Identification And Expression Analysis Of RUB1in Dongxiang Wild Rice

Dongxiang wild rice, which is so far the northernmost wild rice that can survive thewinter, is precious wild rice resource with extremely high cold tolerance, many otheradversity-resistances, and many other favorable biological properties. So it is of greatsignificance to study the physiological and genetic mechanisms for Dongxiang wild rice.In this study, RUB1gene was cloned and identified from the cDNA Library of root ofDongxiang wild rice, which encode an Ubiquitin-NEDD8-like protein RUB1. TheUbiquitin-NEDD8-like protein, DaRUB1of Deschampsia antarctica Desv, was consideredto be a distinct regulation pattern during the cold-acclimation process. Bioinformaticsanalysis revealed that the sequences of RUB1protein from Dongxiang wild rice showedvariance of only one amino acid compared with DaRUB1and more than99%homologywith cultivated rice. RUB1is a kind of Ubiquitin protein, which may plays a key role inresponses to cold stress in rice. By further analyzing the gene location on a chromosome,the gene promoter, the secondary structure and tertiary structure of RUB1, it seemed thatthe RUB1protein was likely ABA-responsive and may involve in the response in coldstress.In order to further study the function of RUB1protein, RUB1was ligated withpET28a(+) to construct the recombinant plasmid of pET28a(+)-RUB1. The recombinantRUB1protein was expressed in E.coli BL21by IPTG. The SDS-PAGE showed that therecombinant RUB1protein with molecular weight of18kD was expressed successfully.And the fusion protein was purified by affinity column chromatography. The analysis ofprotein thermal stability indicated that RUB1protein wasn’t a heat stable protein. Thiswork lays the foundation for preparing its polyclonal antibody.The mRNA level of RUB1genes in different growth period of the wild rice had beendetected by real-time fluorescence quantitative PCR, The result showed that RUB1genewas mainly detected in both roots and leaves in all periods. It remained relative high levelin leaves in growth periods while obviously increased in roots in winter, suggesting thatRUB1closely related with cold-induced responses in Dongxiang wild rice. The expressionlevels of RUB1under different abiotic stresses were also examined. The transcript level ofRUB1showed that RUB1was induced by cold, exogenous ABA, and strongly by salt, butalmost not affected by PEG. Subcellular localization detection of RUB1showed the proteinof RUB1accumulated mainly in the cytoplasm and nucleus.This study laid the foundation for the further research about the function of RUB1protein in the cold resistance. It was also significant for exploring cold resistant mechanismof the dongxiang wild rice and utilization of its cold resistant germplasm.