Cloning And Functional Analysis Of Os04g37530 And Os02g05710 Of DUF1644 Family Of Rice

As a important food crop worldwide and a model monocot species,rice has great research significance in agriculture and biology.Various abiotic stresses such as high salt,drought,high temperature and cold have an impact on the growth,yield and quality of rice.Therefore,it is of great theoretical and practical significance to isolate and clone rice stress-tolerant genes and to clarify the stress resistance mechanismsThrough bioinformatics analysis,a conserved DUF1644 domain family was found,and a conserved zinc finger structure was contained in this domain.The expression of this family gene was induced by various abiotic stresses,but their functions have not yet been obtained.In this study,two genes of the DUF1644 family Os37 and Os05 and a gene Os28 that interacts with Os05 were studied.The main results are as followsqRT-PCR detected that Os37 was mainly expressed in inflorescences,stamens and stems,and the results of the preliminary obtained transgenic plants containing the promoter-driven GUS reporter gene were complementary to this part;Subcellular localization result showed that Os37 is mainly localized in the nucleus and cytoplasm;Protein structure prediction analysis showed that Os37 contains a DUF1644 and a zinc finger domain,and gene expression is induced by abiotic stresses such as salt,drought and heat;The stress treatment of Os37 transgenic rice showed that the RNAi plants could effectively resist salt stress,while the overexpressed plants showed some resistance to drought stress;Using Os37 as bait protein,the rice cDNA library was screened and the candidate gene was verified by BiFC.The results showed that Os37 can interact with itself to form homodimers,and can also form heterodimers with Os05 or Os35;In addition,Os37 can also interact with OsBCl,a protein in BR signal pathway;The observation of transgenic plants revealed that the Os37 overexpressing plants showed phenotypes of dwarfing,delayed heading and seed variability,while the Os37^RNAi plants were just the opposite;Rice phenotypic recovery experiments showed that exogenous application of BL can restore the dwarf phenotype Os37 overexpression plant,while exogenous application of GA aggravates this phenotype.However,detection of related key genes revealed that they did not show significant change in overexpressing or knocking down transgenic plants.qRT-PCR detected that Os05 is highly expressed in all tissues of rice,and its more significant in roots and leaves;Subcellular localization result showed that Os05 is mainly localized in the cytoplasm or cell membrane,and is not expressed in the nucleus;Protein structure prediction analysis showed that Os05 contains a DUF1644 and a zinc finger domain,and gene expression is induced by abiotic stresses such as high temperature and ABA;The stress treatment of Os05 transgenic rice showed that the leaves of the overexpressing plants produced more IDA after drought stress.Using Os05 as a bait protein,screening of rice cDNA library showed that Os05 can interact with Os37 and Os35,and also interact with a universal stress protein Os28;The observation of transgenic plants showed that Os05 overexpression and Os05RNAi plants showed no obvious phenotypic differences in the aerial part,but the roots of the overexpressing plants were significantly shortened at the seedling stage.qRT-PCR detected that Os28 is highly expressed in leaves,sheaths,stems and roots of rice,and its more significant in leaves;Subcellular localization result showed that Os28 is mainly localized in the nucleus and cytoplasm;Protein structure prediction analysis showed that Os28 has a conserved UspA domain,which involves in the Stressful plants,and it is also predicted that it may encode a Na+/H+ ion channel protein;The stress treatment of Os28 transgenic rice showed that the overexpressed plants have stronger ability to tolerate drought stress;The observation of transgenic plants showed that the roots of Os28 overexpressing plants were significantly shorter than the wild type at the seedling stage,and the flag leaves of 28 overexpressing plants are significantly larger at the maturity stage of rice.In summary,this study conducted a preliminary study on the stress resistance and morphogenesis of two proteins in DUF1644 family and the general stress protein Os28,which laid the foundation for further experimental work.