小麦脱水素wzy1-2基因的遗传转化及干旱胁迫下不同生育期表达水平

根据同源分析,在脱水素wzy1-2基因序列中选择298bp的cDNA序列作为干扰片段,将其插入高效植物干扰表达载体pTCK303的多克隆位点,成功构建含反向重复序列的RNA干扰表达载体。采用基因枪法轰击2512个郑引1号小麦的幼胚愈伤组织,获得再生植株26株,对其T_0代再生植株进行特异引物PCR检测,获得6株阳性植株,阳性转化率为0.24%。对脱水素wzy1-2基因实时定量分析发现,小麦中目的基因表达量显著下降。为进一步了解该基因在小麦整个生长过程中表达模式,选取2个不同抗旱性小麦品种(陕合6号和郑引1号),分别在小麦4个不同生长时期(苗期、分蘖期、拔节期和开花期)进行干旱胁迫处理,分析脱水素wzy1-2基因的表达规律。结果表明,随着干旱程度增加小麦WZY1-2蛋白的表达量升高;苗期小麦WZY1-2蛋白的表达量均高于其它3个生长时期。Western blot分析显示陕合6号和郑引1号的非特异性条带单一,表明小麦脱水素wzy1-2是干旱诱导型基因。

Genetic transformation of wheat with dehydrin wzy1-2 gene and the expression level at different growth stages under drought stress

Based on the principle of RNA interference and the homology analysis, a 298 bp cDNA sequence between 0~298 bp in the dehydrin wzy1-2 gene was selected as the interference fragment, and then cloned interfering fragment was inserted into the efficient plant expression vector pTCK303, clone site, to successfully constructed RNA interference expression vectors containing inverted repeats. Using immature embryos of Zhengyin 1 wheat as explants, 2512 immature embryo calli was bombarded by particle bombardment, and 26 regenerated plants were obtained with a regeneration rate of 1.03%. Using the specific primers of the Hpt gene on the expression vector, 26 T₀ generation regenerated plants were subjected to PCR analysis. Six Hpt gene positive plants were detected, and the positive conversion rate of the Hpt gene was 0.24%. Through quantitative real\|time analysis of the dehydrin wzy1-2 gene, it was found that the expression of the target gene significantly decreased. To lay the foundation for further study of dehydrin wzy1-2 gene function, two drought tolerant wheat varieties(Shaanhe 6 and Zhengyin 1), were treated with drought stress at four growth stages (seedling, tillering, jointing, and flowering), and expression of wheat WZY1-2 protein was studied. The expression level of WZY1-2 protein in wheat incread with increasing drought degree and at the seedling stage it was higher than that in the other three growth stages. Western blot results showed that the size of the non\|specific bands of Shaanhe 6 and Zhengyin 1 were different. The wheat wzy1-2 derived from the molecular level was a drought\|inducible gene.