水稻RNase基因的選殖

Abstract

產生RNase是植物對抗缺磷逆境的一種方式。已知TN5-9水稻懸浮培養細胞，在缺磷培養下，可誘導產生一17kDa之RNase。為了解是否在缺磷培養下，僅產生一種RNase？而被誘導產生之RNase其基因表現特性為何？本研究中採用分生方法，以subtractive cDNA PCR、RT-PCR方式選殖出缺磷處理下，水稻被誘導表現之RNase基因。在16個轉型株中，並沒有發現T2/S family RNase基因，但找到有一可能具有RNase活性之pathogenesis-related protein 10a (PR-10a)基因。之後以北方轉漬法及RT-PCR方式，可以驗證水稻在缺磷狀況下，會誘導PR-10a基因大量表現。為確認PR-10a是否具有RNase活性，將此一基因重組後，轉型至大腸桿菌E. coli BL21 (DE3)，進行重組蛋白的表現。純化所得之重組蛋白，在Nus tag片段切除後，可分離得到17kDa之PR-10a蛋白質。進行RNase活性檢測，發現唯有重組蛋白之native form不具有RNase活性。將重組蛋白以β-ME、DTT、EDTA、CIAP處理後，發現CIAP處理之重組蛋白可表現出RNase活性，可分解rRNA及tRNA。

In the previous study, we have known that rice TN5-9 suspension cultured cells will secrete a 17kDa RNase to the medium upon phosphate-starvation. It indicates that RNase production can be induced to reuse the organic phosphate during phosphate-starvation. However, it is unclear, how many RNase genes in the rice genome and what is the gene expression pattern of RNases upon phosphate-starvation. In this study, we try to clone RNase gene from rice by subtractive cDNA PCR and RT-PCR. Sixteen clones induced by phosphate-starcation were isolated. After sequences alignment, none of 16 clones belonged to RNase of T2/S family, but one of them was a putative RNase gene shich was very similar to pathogenesis-related protein 10a gene (PR-10a). Northern and RT-PCR results indicate that rice PR-10a gene is highly expressed during phosphate-starvation, especially from 6 hour to 24 hour after treatment of phosphate-starvation. In order to check whether PR-10a protein has RNase activity. We use E. coli BL21 (DE3) to expressed recombinant PR-10a protein. After protein purification and enterokinase digestion for removing of Nus tag fragnment, a 17kDa recombinant rice PR-10a protein was obtained. However, this mature recombinant PR-10a protein was inactive for RNA digestion. After β-ME, DTT, EDTA, or CIAP treatment, only recombinant PR-10a protein that treated with CIAP exhibited RNase activity in digestion of rRNA and tRNA.