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多基因共转化有利于对植物复杂性状进行改良。本研究通过在pXCS-HAStrep质粒中引入一段间隔序列,构建了双基因共表达载体pXCS-Dgene-HAStrep,实现了在一套表达盒内独立表达两个基因,并将克隆自橡胶树(Hevea brasiliensis)的HbWRKY11和HbWRKY7基因克隆入该载体,利用农杆菌(Agrobacterium tumefacien)介导法将构建好的共表达载体pXCS-HbWRKY11-HbWRKY7-HAStrep转入野生型拟南芥(Arabidopsis thaliana),共获得了84株抗Basta除草剂的拟南芥转基因植株。对随机选择的8株转基因植株进行半定量和荧光定量PCR分析表明,HbWRKY11和HbWRKY7基因能同时独立表达,HbWRKY7的表达量略高于HbWRKY11,但差异不显著(Pr=0.4867>0.05);而在不同的转基因植株间,基因的表达差异显著,其中单拷贝的转基因株系S2和S5表达量最高。对双基因共表达株系S2和S5及HbWRKY11和HbWRKY7的单基因转基因株系L3、L4、J1和J5进行低温和茉莉酸胁迫处理,结果表明,冷应答基因AtCOR47和茉莉酸应答基因AtCOI1及可溶性糖含量在双基因共表达株系中的变化特征与其在单基因转基因株系中的变化特征均表现出明显的差异。实验证明,本研究所构建的双基因共表达载体可以在植物转化中实现一个表达框内独立表达两个基因。
Multi-gene co-transformation is conducive to the improvement of plant traits. In this study, a double-gene co-expression vector pXCS-Dgene-HAStrep was constructed by introducing a spacer into the pXCS-HAStrep plasmid. Two genes were independently expressed in a set of expression cassettes and cloned from Hevea brasiliensis HbWRKY11 and HbWRKY7 genes were cloned into this vector. The constructed co-expression vector pXCS-HbWRKY11-HbWRKY7-HAStrep was transformed into wild-type Arabidopsis thaliana by using Agrobacterium tumefacien-mediated method to obtain 84 Arabidopsis thaliana transgenic plants resistant to Basta herbicides. Semi-quantitative and quantitative PCR analysis of eight randomly selected transgenic plants showed that both HbWRKY11 and HbWRKY7 genes expressed independently at the same time. The expression level of HbWRKY7 was slightly higher than that of HbWRKY11, but the difference was not significant (Pr = 0.4867> 0.05) Among different transgenic plants, the expression of genes was significantly different, of which the single copy copy number of transgenic lines S2 and S5 were the highest. The cryogenic and jasmonic stress treatment of transgenic lines L3, L4, J1 and J5 with double gene co-expression lines S2 and S5, HbWRKY11 and HbWRKY7 were carried out. The results showed that cold stress gene AtCOR47 and jasmonic acid responsive gene AtCOI1 and soluble The characteristics of sugar content in the double gene co-expression lines and their characteristics in single-gene transgenic lines showed significant differences. The experiment proves that the double gene co-expression vector constructed in this study can express two genes independently in one expression box in plant transformation.