紫花苜蓿柠檬酸合成酶基因的克隆及对烟草的转化

Cloning of MsCS Gene from Medicago sativa L. and Transformation of Tobaccos

柠檬酸合成酶是调节植物体内有机酸的活性酶之一,增加植物体内的柠檬酸可以提高植物对土壤中磷的吸收以及抗铝毒性。通过从紫花苜蓿(Medicago sativaL.)中克隆柠檬酸合成酶基因(MsCS),以期获得该基因全长,并为将来研究紫花苜蓿耐铝性以及对磷的吸收方向提供基础。首先利用cDNA末端快速扩增方法(RACE),克隆获得MsCS的cDNA全序列,然后利用pBI121构建植物超表达载体pBI-MsCS,通过农杆菌介导的叶盘法转化烟草(Nicotiana tabacumL.)。结果显示:该基因序列全长为2031 bp,开放阅读框1551 bp,编码517个氨基酸,与其他物种的柠檬酸合成酶具有高度的同源性。获得17株卡那抗性植株。经PCR检测有6株为阳性植株,初步表明该基因已整合到烟草的基因组中。

The citrate synthase,an active enzyme,can regulate organic acids in plants.Increasing citric acid can improve plant capabilities of absorbing soil phosphorus and also affect resistance to aluminum toxicity.The gene named MsCS was cloned from alfalfa（Medicago sativa L.） for alfalfa to proceed with basic research of aluminum tolerance and soil absorption for phosphorus.First a full-length cDNA of MsCS was cloned by rapid amplification of cDNA ends（RACE）.Then the plant expression vector named pBI-MsCS was constructed on pBI121 vector,and transformed the tobacco by using agrobacterium-mediated.Results indicate that the length of cDNA sequence was 2031 bp and included a 1551 bp open reading frame which encoded a deduced protein of 517-amino-acid polypeptide.The amino acid sequence comparison revealed high homology with citrate synthase of other plants.We obtained a total of 17 kanamycin resistant transgenic plants.PCR detection confirmed 6 positive plants,preliminary studies showed that the MsCS had been integrated into the tobacco genome.