花生抗黄曲霉相关ARAhPR10基因克隆及其原核表达

Cloning and Prokaryotic Expression of ARAhPR10 Gene with Resistance to Aspergillus flavus in Peanut

花生黄曲霉污染已成为制约我国花生及花生制品出口贸易的关键因素。基于花生抗黄曲霉相关EST序列,RT-PCR法克隆花生ARAhPR10基因(gb|EU661964.1),开放读码框471bp,编码157个氨基酸,分子量16.9kD,等电点5.03。与已报导AhPR10(gb|AY726607)相似性为49.3%。推导氨基酸序列含有PR10家族的高度保守"P-loop"基序(G*GG*G)和Betv1保守疏水结构域"GVALP PTAEK ITFET KLVEG PNGGSIGKLT LKY",推测ARAhPR10是该花生PR10家族的新成员。其基因组扩增序列长度561bp,两个外显子间存在一个长度为87bp的内含子。原核表达ARAhPR10的融合蛋白约25kD,Ni+-NTA树脂亲和纯化后获得电泳单一条带。纯化的ARAhPR10融合蛋白具有体外核酸酶活性,预测其具有抗黄曲霉活性。该基因的克隆及其原核表达融合蛋白的获取为ARAhPR10功能研究奠定了基础。

A crucial factor restricting the export of the peanut produce in our country was some peanut with the aflatoxin contamination. Based on the EST related Aspergillus flavus resistant, we obtained a cDNA sequence of ARAhPR10（gb｜EU661964.1） from the peanut by RT-PCR. The cDNA of ARAhPR10 contained a single ORF spanning 471bp. The ORF encoded a predicted polypeptide of 157 amino acid residues, with a predicted molecular mass of 16.9kD and pI of 5.03. Sequence alignment, using DNAStar MegAlign, of the predicted amino acid residues of ARAhPR10 in different members of the PR10 family clearly established that ARAhPR10 had similarity with other PR10 proteins. ARAhPR10 showed high identity with the reported AhPR10（gb｜AY726607） by 49.3% and with other PR10 proteins above 40%. The deduced amino acids sequence contained highly conservd “P-loop” motif （G＊GG＊G） and conservative hydrophobic domain “GVALP PTAEK ITFET KLVEG PNGGS IGKLT LKY” throughout the PR10 proteins, we conclude that ARAhPR10 is a new member in this peanut family. Amplification by PCR as genome DNA template gained 561 bp including a 87 bp intron between two extrons. In our study, we found a single strap of the recombination ARAhPR10 protein, expressed in BL21 and purified by Ni^＋-NTA resin, about 25 kD in SDS-PAGE gel. RNase activity of the purified protein in vitro was validated and we predict it has the activity against Aspergillus flavus. The gene cloning and the harvesting recombination protein have established the foundation for studying the function of ARAhPR10.