| Plant type is closely related to the resistance and yield of crops.The ideal plant type is an important direction for crop breeding.The length of hypocotyl and leaf shape are important factors for the plant type of cucumber.It's of important significance in the construction of ideotype and plant type improvement of cucumber through the research of its regulatory genes in this study.Our team obtained two candidate genes related to the plant type traits of cucumber through fine genetic mapping in the previous study.They were CsLH1(LONG HYPOCOTYL 1)and CsRL(ROUND LEAF),genes that control the elongation of cucumber hypocotyls and round leaf,respectively.In this study,these two candidate genes were cloned and related expression vectors were constructed.The function of these two genes was initially verified by genetic transformation of Agrobacterium.The main results are as follows:1.Construction of overexpression vector and gene knockout vector of CsNABP and genetic transformation in cucumberThe knockout expression vectors of the transcriptional start site and core functional region of CsNABPgene were constructed using CRISPR/Cas9 technology,hereinafter named sgRNA1-Cas9-CsNABP and sgRNA2-Cas9-CsNABP,respectively.Genetic transformation of CsNABP Gene Knockout Vector by Agrobacterium-mediated in Cucumber was carried out.Two strains of sgRNA1-Cas9-CsNABP positive strains and nine strains of sgRNA2-Cas9-CsNABP positive strains were obtained by PCR.It was presumed that the transformation rates of the knockout vector in the CsNABP gene start site and core functional regions were 1.7% and 0.95%,respectively.The 35S-CsNABP expression vector was constructed by homologous recombination.Sequencing and nucleotide sequence comparisons revealed that there were multiple variable cleavages in CsNABP,so genetic transformation of 35S-CsNABP was not conducted.2.Morphological traits and photosynthetic characteristics of round leaf cucumber mutants regulated by CsPID geneThe morphological indexes such as plant height and stem diameter of round leaf mutant C949 were significantly lower than those of wild type,indicating that CsPID gene not only controlled the leaf shape of cucumber,but also affected the growth and development of other cucumber organs.In terms of photosynthetic characteristics,the chlorophyll content and net photosynthetic rate of the mutants were significantly higher than those of the wild type.3.Functional deletion analysis of promoter of CsPID geneDifferent length fragments of CsPID gene promoter in round leaf mutant was cloned,and then insert the plant expression vectors by homologous recombination.Agrobacterium carrying promoters was injected into tobacco leaves.The promoter activity was verified by transient expression of GUS gene.The results showed that the promoter fragments above 195 bp are active;the expression vectors of different length fragments of CsPID gene promoter were transformed into Arabidopsis thaliana,and GUS staining was performed on T1 plants.The results showed that the promoters were mainly expressed in roots,leaves,floral twigs,and seed pods.4.Construction of CsPID Overexpression and Knockout Vectors and Their Genetic Transformation in Cucumber and Arabidopsis The cDNA of CCMC was used as a template to amplify the CDS sequence of CsPID gene.The 35S-CsPID overexpression vector was constructed by homologous recombination and the genetic transformation of cucumber was carried out.Thirty-two 35S-CsPID positive plants were obtained by PCR.The conversion rate of 35S-CsPID was 3.4%.CsPID knockout vector was constructed using CRISPR/Cas9 technology.Eleven sg RNA-Cas9-Cs PID positive plants were obtained by PCR.These positive transgenic strains were sequenced and the results showed that they were No mutations occur near the target sequence.The 35S-Cs PID overexpression vector was transformed into Arabidopsis,and the T2 generation positive transgenic lines showed phenotypes such as increased lateral primary inflorescences,clustered rosette leaves and smaller leaves. |
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