香蕉4-CL基因Mu4CL15的功能鉴定

Functional Identification of Mu4CL15 in 4CL Gene in Banana

在香蕉基因组中寻找具有抗倒伏功能的基因,为培育抗倒伏香蕉新品种奠定基础。香豆酸:Co A连接酶(4CL)是植物木质素合成的限速酶。香蕉A基因组中有25个4CL基因同源序列。高粱基因bmr2是一个已经被鉴定的具有4CL功能的基因。与bmr2进行序列比对,将候选基因在大肠杆菌中过量表达,提取重组蛋白,研究其催化活性。将候选基因在烟草中过量表达,研究转基因植株与对照的木质素含量和茎秆物理强度。香蕉4-香豆酸:Co A连接酶基因Mu4CL15预测的底物结合域序列、二级结构、三级结构都与bmr2的相应序列具有高度的相似性。将Mu4CL15在大肠杆菌中表达并提取纯化,重组蛋白质催化效率最高的底物是4-香豆酸,其次是咖啡酸、阿魏酸和芥子酸。将Mu4CL15在烟草中进行转化后发现,转基因植株茎秆的木质素含量显著高于只转化空载体的野生型。转基因幼苗的茎秆强度显著高于只转化空载体的野生型。在烟草中过量表达Mu4CL15能够增加烟草植株茎秆的木质素含量和抗倒伏能力。通过过量表达基因Mu4CL15极有可能能够提高香蕉植株假茎的抗倒伏能力。

To find lodging resistance genes from banana genome, it will make a foundation for breeding new banana varieties with lodging resistance. 4-coumarate： CoA ligase （4CL） was a rate-limiting enzyme for lignin sy- nthesis. There were 25 4CL genes in banana A genome. Sorghum gene bmr2. has been identified to have 4CL function. To compare with the sequence bmr2, the candidate gene was over expressed in E. coli, recombinant protein was extracted and the catalytic properties of the recombinant protein was studied. The candidate gene was over expressed in tobacco. The lignin content and the physical strength of the transgenic plant and the control group were studied. Banana 4-coumaric acid： CoA ligase gene Mu4CL15 has been identified to have highly similar substrate-binding-pocket motifs, secondary structural pattern and 3D structures with bmr2. Mu4CL15 was expressed and operated with extraction and purification. The substrate that Mu4CL15 preferred could be ordered as the followed： 4-coumaric acid, caffeic acid, ferulic acid and sinapic acid from the most to the least. After Mu4CL15 was transformed into tobacco, the lignin content in the stem of the transgenic plant was significantlyhigher than that of the wild type. The stem strength of the transformed seedlings was higher than that of the wild type. Over-expressing Mu4CL15 could increase plant＇s lignin content and lodging resistance. Banana varieties with high lodging resistance might be cultivated by transforming Mu4CL 15.