冬凌草IrCYP71基因的克隆和功能

Gene Cloning and Functional Characterization of IrCYP71 Gene in Isodon rubescens Leaves

目的：克隆冬凌草二萜类化合物生物合成途径下游关键酶基因Ir CYP71,进行序列特征分析,对此基因编码的蛋白进行原核表达分析以及亚细胞定位,并对蛋白在宿主细胞表达的条件进行了优化。方法：根据转录组测序所得的Ir CYP71基因片段,克隆出全长c DNA序列;构建p ET28a（＋）-Ir CYP71重组质粒,转化到Rosetta感受态细胞,小量表达和大量表达蛋白并鉴定,进行包涵体蛋白的纯化与复性,再对复性蛋白纯化鉴定分析。基于gateway克隆技术构建出PCR8/GW/TOPO-Ir CYP71载体之后与改造Pearleygate104载体重组,之后与农杆菌GV3101重组,转入烟草中瞬时表达,从而进行蛋白亚细胞定位。结果：克隆得到的Ir CYP71基因全长c DNA为1 593 bp,编码530个氨基酸,并在Gen Bank注册（登录号MG800628）。经大肠埃希菌表达系统表达的重组蛋白相对分子质量正确,在62 k Da左右,纯化后的重组蛋白总量有1 mg,蛋白纯度为85%。此蛋白亚细胞定位在细胞核。结论：对冬凌草Ir CYP71基因进行了初步验证,并对基因表达的蛋白进行原核表达和亚细胞定位,使该基因在冬凌草二萜成分的生物合成过程中的作用得到了进一步的阐述。

Objective： To clone the downstream key enzyme Ir CYP71 gene in diterpenoids biosynthesis of Isodon rubescensfor sequence analysis,carry out prokaryotic expression analysis and subcellular localization for the protein encoded by this gene,and optimize the conditions for protein expression in host cells. Method： The fulllength c DNA sequence was cloned according to the Ir CYP71 gene fragment obtained from transcriptome sequencing;the recombinant plasmid of p ET28 a（ ＋）-Ir CYP71 was constructed and transformed into Rosetta receptive cells,small amount and large amount expressed proteins before identification. The inclusion body proteins were purified and renaturated,and then the renaturated proteins were purified,identified and analyzed. refolding of inclusion body protein,and analyzed the purification and identification of complex protein. The vector PCR8/GW/TOPOIr CYP71 was constructed by gateway cloning technology,recombined with transformed Pearleygate104 vector,and then introduced into tobacco epidermal cells by agrobacterium-mediated（ GV3101） transformation for protein subcellular localization. Result： The full-length c DNA of cloned Ir CYP71 gene was 1 593 bp,encoding 530 amino acids,whichwas registered in Ge Bank（ Accession No. MG800628）. The recombinant protein expressed via Escherichia coli showed relatively correct molecular weight, about 62 k Da. The total amount of purified recombinant protein was 1 mg and the protein purity was 85%. Green fluorescence was tested and targeted to nucleus under a laser scanning confocal microscope. Conclusion： The preliminary validation of Ir CYP71 gene in I. rubescens revealed the prokaryotic expression and subcellular localization of the expressed proteins, laying foundation for further elucidating the function of the gene inditerpenoidsbiosynthesis.