A Preliminary Analysis Of The Function Of The CsPHB Gene In Cucumber(Cucumis Sativus L.)

Class Ⅲ homeodomain-leucine zipper（HD-ZIP Ⅲ）transcription factor is a class of transcription factors unique to higher plants,regulating the differentiation of various plant cells,such as the formation of meristems,the establishment of the polarity of lateral organs,and the differentiation of vascular tissue cells.So far,most of the research on the HD-ZIP Ⅲ gene has mainly focused on model plants such as Arabidopsis,rice and corn,while there have been few reports on melon crops.Through the precise mapping of the period genes,the research team identified the candidate gene of cucumber leaf curl mutant as CsPHB（PHABULOSA）.In this study,the gene was cloned,and a plant expression vector was constructed.Agrobacterium-mediated genetic transformation was used to initially verify the function of the gene.The main results are as follows:1. Phenotype analysis of cucumber leaf curl mutant regulated by CsPHB geneCompared with the wild type,the cucumber leaf curl mutant has obvious differences in different growth stages,the leaf curl of the homozygous is more obvious,and the heterozygote belongs to the intermediate type,indicating that there is a dose effect of CsPHB.The root length and the number of lateral roots of the mutants at the seedling stage were significantly higher than those of the wild type,but there was no significant difference in plant height,stem thickness,leaf area and leaf perimeter.In the fruiting stage,the plant height,stem thickness,leaf area,leaf perimeter is lower than the wild type.2. Observation of stem tissue slices of cucumber leaf curl mutant at the adult stageObservation of tissue sections revealed that the mutant had the same basic structure as the wild-type stem,and each had 9 bifilar vascular bundles of different sizes.Counting the number of catheters with a diameter greater than 50um in the 500um field of view,it was found that the number of xylem catheters in the mutant increased by 11%compared with the wild type.3. Construction of CsPHB Gene Overexpression Vector in Cucumber Leaf Rolling Mutant and Genetic Transformation in ArabidopsisUsing the cDNA of cucumber leaf curl mutant as template,amplify the CDS sequence of CsPHB gene and construct overexpression vector with seamless cloning kit.Using the dipping method,the mutant CsPHB gene was overexpressed in Arabidopsis thaliana to obtain T₃ generation homozygous plants.RT-PCR results showed that the expression level of CsPHB gene in transgenic Arabidopsis CsPHB-OE was significantly higher than that of wild type,which further indicated that CsPHB is a candidate gene for controlling cu L of leaf curl mutant.After drought stress treatment,after 16 days of water control treatment,some leaves of CsPHB-OE Arabidopsis plants were dried and yellowed,and all leaves of wild-type Arabidopsis plants were dried and shrunk to death.After rehydration,wild-type Arabidopsis did not Changes,and the CsPHB-OE Arabidopsis plants return to green and grow normally,showing obvious drought resistance;The relative water loss rate of transgenic Arabidopsis thaliana is lower than that of wild type,indicating that CsPHB gene improves the water retention capacity and drought resistance of Arabidopsis thaliana.4. Construction of CsPHB gene knockout vector and genetic transformation in cucumberThe Agrobacterium-mediated method was used to overexpress the mutant CsPHB gene on cucumber.After screening and PCR identification of resistance marker genes,T₀generation obtained 16 PCR positive plants in 1107 explants with a conversion rate of 1.4%;Using the online tool CRISPR-P 2.0 to design the target site,connect the vector p KSE401-cas9,construct the CsPHB gene knockout vector and perform genetic transformation,72 gene-knockout PCR positive plants were obtained from 2959 explants.After sequencing,it was found that only one strain had a base insertion near a single target,and mutation occurred.The mutant plants grow slowly and have a deformed leaf phenotype.The gene editing efficiency is only 0.3‰.