青天葵中SKP1同源基因的克隆及原核表达

Cloning and prokaryotic expression of SKP1 homologous genes from Nervilia fordii

目的从青天葵Nervilia fordii克隆SKP1的同源基因NSKs,并对编码的蛋白进行生物信息学分析及原核表达,为了深入研究青天葵中SCF复合体的功能奠定基础。方法从青天葵转录组中筛选得到5条SKP1同源基因序列(NSK2、NSK3、NSK5、NSK7、NSK11),构建pGEX-4T-NSKs原核表达载体于大肠杆菌Rosetta (DE3)中诱导表达GST-NSKs融合蛋白并进行纯化。结果 NSK2、NSK3、NSK5、NSK7、NSK11的开放阅读框(ORF)分别为495、483、489、498和486 bp,编码164、160、162、165和161个氨基酸,蛋白相似性达到73.65%,均属于SKP1蛋白家族,具F-box和Cullin蛋白结合位点。亚细胞定位预测NSK2、NSK3和NSK5定位于细胞核,而NSK7和NSK11定位于细胞质和细胞核。系统进化树结果表明,NSK2、NSK3和NSK5聚为一簇,与小麦和玉米等的同源性较高;NSK7和NSK11聚为一簇,与多头绒泡菌的同源性较高。通过构建原核表达载体,确定了在大肠杆菌Rosetta (DE3)中的有利诱导条件,成功诱导表达并纯化得到5个GST-NSKs重组蛋白。结论成功克隆5个SKP1同源基因NSKs,获得NSKs蛋白,为深入研究青天葵中SCF复合体的功能提供了一定的基础。

Objective In order to study the function of SCF complex in Nervilia fordii,five SKP1 homologous genes,the NSKs were cloned,and the encoded proteins were analyzed by bioinformatics,and the recombinant proteins were obtained by prokaryotic expression. Methods Sequences of five SKP1 homologous gene were screened from the N. fordii transcriptome,designated NSK2,NSK3,NSK5,NSK7,and NSK11. The open reading frames(ORFs) of these five genes were amplified by RT-PCR using specific primers. The genes were introduced into the prokaryotic expression vectors p GEX-4 T-NSKs for expression of the recombinant proteins in Escherichia coli Rosetta(DE3) cells under IPTG induction. Results The ORFs of the NSK genes were 495,483,489,498,and 486 bp,encoding 164,160,162,165,and 161 amino acids,respectively. The protein similarity reached 73.65%,all belong to the SKP1 protein family,containing F-box and Cullin protein binding sites. The prediction of subcellular locations showed that NSK2,NSK3,and NSK5 were located in the nucleus,while NSK7 and NSK11 were located in the cytoplasm and nucleus. The phylogenetic tree showed that NSK2,NSK3,and NSK5 clustered together with Triticum aestivum and Zea mays,and NSK7 and NSK11 clustered together sharing high homology with Phytophthora polycephalum. By constructing a prokaryotic expression vector,the favorable induction conditions in E. coli Rosetta(DE3) were determined,and GST-NSKs recombinant proteins were successfully induced and purified. Conclusion Five SKP1 homologous genes,NSKs were cloned and the correspondent NSK proteins were obtained,which provided a reference for further study on function of SCF complexes in N. fordii.