摘要
【目的】类受体蛋白激酶在植物的生长发育及果实成熟过程中发挥重要作用,而植物中已鉴定的类受体激酶绝大多数属于LRR型。为了得到草莓中LRR型蛋白激酶Fa RIPK1,从而进一步研究其功能。本实验采用真核表达的方法诱导表达Fa RIPK1蛋白质,以期确定其最佳的诱导表达时间。【方法】以‘童子一号’草莓果实为试验材料,扩增出Fa RIPK1基因的功能区序列,构建真核表达载体p PICZB-RIPK1后转入酵母表达菌株X-33诱导表达,通过在0,12,24,36 h处取样检测Fa RIPK1蛋白质的表达情况并采用Ni-NTA琼脂糖树脂亲和层析柱纯化得到目的蛋白,纯化后的Fa RIPK1蛋白质再进行Western-blot验证。【结果】重组表达菌株经过BMMY诱导培养基诱导24 h后Fa RIPK1蛋白质的表达量达到最高值,此时最利于Fa RIPK1蛋白质的纯化。Western-blot验证表明所得蛋白质确为Fa RIPK1蛋白质。【结论】确定了诱导表达后24 h为Fa RIPK1蛋白质真核表达的最佳时间,为进一步研究Fa RIPK1蛋白质的功能奠定基础。
【Objective】Receptor like protein kinases play an important role in plant growth,development and fruit ripening. Most receptor like kinases which have been identified belong to LRR type. The Fa RIPK1 protein of strawberry belongs to LRR protein kinases. To get the Fa RIPK1 protein,we expressed the Fa RIPK1 protein by eukaryotic expression system and identified the optimal expression time.【Methods】In this study,we cloned the function sequences of Fa RIPK1 gene from the fruits of‘tong zi yi hao＇,constructed eukaryotic expression vector p PICZB-RIPK1,and transformed p PICZB-RIPK1 into yeast strain X-33 to express the Fa RIPK1 protein. Then we detected the expression of Fa RIPK1 in 0,12,24,36 h to identify the optimal expression time,used Ni-NTA resin agarose to purify protein and verified the Fa RIPK1 protein by Western-blot. 【Results】The highest expression of Fa RIPK1 protein in recombinant strain was appeared after 24 h on BMMY induction medium. It was the best time to purify Fa RIPK1 protein. And the purified protein was identified by Western-blot. 【Conclusion】We cleared that 24 h is the best time for eukaryotic expression of Fa RIPK1 protein. It provided a basis for further study on Fa RIPK1 protein＇s function.
出处
《北京农学院学报》
2016年第2期42-45,共4页
Journal of Beijing University of Agriculture
基金
北京市属高等学校创新团队建设与教师职业发展计划项目(IDHT20140509)
科技北京百名领军人才培养计划
