Identification And Analysis Of Gene SRFP1 In Dongxiang Wild Rice

Dongxiang wild rice is rich in excellent traits and genetic resources, such as drought tolerance, cold resistance to disease and so on, which is known as "the plant panda", and of great significance in the improvement of rice germplasm. The plant ubiquitin protein degradation pathway is closely related to the regulation of stress response. The E3 ubiquitin ligase is one of key enzymes in protein degradation by ubiquitination pathway that is proved to involve in regulation of plant abiotic stress response at the levels of transcription and translation. Focuing on the key genes in protein ubiquitin degradation pathway of Dongxiang wild rice has important significance in studies on mechanisms in stress resistance and the utilization of favorable genes.In this study, a SRFP1 gene with a complete ORF of 783 bp was cloned from the the cDNA library of Dongxiang winter wild rice root, which encoded a polypeptide of 260 amino acids of E3 type ring finger protein.Bioinformatics analysis revealed that the sequences of protein from Dongxiang wild rice showed 63% homology with that of Arabidopsis. The analysis of promoter cis element showed that the the promotor of SRFP1 gene included a series of response elements such as ACE, HSE, MSA-like, ABRE and et al, suggesting that the gene may be regulated by light and involved in protein metabolism, cell cycle process and responses in the defense and stresses.The recombinant plasmid was constructed by ligating with the SRFP1 and tranformed into E.coli BL21. The recombinant SRFP1 protein was expressed in E.coli BL21 induced with IPTG. The recombinant SRFP1 protein with molecular weight of 29.8 kD was expressed purified by Ni-IDAE chromatagraphy column. The detection results showed the protein SRFP1 was not thermal stable.The mRNA expression level of SRFP1 gene expression in root and leaf in the different growth period of Dongxiang wild rice was detected by realtime quantitative PCR.The results showed the gene expression level was highest in the flowering stage. The gene expression levels of Dongxiang wild rice seedling by the different stress treatments such as cold stress, ABA, H2O2, NaCl and PEG6000 were further analyzed, the result showed the expression of SRFP1 was markedly induced ABA, which reached 11 times that of the control, suggesting that SRFP1 is an effector protein in ABA-dependent signaling pathway in Dongxiang wild rice.This study reported SRFP1 gene in Dongxiang wild rice and analyzed the basic characteristics of the SRFP1 gene and protein by bioinformatics analysis. The protein of the SRFP1 was expressed prokaryotically, purified and proved to be non-thermal stable.Realtime-PCR analysis showed that the gene in Dongxiang wild rice was expressed in an high level in flowering stage and induced by cold,stress, ABA, H2O2, NaCl and PEG6000.The result that the SRFP1 expessed with significant increase level by ABA treatment suggested SRFP1 protein was involved in ABA signal transduction. This study laid the foundation for the utilization of the favorable genes of Dongxiang wild rice.