studied further. These transgenic tobacco plants accumulated 25% more solutes than wild-type plants without NaCl stress and 20-32% more Na+ under salt stress conditions. Although transgenic tobacco lines TL3 and TL5 accumulated more Na+ in leaf tissues, the malondialdehyde content and cell membrane damage were less than those of the wild type under salt stress conditions. Presumably, compartmentalization of Na+ in vacuoles reduces its toxic effects on plant cells. This result supports the hypothesis that overexpression of H+-PPase causes the accumulation of Na+ in vacuoles instead of in the cytoplasm and avoids the toxicity of excessive Na+ in plant cells.