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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1131bp,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/LNaCl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。
In this paper, total RNA was extracted from leaves of Kandelia candel using Kandelia candel seedlings treated with 200 mmol / L NaCl for 24 h. The full-length cDNA of KcRD22 gene was cloned by RT-PCR. The pCAMBIA-2300-KcRD22 overexpression vector was constructed, Way to get over-expression of KcRD22 tobacco transgenic lines, and salt-tolerant transgenic lines to make a preliminary analysis. The experimental results showed that the ORF of KcRD22 gene was 1131bp in length and encoded a protein with an isoelectric point of 9.07 and a molecular weight of 39.8kD consisting of 375 amino acids. The results of PCR and RT-PCR showed that the KcRD22 gene was integrated into the chromosomes of eight tobacco plants and expressed in two strains. The photosynthesis of transgenic tobacco was measured. The results showed that 100 mmol / L NaCl treatment significantly reduced the net photosynthetic rate of wild-type tobacco, while the photosynthesis of transgenic plants was less affected. When the salt concentration reached 200mmol / L, the net photosynthetic rate of transgenic plants and wild type tobacco were significantly reduced, but after salt stress was relieved, the recovery of photosynthesis of transgenic tobacco was significantly better than that of wild type tobacco, indicating that KcRD22 overexpression increased the photosynthetic rate Salt resistance. This paper initially determined the contribution of KcRD22 gene to salt tolerance in plants, which laid a good foundation for further study on the function of this gene in salt tolerance mechanism.