Study On The Function Of The Interaction Protein CIP2 Of Chloroplast Relocation Gene CRD1

China is a big country,grain production has always been the focus of public concern,and 60% of China's crop cultivation is mainly rice.Therefore,how to effectively increase rice yield has become the object of scientists' research.There are many ways to increase rice yield.Among them,it is the most effective to enhance the ability of the leaves to capture light energy and to produce organic matter by photosynthesis.Recently,the isolation and charaterization of leaf color mutants has greatly improved our understanding for the mechanism of photosynthesis and chloroplast biogenesis,which is also contributed significantly for the study of plant growth and development.To elucidate the molecular mechanism of photo signaling pathway in rice,we had isolated several mutants through forward genetics approach.One of them,named crd1,showed deficency in chloroplast removement ability.To further explore the molecular mechanism of CRD1 in chloroplast movement and cell elongation,we attempt to isolate the interacting protein of CRD1 by immunoprecipitation.Through mass spectrometry analysis,we found that CIP2(CRD1 interacting protein 2)is belong to a member of chloroplast GTP binding factor,which plays an important role in regulating chloroplast biosynthesis and chloroplast movement under high light.In the first,in order to verify the interaction between CIP2 and CRD1,we performed bimolecular fluorescence complementation,Pull-down and yeast double hybrid assays and these results were consistent with immunoprecipitation.Then,the realtime-PCR assay revealed that CIP2 is highly expressed in rice leaves and sheaths and with a lower expression in rice roots.Subcellular localization assay revealed that CIP2 is localized in the chloroplast and colocalized with CRD1.Moreover,the CIP2 fluorescence signal could be transferred from the chloroplast to the membrane.To further explore the function of CIP2,we created the cip2-ami mutant by artifical small RNA and the mutant showed a yellow leaf phenotype,that might caused by the decreased chlorophyll content.The RNA and Protein blot assay revealedthat the expression level of CRD1 gene is up-regulated in cip2-ami mutant,similarly,the expression level of CIP2 is also up-regulated in the crd1 mutant.Furthermore,the white band assay showed that the white band appeared on cip2-ami leaf is earlier than that of on the wild type,indicating that chloroplast movement in cip2-ami mutant is more sensitive to high light stimulation.However,the epistasis relationship between CIP2 and CRD1 remains to be further clarifed.All together,our results suggestted that we identified a novel CRD1 interacting protein,which is involved in CRD1-mediated chloroplast relocation upon high lght stimulation.Our results should provide theoretical base to high-efficiency breeding and achieve crop yield increase.