秋茄KcRD22基因的克隆与功能分析

本文以用200mmol／LNaCl处理24h后的秋茄幼苗为材料提取秋茄叶片总RNA，利用RT-PCR方法克隆获得KcRD22基因的全长cDNA，通过构建pCAMBIA-2300-KcRD22过表达载体，利用农杆菌侵染的方式获得过表达KcRD22的烟草转基因株系，并对转基因株系的耐盐性做出初步分析。实验结果显示：KcRD22基因的ORF长1131bD，编码1个等电点为9．07、分子量为39．8kD、由375个氨基酸组成的蛋白。PCR及RT—PCR鉴定结果表明，KcRD22基因已经分别整合到8株烟草的染色体中，并在两个株系中获得表达。对转基因烟草进行光合作用测定，结果显示100mmol／LNaC1处理显著降低了野生型烟草的净光合速率，而转基因植株叶片的光合作用受到的影响较小。盐浓度达到200mmol／L时，转基因植株及野生型烟草净光合速率都明显降低，但盐胁迫解除后，转基因烟草光合作用的恢复情况明显好于野生型烟草，说明KcRD22的过表达提高了烟草的抗盐性。本文初步确定了KcRD22基因对植物耐盐性的贡献，这为进一步深入研究该基因在耐盐机制中的功能奠定了良好基础。

Cloning and Functional Analysis ofKcRD22 Gene ofKandelia candel

By using RT-PCR, the full length cDNA ofKcRD22 gene was cloned from leaves ofKandelia candel seedlings which were treated with 200 mmol/L NaC1 solution for 24 hours. Expression vector ofpCAMBIA-2300- KcRD22 was successfully constructed and introduced into tobacco by the A grobctcterium mediated transformation system. And the salt-resistant phenomenon of the transgenic lines was analyzed. Our results indicated that the ORF of KcRD22 had a size of 1 131 bp, encoding for a protein of 375 amino acids with an isoelectric point of 9.07 and a molecular weight of 39.8 kD. The PCR data showed that we successfully got 8 transgenetic lines. While in two of these lines, KcRD22 was detected over-expressed compared to widetype based on RT-PCR results. Furthermore, net photosynthetic rate of transformation plants were higher than that of wild-type plants under 100 mmol/L and 200 mmol/L of NaCl stress although both their leaf photosynthesis was reduced by salt treatment. It was noteworthy that the photosynthetic rate of the transgenic plants recovered faster than that of the wildtype plants after the salinity relief. Our results suggested that KcRD22 gene played an important role in plant salt tolerance. In conclusion, our study demonstrated that KcRD22 gene cloned from Kandelia candel was able to enhance the capacity for salt tolerance of model plants, although the role of KcRD22 in salt resistance needed further study.