表达外源DsCOR转基因拟南芥的筛选及其抗寒性

Heterologous expression of DsCOR enhances cold resistance in Arabidopsis

为验证播娘蒿抗寒基因DsCOR的抗寒功能,在实验室前期DsCOR基因大量研究的基础上,本研究通过农杆菌介导花序浸染法把诱导型表达载体PBI121-DsCORpro681-DsCOR转入COR15a缺陷型拟南芥中(A-S),筛选获得转DsCOR基因阳性苗(A-I),并测定了A-I、A-S和野生型(A-W)拟南芥在4℃低温胁迫下的叶片脯氨酸含量、电解质渗透率和幼苗在-4℃下存活率等抗寒生理指标;使用Real-time quantitative RT-PCR检测A-I在低温胁迫下DsCOR基因的表达规律.结果显示,A-W和A-I两种植株叶片脯氨酸含量在低温处理过程中显著增加,从处理前70μg g^(-1)到处理后12h分别升至453μg g^(-1)和406μg g^(-1)并趋于稳定,A-S叶片脯氨酸含量变化不大(30^(-1)05μg g^(-1)).随着处理时间增加,A-S叶片电解质渗透率升高较明显,36 h后达到45.2%;而A-W和A-I两种植株叶片电解质渗透率最高分别达到14.3%和16.3%.幼苗存活率统计结果显示,A-S缺失COR15a后,在-4℃处理后该株系幼苗全部死亡,而A-W和A-I两个株系都有较高的存活率,分别达到53%和83%.抗寒生理指标结果表明转DsCOR基因拟南芥的抗寒能力已得到显著提高,达到野生型拟南芥水平.Real-time quantitative RT-PCR检测显示DsCOR基因在A-I中表达明显受到低温胁迫诱导.综上,DsCOR基因在COR15a缺陷型拟南芥中实现了成功表达,并显著提高了其抗寒性,最终验证了播娘蒿抗寒基因DsCOR的抗寒功能.

This study aimed to identify the cold-resistance function of Descurainia sophia COR gene（DsCOR）. On the basisof many research about DsCOR gene in our lab before thisstudy, the inducible DsCOR expression transgenic vector PBI121-DsCORpro681-DsCOR wastransferred into COR15 a defect mutantsof Arabidopsisthaliana（A-S） by the way of Agrobacterium-mediated inf lorescence infection. One positive seedling named asA-I wasobtained by selection based on kanamycin and PCR. Cold-resistance physiological propertiesincluding free proline content, leaf ion leakage and seedlingssurvival rate were determined to assessthe cold-resistance abilitiesof the wild type A. thaliana（A-W）, A-S and A-I under 4 °C treatment. Real-time q uantitative RT-PCR wasused to detect the expression pattern of DsCOR in A-I before and after the chilling treatment. The free proline content of A-S kept stable（30（-1）05 μg g（-1）） during the chilling treatment, while that of A-W and A-I increased significantly up to 453 and 406 μg g（-1） respectively after 12 h. The leaf ion leakage of A-S increased dramatically compared to that of A-W and A-I, to about 45.2% after 36 h chilling treatment. The statistical result of seedlingssurvival ratesshowed that all A-S seedlingsdied after-4 °C chilling treatment, but A-W and A-I seedlingshad high survival rates, about 53% and 83% respectively. Real-time quantitative RT-PCR showed that the expression of DsCOR wasobviously induced by chilling treatment in A-I. Thisresearch achieved inducible DsCOR expression in COR15 a defect mutantsof A. thaliana, which enhanced cold-resistance abilitiesof transgenic A. thaliana. The resultsconfirmed the cold-resistant function of DsCOR gene in Arabidopsisthaliana.