Identification And Functional Analysis Of Dwarf Mutant Gene Csdw In Cucumber (Cucumis Sativus L.)

Cucumber(Cucumis sativus L.2n = 14)belongs to Cucurbitaceae,it is one of the important edible and economic vegetables all around the world.In this study,we identified a dwarf mutant named Csdw,whose mutagenesis was induced by EMS in cucumber.Firstly,the physiology experiments including phenotypic and microscopical observation,exogenous hormone treatment and endogenous hormone determination were performed to do a preliminary analysis of Csdw.Secondly,the candidate gene of Csdw was identified by Mutmap and KASP.Further analysis of the gene sequence,protein structure,expression level and function of candidate gene were carried out by bioinformatics technologies to elucidate the regulation mechanism of Csdw in cucumber.Following are the main findings:(1)The average plant height of Csdw is 42.4 ± 2.30 cm,which was only 28.15% of the wild type’s(150.60 ± 3.20 cm),but the number of internodes was both about 20 which showing no significant difference between Csdw and wild type,so the average internode length decreased from 7.5 ± 0.29 cm in the wild type to 2.21 ± 0.25 cm in Csdw,which led to the conclusion that Csdw was mainly caused by the shortening of the average internode length.(2)Microscopic observations of the main stem showed that the number of longitudinal section cells was significantly reduced from 125.7 ± 3.50 in the wild type to 76 ± 3.41 in Csdw.So the division process of the main stem cells of Csdw is inhibited which might affected the internode length.(3)Exogenous hormone treatment showed that the application of GA3 could partially rescue the plant height and average internode length of Csdw,and the maximum recovery of Csdw appeared after treatment with 400 mg·L-1 GA3.The plant height of the Csdw control group was 42.4 ± 2.30 cm,and it become 64.2 ± 4.40 cm after treatment with 400 mg.L-1 GA3,which was significantly increased by 51.4% compared with the Csdw control group.However,it did not return to the wild type(150.6 ± 3.20 cm);The average internode length of the wild type and Csdw control group was 7.50 ± 0.29 cm and 2.21 ± 0.25 cm,respectively,the average internode length of Csdw after treatment with 400 mg.L-1 GA3 was 3.24 ± 0.08 cm,it increased by 46.6% significantly compared with the Csdw control group and it was not completely restored to the wild type as well as.There were about 20 internodes among wild type,Csdw control group and treatment groups,which showed no significant difference among them.Endogenous hormone assay showed that the endogenous GA3 content of wild type was 4.87 ± 0.44 ng.g-1.FW-1,while it decreased to 2.89 ± 0.25 ng.g-1.FW-1 in Csdw.The contents of endogenous BR,IAA and ZR did not differ between wild type and Csdw.In addition,there was no recovery of Csdw after treatment with different concentrations of exogenous BR and IAA,indicating that Csdw was insensitive to the treatment of exogenous hormones BR and IAA.In summary,Csdw has certain sensitivity to GA3 treatment and GA3 content of Csdw is lower obviously than that of wild type.(4)Genetic analysis indicated that Csdw was regulated by a single recessive gene as a quality trait.The Mutmap and KASP genotyping results localized to a mutation site SNP36493306 whlich is locating on gene Csa3G872760 for Csdw,so Csa3G872760 was considered as the candidate gene for Csdw.Membrane protein prediction and phylogenetic tree analysis indicated that Csa3G872760 encodes CLAVATA1 type receptor kinase,so Csa3G872760 was renamed as CsCLAVATA1.CsCLAVATA1 was expressed in different organs of wild type,and the expression of this gene in Csdw stem is significantly lower than that of wild type.(5)The RNAi vector ligated with the target fragment of CsCLAVATA1 was transformed into wild type Arabidopsis thaliana by agrobacterium tumefaciens-mediated inflorescence soaking,5 transgenic lines were obtained by resistance screening with hygromycin and PCR identification.By comparing the growth of wild type and 5 homozygous transgenic lines,the plant height of the transgenic lines was significantly higher than that of the wild type after colonization for 17-38 d,and the plant height of transgenic lines gradually became consistent with that of the wild type over time.By comparing the bolting time of wild type and 5 homozygous transgenic lines,it was found that all the transgenic lines entered the bolting stage 6 d earlier than wild type.Moreover,the number of lateral branches coming from cauline leaves of wild type was about 4,while it was more than 6 and up to 9 in the transgenic lines,which showed a significant increase.Additionally,scanning electron microscopy showed that the size of the apical inflorescence was 1.5 mm in the wild type,while the size of the apical inflorescence of the transgenic lines was more than 2 mm and the maximum was close to 4 mm,therefore,the size of apical inflorescence of the transgenic lines was significantly increased compared with wild type.The expression level of the Arabidopsis thaliana gene(LOC9329593)with the highest homology to CsCLAVATA1 was significantly inhibited by more than 3.13 times in the transgenic lines,it indicated that the interference vector plays a role in inhibiting the expression of homologous genes in transgenic lines.In conclusion,it was confirmed that CsCLAVATA1 is involved in the regulation of the growth and development of SAM.