Functional Analysis Of Transcription Factor OsBBX4 In Rice

The BBX gene family is a class of zinc finger protein transcription factors involved in plant photomorphogenesis,shade avoidance,flowering regulation,biological and abiotic stress and so on.As one of the major food crops in the world and the important model organism,the study on the function of rice BBX gene is important for enriching plant gene function.We identified 1070 differentially expressed genes(DEGs)between young panicles in stage 5 and stage 6 from rice TGMS line Zheda13 S under restrictive temperature(30?)and permissive temperature(22?)by RNA-Seq,and within them the expression of OsBBX4 gene is induced by temperature.In this study,the characteristics,expression patterns and subcellular localization of OsBBX4 gene were analyzed,and 13 OsBBX4 gene knockout transgenic events were obtained through CRISPR/Cas9 and transgenic technology to validate the function of OsBBX4 gene on the rice growth and development combinated with agronomic traits.The main research results are as follows:(1)The OsBBX4 gene contains two exons and one intron,and encodes the zinc finger protein that contains two B-box domains,which was highly expressed in leaves and induced by temperature by qRT-PCR.The promoter of OsBBX4 contains 41 cis-regulatory elements,including low temperature response elements,light response elements,stress response elements and plant hormone response elements.A total of 20 OsBBX4 homologous genes are identified from five poaceaes and four dicotyledonous,among which OsBBX11 is highly homologous with OsBBX4.Protein interaction network analysis revealed that OsBBX4 was predicted to interact with six proteins,which included OsHY5L1,OsHY5L3 and OsCOP1 three proteins co-interacted with OsBBX11.(2)A total of 13 T0 generation transgenic mutants were obtained through CRISPR/Cas9 editing and transgenic technology,and secondary editing was occurred at T1 generation.Homozygous T3-generation osbbx4 mutants without T-DNA insertion were obtained through self-crossing.Compared with wild type,the plant height,grain number per panicle,seed setting rate and grain traits were different in mutant plants.The significantly reduced length of first internode and panicle was the major cause of the significant reduction of plant height in mutants,the grain length and grain weight were also significantly reduced.All these suggested that OsBBX4 gene may also participate in regulation of rice vegetative and reproductive growth.(3)One of the transgenic mutants,bbx4-8,was fertile at restrictive temperature and sterile at permissive temperature,acting as reverse thermo-sensitive genic male sterile(TGMS).However,the setting rate of bbx4-8 is very low at restrictive temperature.Inverse PCR result shows that T-DNA was inserted into the intergenic region between LOC_Os08g08330 and LOC_Os08g08340.In addition,no off-target was detected.The reverse of fertility of bbx4-8 possibly due to the deletion of 84 bp in OxBBX4.(4)The OsBBX4 gene regulates the response to abiotic stress.The expression level of OsBBX4 decreased under salt stress,but the mutant and wild type showed the same growth state.Under drought stress simulated by mannitol,the expression level of OsBBX4 decreased gradually,and the growth of knockout mutant was better than that of wild type.Under permissive temperature,OsBBX4 was upregulated,seed setting rate and grain number per panicle were decreased,while grain length and grain weight increased.In summary,the OsBBX4 gene was conserved in monocot and dicot.The OsBBX4 gene was involved in the regulation of plant height and grain traits,as well as responded to salt and drought stress.At the same time,a reverse thermo-sensitive genic male sterile mutant bbx4-8 was obtained,which was fertile at restrictive temperature and sterile at permissive temperature,providing certain value for rice crossbreeding.