Functional Analysis Of A Spliceosome-associated Protein SAPH In The Regulation Of Exogenous Gene Expression In Arabidopsis Thaliana

Transgenic technology is an efficient tool for improving agricultural traits of crops and also widely used in identification of gene functions.The expression of foreign gene(transgene)is suppressed by the internal genetic mechanism of plants which leads to the decrease of the expression of foreign gene with increased generations of propagation,and this phenomenon is called gene silencing.At present,it is accepted that chromatin modification,DNA methylation and small RNA are involved in gene silencing.However the detailed molecular mechanism is still not clear.In this thesis,the role of a spliceosome-associated protein(SAPH,Spliceosome-Associated protein in Heat)in gene silencing was investigated.In previous work in our lab,the saph muant(numbered hl1401)was identified through screening mutants with increased expression of foreign gene LUC(encoding luciferase)driven by the promoter of HSP18.2 and map-based cloning.However we found that the expression of endogenous HSP18.2 gene in hl1401 was not significant different from that in the wild type(P-LUC).It seems that SAPH repressed the expression of exogenous LUC driven by HSP18.2 promoter.As in both hl1401 and its wild type P-LUC,there is another transgene,hygromycin resistance gene(HYG),we checked the hygromycin resistance of hl1401,and found that the mutant hl1401 was more resistant to hygromycin than the wild type.On agar plates containing 20 mg/L hygromycin the mutant showed significant stronger resistance to hygromycin than the wild type.Consistently,the expression level of HYG driven by 35S promoter was eight times higher in hl1401 than in the wild type.Moreover,both of the expression of exogenous genes(LUC and HYG)and the resistance to hygromycin were restored to the wild-type level after introducing wild type SAPH gene into hl1401 mutant(complementary plant,numbered 4-212-63-8).These results indicate that SAPH preferentially inhibits the expression of exogenous genes.Further analysis showed that the DNA methylation of endogenous HSP18.2 in hl1401 is at the similar level to the wild type P-LUC.However DNA methylation of exogenous genes such as LUC and HYG is significantly lower in mutant hl1401 than in wild type P-LUC,consistent with the transcription result aforementioned.Furthermore in complementary plant 4-212-63-8,the DNA methylation is restored to the wild-type level.All these results suggest that SAPH gene is involved in exogenous gene silencing by improving the DNA methylation.Meanwhile,we found that the enrichment of histone variants H2A.Z in chromatin of exogenous genes in mutant hl1401 is significantly higher than that in the wild type P-LUC.The increasing in enrichment of histone variant H2A.Z is associated with gene activation and H2A.Z antagonizes the DNA methylation in regulation of gene expression.Therefore our results are consistent to these previous findings.In addition,we also found that the level of histone H3 acetylation of exogenous genes in mutant hl1401 was significantly higher than in wild type,which means that SAPH may repress the expression of exogenous gene via regulating the acetylation of histone as the increasing in histone acetylation level is associated with the activation of gene expression.To explore the molecular mechanisms of SAPH protein on inhibiting exogenous gene expression,we further identified partner proteins which work together with SAPH protein by yeast two-hybrid verification and bimolecular luciferase complementation analysis.At present,in total nine proteins were identified as candidate partners interacting with SAPH,including six components(DMS3,SUVH2,SUVH9,MET1,DRM2 and CMT3)of RNA-mediated DNA methylation pathway and three subunits of SWR1(SWC6?SWC2 and PIE1)that catalyzes replacing H2A with H2A.Z in chromatin.These data are consistent with the conclusion that SAPH inhibits the expression of exogenous gene by regulating DNA methylation,H2A.Z incorporation in chromatin and histone modification.Our work provided the community with novel clues for exploring the biological functions of SAPH in plants and revealing the molecular mechanism on exogenous gene silencing.