枯草芽孢杆菌(Bacillus subtilis)cspB基因转化烟草(Nicotiana tabacuum)的研究

The Study of Tobacoo Transformation with cspB Gene from Bacillus subtilis

细菌冷休克蛋白cspB是原核生物RNA的分子伴侣,含有原始型的冷休克结构域,具有与核酸结合功能,可以防止RNase对mRNA的降解,也能纠正mRNA的折叠错误。为了寻找作物改良的潜在基因资源,从枯草芽孢杆菌XS-01基因组中克隆出cspB基因,并与pBI121构建成pBI121-cspB植物表达载体。利用叶圆盘转化法转化烟草,通过卡那霉素筛选和PCR、Southern杂交鉴定5个转化体株系。除TN010外,其他转基因株系在外观形态与野生亲本的没有差别,但TN001和TN011育性降低,TN010和TN012则完全丧失育性。干旱处理结果表明,转基因植株在土壤水分恢复后10 d,其平均单株干物质质量较之野生亲本的显著增加;叶片光合速率测定结果表明,在干旱处理时,转基因植株和对照亲本叶片光合速率均显著下降,在土壤水分恢复后,转基因植株的光合速率能快速恢复,但对照亲本的恢复缓慢。实验结果说明,cspB能够促进植物细胞从逆境伤害中快速恢复功能。

cspB, one of RNA chaperon in bacteria, has a prototypical cold shock domain（CSD） with a polynucleotide binding function which prevents mRNA from digestion by RNase and rescues misfolded mRNA. In search of candidate genes for crop improvement, cspB gene was cloned from the genome of Bacillus subtilis 168 and ligased to pBI121 to form pBI121-cspB construct. With leaf disc transformation protocol, 5 transformant plants were selected by Kanamycin and i- dentified by PCR and Southern blot. There are no difference between the transformant and the wild type plant in appear- ance except TN 010. However, fertility of TN 001 and TN011 decreased, and TN010 and TNO12 were completely ster- ile. In the tests of resistance to drought stress, the mean dry matter weight of each transfomant was significantly higher than that of wild type plant 10 d after soil water recovery. Meanwhile, the test results also showed that photosynthetic rate of leaves in transformts and wild type plants both dramatically decreased under soil water-deficit condition. When soil water was restored, photosynthetic rate of leaves increased more quickly in transformants compared with wild type plants. The results indicate that cspB promotes function recovery from stress harms for plant cell.