Molecular Mechanism Of C2H2 Zinc Finger Transcription Factor Regulating Leaf Width Development In Rice

Leaf is the most important organ of photosynthesis in rice,which plays a very important role in the formation of yield and quality.The leaves convert carbon dioxide into organic matter through photosynthesis to promote plant growth and development.Appropriately increasing the leaf area is conducive to the photosynthesis of the leaves and the improvement of CO₂utilization,so as to increase the rice yield.Therefore,it is of great significance to study the molecular mechanism that regulates the development of leaf width in rice.In the study,the WIDE LEAF（WL1）gene was cloned,which invoved in the development of leaf width in rice.We carried out observation and histological analysis of the mutant phenotype in detail,analyzed the expression pattern,protein function of WL1,and the molecular mechanism of WL1 regulating leaf width.The results are as follows:1.Phenotype observation and histological analysis of wl1 mutantCompared with the wild type Xinong 1B,the leaves of the wl1 mutant gradually became wider and reached an extremely significant level during the whole growth period,but there was no significant change in leaf length.Histological analysis showed that the number of small vascular bundles between the great vascular bundles of wl1mutant was significantly increased,and the spacing between the two adjacent small vascular bundles was significantly increased.Further analysis of leaf primordium revealed that the size of stem apical meristem of wl1 mutant was not significantly different from that of the wild type,but the width and area of leaf primordium of P1,P2 and P3 of the mutant were significantly increased.2.Cloning and Functional analysis of WL1 geneThe DNA and c DNA sequencing analysis of the annotated genes related to the possibility of the location interval revealed that a gene encoding the C2H2 zinc finger protein,LOC＿Os03g57240,had a C base to T base mutation in the coding region of the wl1 mutant,and caused the early termination of translation.We initially concluded that the LOC＿Os03g57240 is a candidate gene for WL1 gene.In order to confirm that the LOC＿Os03g57240 is our target gene,the genomic fragment of LOC＿Os03g57240 was transformed into wl1 and the mutant phenotype was completely rescued in the transgenic plants.We have transformed RNAi vector into Xinong 1B,the expression of LOC＿Os03g57240 was significantly reduced,and the leaves became wider.The above results clarified that the LOC＿Os03g57240 is the WL1 gene.3.Expression pattern analysis of WL1 geneq PCR was used to analyze the expression pattern of WL1 gene,the results showed that WL1 gene was expressed in different tissues at the same period including roots,stems,leaves,leaf sheaths and panicle,especially high in leaves,followed by leaf sheath.In situ hybridization showed that the WL1 gene was mainly expressed in the SAM and the leaf primordia.With the differentiation and development of the leaf primordia,the signal of the WL1 gene was mainly concentrated in the vascular procambium.In mature leaves,WL1 gene signals are mainly concentrated in vascular bundles.4.Function analysis WL1 proteinPhylogenetic tree analysis showed that WL1 encodes a C2H2 zinc finger protein,and is highly conserved in monocotyledonous and dicotyledonous.The WL1 protein contains three domains,the Lx Lx L EAR domain,the conserved C2H2 zinc finger domain,and the DLNxx P EAR domain from the N-terminus to the C-terminus.5.Analysis of the molecular mechanism of WL1 regulating leaf widthThe expression frequency of the cell cycle related genes His4 gene and CDKB2 gene in the leaf primordium of wl1 mutant was significantly increased,indicating that WL1may regulate the development of leaf width by regulating the expression of cell cycle related genes.The transcriptional activation test showed that WL1 protein has transcriptional activation activity.It was found that the N-terminal EAR domain is necessary for transcriptional activation activity.Yeast two-hybrid results showed that both the N-terminal and C-terminal EAR domains can interact with the co-inhibitor TPR,indicating that WL1 protein may interact with TPR to regulate the development of rice leaf width.