Map Based Cloning And Functional Analysis Of Round Leaf And Micro Plant Mutant Genes In Cucumber

Plant architecture is the key factor affecting the photosynthetic efficiency and cultivation pattern of crops.The ideal plant architecture can make the plant has a better form to receive light and can improve the planting density,hence the plant population could increase its utilization of light energy during the growth and development period,and achieve the purpose of improving the economic yield.For vegetable crops,the ideal plant architecture can also save labor cost.Plant architecture of cucumber(Cucumis sativus L.)is complex.Cucumber cultivars in current production are mainly vine cultivars with dense growth and large stacking degree of leaves,which usually affect photosynthesis and reduce photosynthetic yield.In addition,more manpower is needed to carry out plant management,which increases production cost.Therefore,the selection and breeding of ideal plant architecture with suitable plant height,reasonable leaf distribution and meeting different cultivation conditions is an important direction of cucumber high-yield breeding,and has important theoretical and practical significance for efficient cucumber production.The key to ideal plant architecture breeding is to cultivate cucumber varieties with different plant architectures to meet the needs of various cultivations,which requires continuous digging of new plant architecture materials,cloning of regulatory genes and analyzing the formation mechanism of ralted traits.In this study,we identified two novel plant architecture materials in cucumber EMS mutant population,including round leaf mutant(rl)and micro-plant mutant(mp),and performed mapping cloning and analytical related studies on candidate genes of these two plant architecture traits.The main research results are as follows:1.Phenotype identification and gene mapping of cucumber rl mutant1.1 Phenotypic observation showed that rl has nearly round leaf shape,a few tendrils,disorderly root development,absence of calyx,defective fruit heart cavity,and produced no viable seeds.Genetic analysis showed that a single recessive gene rl regulated the mutation.The gene was further mapped to 165 kb region on chromosome 1 of cucumber genome by using Mut Map analysis and linkage molecular marker analysis.The candidate gene Csa1G537400 was identified through screening the sequencing data of two bulked pools.There was a single base G to A mutation in the coding region of the gene at the 1092 ed bp,which converted the coding amino acid from valine to methionine.Allelism test of the candidate gene in 74 cucumber materials also confirmed that the mutation site of this gene was closely linked to the rl trait.1.2 Functional annotation of rl gene indicated that this gene could encode the auxin transportation related protein PINOID.Sequence alignment and evolutionary relationship analysis showed that this protein was conserved in monocotyledonous and dicotyledons plants,but there were also some differences,suggesting that this protein may have functional differences in different plants.q RT-PCR analysis showed that the expression of rl gene was the highest in the stem of the wild type and the ovary of the mutant,and the expression of rl gene in all organs of the mutant was significantly lower than that in the wild type.Subcellular localization showed that the protein was expressed in the cytoplasm,cell membrane and nucleus of onion epidermis cells.2.Phenotype identification and regulatory mechanism analysis of cucumber mp mutant2.1 Compared with wild-type plant,the mp mutant had the phenotypic characteristics of dwarf,miniaturization of organs,no stacking of leaves,short fruits,no obvious lateral branch development,and female sterile.Scanning electron microscope(SEM)observation showed the shape and size of stem epidermis cells of mp mutant were changed.Cs KTN1,a regulatory gene that controls mp trait,was cloned by fine mapping.The expression level of the gene was down-regulated in all organs of the mp mutant.Subcellular localization showed that both wild-type and mutant Cs KTN1 proteins were expressed in the cell membrane,cytoplasm and nucleus of tobacco leaf cells.Knockdown the expression of Cs KTN1 by RNAi in cucumber led to plant growth and development defect such as dwarf,indicating that Cs KTN1 gene could regulate the development of cucumber plant architecture.2.2 Cs KTN1 could encode a Katanin P60 protein,contains an AAA+ conserved domain,and could shear microtubules.Microtubule co-precipitation and shearing assay in vitro showed that Cs KTN1 protein could bind and shear microtubules in vitro,and the protein domain conformation was changed because of the amino acid mutation,which lost its ability to bind and shear microtubule.Microtubule related reagent spraying test confirmed that the microtubule status could change the plant architecture of wild-type and mp mutant.2.3 Five proteins that could interact with Cs KTN1 were obtained through screening of cucumber yeast c DNA library.Function annotation showed that these proteins were involved in terpenoid synthesis,MAPK signaling cascade,RNA transport and methylation dependent chromatin silencing pathways.Quantitative analysis showed that the expression levels of several interacting genes were changed in mp mutant.Yeast one-to-one hybridization and bimolecular fluorescence complementation confirmed that Cs KTN1 could interact with the rate-limiting enzyme HMGR of terpenoid synthesis in yeast and tobacco cells.2.4 The transcriptome and metabolome profiling identified differentially expressed genes(DEGs)and differentially accumulated metabolites(DAMs)between mp mutant and wild-type,suggesting that the regulation of cucumber plant architecture by Cs KTN1 involves the joint regulation of multiple genes and multiple metabolites.Enrichment analysis shows that the Cs KTN1 mutations affect multiple metabolic pathways related to plant growth and development,including terpenoids synthesis and accumulation of plant hormones,cell growth,photosynthesis regulation pathways,etc.