Functional Study Of Sheathed Panicle Candidate Gene Of Shpx And Zinc Finger Gene Of OsBBX24 In Rice

Sheathed panicle of rice is a phenomenon thatthe young panicle cannot be stretched from the sheathin the period of panicle differentiation in rice.In this study,Genome sequencing and comparative analysis were performed on a sheathed panicle mutant M893 and corresponding wild type T893,and we found the mutant site of T893.The corresponding gene of the mutant site is named shpx(sheathed panicle gene X).Therewith bioinformatics analysis,gene expression analysis and subcellular localization for shpx were carried out,and overexpression vector and gene editing CRISPR/Cas9 vector for shpx was constructed,as well as transgenic rice were obtained.The results of this study are followed:1.By genome sequencing and comparative analysis,a mutation site of the base G/C change to T/A was found on 5799448 of chromosome 2 in M893.Besides,through designing specific primers of shpx and PCR product sequencing,the mutant site of shpx in M893 was verified.2.By analyzing the characteristics of shpx gene structure,we found that the site 5799448 was just in the splice of second intron and third exonof shpx.Compared to T893,shpx could not be expressed completely in M893.This result showed that the mutation of shpx in M893 affected the normal splicing of shpx.So we speculated that the structural change of transcriptional product of shpx in M893 leads to sheathed panicle phenotype.3.Through bioinformatics analysis of shpx,we found that shpx consists of 4 exons and 3 introns,belongs to the C2H2 type zinc finger protein family.The DNA length of shpx is 7616 bp,encoding 173 amino acids,and SHPX contains 3 zinc finger domains.By homology comparison analysis of SHPX,we found that SHPX have a conserved domains in many species and a highly homologous members from maize,sorghum and brachypodium.4.Based on the rice microarray analysis,we found that shpx was lowest expression in roots,highest expression in young panicle,the expression of shpx in stem and leaf was second and third highest.By fluorescence quantitative PCR analysis,the results showed that the expression of shpx is hightest leaves,second highest in stems,third highest in roots and in lowest young panicle.5.By transient expression analysis using pCAMBIA2300-SHPX-GFP and rice protoplast,the result of observed by confocal laser scanning microscopy indicated that the SHPX protein was localized in the nucleus.6.Through constructed pUbi130-shpx and pYLCRISPR/Cas9-shpx,then transformed into rice callus and screened by hygromycin resistance gene detecting and PCR amplification confirmed,related positive transgenic rice of corresponding transformants were obtained.These results provided the basis for further investigations on the shpx and illuminated the mechanism of sheathed panicle in rice.Beside,we also carried out a research that OsBBX24-RNAi transgenic rice responsed to heat stress.We found that the heat resistance of OsBBX24-RNAi transgenic lines at seeding stage was lower than wild type Kitaake after heat stress.These results showed that OsBBX24 can enhance the thermotolerance of rice seeding.