Functional Analysis Of Hairy Leaf Gene HL1 And Mapping Of QTL For Panicle Size-Related Traits In Rice(Oryza Sativa L.)

The surface of the plant is covered with single-cell or multi-cellular epidermal hairs,which is differentiated from epidermal cells.It is a trait evolved by plants in response to environmental stress.Epidermal hair not only increases the thickness of the epidermal layer of plants,but also plays an important role in the protection of plants against biotic and abiotic stresses.Therefore,research on the development and regulation mechanism of plant epidermal hair has important scientific significance and application value.In the previous study,the hairy variety SWWR and the hairless variety Nipponbarethe were selected to construct the mapping population.The hairy gene HL1 was located on chromosome 6,and finally an AP2/ERF transcription factor was selected as the candidate gene.Based on this research,we conducted a study on the function of the hairy gene HL1.The findings are as follows:1 Sequencing alignment revealed that there are 2 SNPs and 3 base deletion in the CDS region of SWWR,resulting in the amino acid of the 330 protein from methionine to isoleucine,the 356 glutamine deletion.There are 12 SNPs and 7 base deletions in the promoter region,resulting many cis-binding elements differences such as NF-YB、SBP and AT-hook.Expression analysis showed that the expression of HL1 in leaves and roots of SWWR is higher than Nipponbare.2 Constructing PROSWWR:HL1SWWRvector,we transferred it to Nipponbare.The leaves and leaf sheath of the transgenic plants showed hairy and the expression of the transgenic plants was significantly higher than wild type.Constructing PROSWWR:HL1NIP and PRONIP:HLSWWR vector,transgenic plants showed hairless.It explain that PROSWWR and HLISWWR are indispensable for hairy leaf.HL1 was knocked out in the hairy parent SWWR by CRISPR/Cas9 site-directed mutation method.The mutant leaves is hairless and hairy leaf turn into bristle leaf.The results demonstrate that coding region of HL1 is functional.Observing of the transgenic lines under scanning electron microscope found that both the trichomes and the bristles are placed in the corresponding positions of the leaf veins and arranged in parallel.It is believed that the HL1 gene promotes the elongation of the bristles into trichomes.3 Sequencing analysis of 22 varieties revealed that there were 18 variations in the coding region of the HL1 gene.Deletion in the 356 amino acid,frameshift in 347 amino acid and deletion in 388-389 asparagine and serine may be associated with trichome growth.Difference analysis of HL1 promoter cis-binding elements revealed that AT-hook and bHLH binding elements were more frequently found in the promoter of the hairy leaf varieties.NFYB and SBP elements were more frequently found in the hairless leaf varieties.4 Subcellular localization analysis indicates that HL1 is localized in the nucleus.Yeast cell in vivo and bimolecular luciferase reporter experiments showed that the HL1 protein has transcriptional activation activity.Real-Time PCR found that the expression level of HL1 gene was high in young roots and seeds,and the expression abundance was low in leaves,stems,sheaths and panicles.5 The HL1 interaction protein was screened by yeast two-hybrid method from rice seedling cDNA library and 8 positive clones were obtained.Selecting the histidine transferase OsAHP2 for subsequent analysis,yeast cell in vivo and BiFC assay suggested that OsAHP2 and HL1 could interact.Subcellular localization results showed that the OsAHP2 protein was distributed in the cell membrane and nucleus.6 Transcriptome sequencing analysis of hl1 mutant and HL1 complementary material revealed that 1415 differential genes were detected in the mutant/wild-type group and 1010 differential genes were detected in the complementary material/control group.There were 204 differential genes in the two groups.Among them,81 genes were differentially expressed in opposite directions in the two groups.Real-time PCR verified that most differential gene expression were consistent with transcriptome analysis.The KEGG pathway analysis showed that the differentially expressed genes in both groups were significantly enriched in secondary metabolite biosynthesis,plant pathogen interaction and plant hormone signal transduction.Panicle size are important agronomic traits,which associated with grain yield.Revealing the genetic mechanism of rice panicle formation will not only strengthen our understanding of regulatory mechanisms of these traits,but also be valuable for high-yield breeding in rice.1 QTL mapping of panicle:In this study,we choose two different parent materials in the target phenotype,YR1 and NJ1(recurrent parent),to construct the population for a genetic analysis of the panicle size.A total of 3 QTLs were detected from QTL analysis of five phenotypes including panicle length,primary branch number,secondary branch number,spikelet number,and seed setting rate in BC2F2 population.The QTL qGNP1 and QTL qPBN8 were identified between marker RM8105-RM1220 on chromosomes 1.The LOD values were 6.93 and 6.14,which explained 30.6%and 25.4%of phenotype variation,respectively.This interval contains the GN1a controlling spikelet number,which has been reported.The QTL qPBN8,between marker S4 and S21,showed logarithm of the odds scores of 9.43 and phenotype variance of 39.57%.The additive effect of both loci comes from the YR1.Selecting the remaining hybrids from the BC2F2 population,we construct a segregating population and further validated qPBN8.The result showed that the locus was consistent with the initial positioning result.2 Fine mapping of qPBN8 and candidate genes analysis:BC2F4 plants derived from BC2F3 plants heterogeneous in the target region were selected to screen recombinant plants.To fine mapping qPBN8 locus and progeny of key recombination verification,new polymorphic molecular markers were developed.Finally,qPBN8 was located within the 51.8 kb interval between markers S30 and S10.According to Rice Genome Annotation Project,six Opening Reading Frames(ORFs)exist there.Among these candidate genes,ORF3 is previously reported OsSPL14,which regulates branch number and increase grain number.Sequence analysis revealed that there was no difference in the coding region of ORF3 between YR1 and NJ1.The expression level of OsSPL14 at the 1-2 mm panicle was about ninefold higher in YR1 than in NJ1.But,there is no difference at the 5-10 cm panicle.3 Yield analysis of qPBN8 near isogenic lines:The investigation of yield structure in six pair of NILs found that the qPBN8YR1 large panicle line showed fewer tiller,more spikelets,slightly larger grain and lower seed setting rate.The effects of qPBN8 on grain yield were related to the length of vegetative period.The NIL with late heading date can reflect the yield increasing of qPBN8 YR1.The qPBN8YR1 is more suitable for late-maturing varieties to improve yield.