Functional Analysis Of OSPP2C Gene Related To Mercury Tolerance

As a highly toxic metal in the environment,mercury can accumulate in rice,and induce plant damage and cause yield loss.In previous study,one OsPP2 C gene in the interval of QTL analysis was selected as one candidate gene for mercury tolerance in rice.In this study,in order to explore the mechanism of mercury tolerance in rice,the expression characteristics of OsPP2 C was analyzed,gene function of OsPP2 C was validated by analysis mercury content in the over-expressed and interferred transgenic rice plants of OsPP2 C,and the OsPP2C-medidated mercury tolerance in rice was also analyzed by RNA-sequencing.The main results are listed as follows:1.Analysis of expression characteristics.Using Nipponbare seedlings at the three-leaf stage as tested materials,the expression of OsPP2 C in leaves was analyzed under the lighting at intervals of two hours from 0 h(ZT 0 h)to 22 h.The results showed that the expression peak of OsPP2 C in Nipponbare was at ZT10h;the expression of OsPP2 C in Nipponbare were stable under the dark,therefore,the expression of OsPP2 C had a circadian rhythm at rice seedling stage.The OsPP2 C was expressed in all tissues under normal growth conditions,and expression in leaves was the highest.The expression of OsPP2 C increased significantly in the rhizome junction,roots and stems but decreased significantly in the leaves after mercury stress.The fusion vector of OsPP2C-GFP was constructed and infected with Agrobacterium tumefaciens GV3101 to infect N.benthamiana leaves.It was found that OsPP2 C may be localized to the cell membrane and cytoplasm.2.Functional verification.After mercury stress for 4h,the expression level of OsPP2 C in the leaves of the over-expressed and silenced transgenic rice plants showed no significant change.After mercury stress for 6h,the expression levels of OsPP2 C decreased in the leaves of wild-type and over-expressed lines but not in the leaves of the interferred lines.Unlike the expression partterns of OsPP2 C in the leaves,the expression level of OsPP2 C in the roots of all the transgenic rice plants significantly increased after mercury stress for 4 h,and the expression of OsPP2 C in the roots of the interferred lines also increased after mercury stress for.After mercury stress for 72 h,the over-expressed lines had the lowest mercury content in the roots;and the mercury content in the leaves of RNAi lines was the lowest and was much lower than that in the roots of the RNAi lines.3.RNA-sequencing.Transcriptome sequencing analysis was performed by using the transgenic lines of OsPP2 C and Nipponbare which was treated with 20 μM and no mercuric chloride solution for 12 h.The differentially expressed gene(DEG)was selected(with Qvalue < 0.05,Log2 FC > 1)2978 DEGs were detected in Nippon(Nt vs Nck),among the 2978 DEGs,2066 genes were significantly up-regulated and 912 DEGs s were significantly down-regulated;2034 DEGs were detected in over-expressed lines(Ot vs Ock),1240 DEGs were significantly up-regulated,and 794 DEGs were significantly down-regulated;3327 DEGs were detected in interferred lines(Rt Vs Rck),2307 DEGs were significant up-regulated and 1020 DEGs were significant down-regulated.KEGG Pathway analysis of the mercury stress responsive genes revealed two pathways related to self-defense,(the phagosome pathway and the autophagy regulatory pathway).Functional annotation of three of the two pathways indicates that two genes are presumed to encode tubulin and one encodes an autophagic protein.The results were consistent with RNA-seq data based by quantitative fluorescence analysis of 7 genes of phagosome pathway was performed by qPCR.