玉米耐热DNA聚合酶基因表达载体构建与花粉介导转化
热稳定性DNA聚合酶是PCR反应中的重要试剂,但是国内尚未报道有关热稳定性DNA聚合酶基因用于转基因的基因工程研究。若将此耐热基因转入某些经济作物中培育耐热新品种,将会有很好的发展前景和实用价值。本实验将构建热稳定性聚合酶的基因表达二元载体。利用转基因方法将热稳定性DNA聚合酶基因利用花粉介导法导入玉米幼胚,获得耐热转基因玉米株系,这将有利于增加经济价值和减少农作物由于热压力造成的损失。首先,我们用热稳定性 DNA聚合酶基因构建表达载体；然后利用花粉介导法进行转化,花粉收集自仲恺自交系玉米甜一号和自交系糯2号,将花粉与构建好的基因表达载体混合,利用花粉介导法进行转化。第三,收集到的T1代种子用PCR凝胶电泳技术和Southern 杂交技术进行检测,选出阳性的转化株。最后,在T2代经过田间耐热压力试验结合测定的玉米生理指标筛选出9个具有良好耐热性玉米株系。
英文摘要:
Thermus aquaticus (Taq) DNA polymerase has been extensively used in DNA amplification procedures, but its application in plant transgenic has not yet been reported. In the present study, we used Taq DNA polymerase gene to develop new heat-tolerant maize lines, which will potentially add to the economic value and aid in controlling crop losses due to heat stress. First, we established an expression vector with Taq DNA polymerase gene. Then, according pollen-mediated transformation, pollen from the maize ZhongKai inbred lines, Zhong sweet No.1 and Zhong glutinous No.2 was collected, which was mixed with the established expression vector and was applied to the maize silks for transformation. Thirdly, the harvested T1 generation seeds were confirmed to be positive transformants by PCR and Southern hybridization. Finally, after screening the T2 generation, by exposure to heat stress conditions in the field combined with physiological indexes, we obtained nine transformed heat-tolerant maize lines.
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