Functional Study Of Rice TFIIB Family Transcription Factor OsBRP1

Transcription is the most important step in gene expression,and it is generally believed that the basic pathway for transcription of eukaryotic nuclear genes is highly conserved.In eukaryotes,TFIIB is a key general transcription factor for RNA Polsymerase II,assisting RNA Polymerase in the initiation of transcription of protein-coding nuclear genes.Usually,there are only two homologous proteins of TFIIB that act as general transcription factors for RNA Polymerase I and III.However,there are a large number of TFIIB family members in plants with 14 and 9 in Arabidopsis and rice,respectively.Several studies in Arabidopsis have shown that the TFIIB transcription factor family has expanded and shown functional diversity during evolution.However,these studies are limited to Arabidopdid and have not been reported in other plants.Rice is one of the most important food crops in the world nowadays,and it is also a model organism for monocotyledonous plant.Elucidateing the differences between rice and other eukaryotic transcription systems is important for improving our current understanding of eukaryotic general transcription factors and transcription systems.This study focused on the function of rice TFIIB-related protein Os BRP1 through analyzing its expression patterns,subcellular localization,function in growth,development and environmental response,and interaction with TBP（TATA-binding protein）.The main research results from the studies are as follows:（1）Systematic expression pattern study of Os BRP1The promoter sequence of Os BRP1 gene was cloned into 1300-NH-GUSPlus vector,and transgenic plants were obtained by genetic transformation,from which positive seedlings were identified and stained for GUS.The results showed that Os BRP1 was expressed at high levels in reproductive organs and young leaves,suggesting that it may play a role in plant reproductive growth.After the wild-type Nipponbare material was subjected to salt stress,it was found that the expression of Os BRP1 significantly increased,suggesting that the expression of this gene was responsive to salt stress.（2）Subcellular localization analysis of Os BRP1The Os BRP1 coding sequence was cloned into 1300UR-s GFP vector,and subcellular localization was examined by laser confocal microscopy after transient expression in Nicotiana bemthamiana.The results showed that Os BRP1 protein was localized to the endoplasmic reticulum（ER）,not to the nucleus.In order to identify the the region responsible for the ER localization of Os BRP1,Os BRP1 protein was divided into C and N-terminal domains（CTD and NTD）,and GFP fusion expression vectors with truncated Os BRP1 were constructed,and the subcellular localization was examined.The results showed that the CTD of Os BRP1 was localized in the nucleus and and the NTD was in the ER.These observations indicated that the NTD of Os BRP1masked the nuclear localization signal of the CTD of the Os BRP1.（3）Construction of CRIPSR/Cas9 knockout vector and isolation of osbrp1mutantsFor determining the function of Os BRP1 protein in growth,development and environmental response,rice CRISPR/Cas9 gene editing recombinant vectors were constructed and transgenic plants were obtained by genetic transformation.Mutations were identified by a combination of PCR and DNA sequencing and homozygous mutants were obtained from self-pertilized progeny.Three different types of homozygous shifting mutants（osbrp1-6,osbrp1-24,osbrp1-50）were isolated.（4）Phenotypic analysis of osbrp1 mutants in growth development and environmental responseThe growth and development phenotypes of the obtained osbrp1 mutant plants were examined,and found to be normal the seedling stage,but significantly altered in the reproductive growth stage.The osbrp1 mutants showed significant reduction in plant height,panicle length,panicle branches,number of tillers,grain length and other related traitsr whens compared with those of wild type.The growth phenotypes,physiological and biochemical changes of rice osbrp1mutants were also compared with wild type under salt stress.The osbrp1 mutants had enhanced leaf yellowing and wilting but reduced height biomass chlorophyll content and survival rates under salt stress when compared to those od wild type.Under salt stress the osbrp1 mutants also had increased activities of SOD、POD、CAT and other antioxidant enzymes and elevated accumulation of H₂O₂.The results indicated that the osbrp1 mutant was more sensitive to salt stress,indicating that Os BRP1 plays an important role in the adaptation to salt stress.（5）Construction of overexpression vector and analysis of overexpression plantsThe Os BRP1-1300s GFP overexpression recombinant vector with the strong Ubiqutin promoter was constructed,and 42 Os BRP1 overexpressed plants were obtained by genetic transformation,the transgenic lines were screened by q RT-PCR method and Western blotting analysis,and several lines with high expression levels of Os BRP1 were identified,which will be useful experimental materials for future study of the function of the Os BRP1 gene.（6）Analysis of the interaction with TBP proteinFor determining whether Os BRP1 is a general transcription factor or has evolved into a specific transcription factor,its interaction with TBP（TATA Binding Protein）protein was analyzed using yeast two-hybrid experiments.The analysis of auxotrophic growth on selective medium found that Os TBP did not self-activate,and did not interact with Os TBP.Analysis using bimolecular fluorescence complementation（Bi FC）also failed to detect interaction between Os BRP1 and Os TBP.These result indicate that Os BRP1 may no longer function as a general transcription factor,but has evolved into a transcription factor with specific roles in plants respond to salt stress.