Map-based Clonging And Functional Analysis Of Short Fruit-Related Gene SF3 In Cucumber(Cucumis Sativus L.)

The cucumber fruit length is the main factor that determines the cucumber fruit shape and size,and it is also an important agronomic trait that affects the yield and quality of cucumber.China is the country with the largest cucumber cultivation area and yield in the world.Recently,the requirements for the specialization of cucumber varieties have gradually increased,for example,the fruit length of North China fresh type cucumber is 30?40 cm,the fruit length of South China type cucumber is about 20 cm,and the fruit length of mini cucumber is about 12?15 cm.Therefore,excavating and cloning genes that control cucumber fruit length and revealing the regulation mechanism of cucumber fruit length,have important theoretical and pratical value for the diversified breeding of cucumber.At present,although some QTL loci regulating cucumber fruit length or control genes related to fruit length traits have been excavated through natural mutation populations or fruit length mutants,but only 4of them have been cloned.Research on the molecular regulation mechanism of cucumber fruit length is still weak.In this study,we identified a short fruit-related mutant C2111(temporarily named its mutant gene SF3)and cloned the candidate gene CsKTN1 by fine mapping,and conducted hormone content determination,transcriptome analysis,interacting proteins screening,etc.The results laid the foundation for the study of cucumber fruit length regulation and KTN1function.The main results of this study are as follows:1.Phenotype analysis of short fruit-related mutant C2111The fruit length of mutant C2111 was significantly shorter than that of the wild type,while the fruit diameter had no significant difference.Paraffin section showed that the cell size in the longitudinal section of the mutant ovary was alomst the same as the wild type,but the cell number was significantly reduced in the mutant.Except for the difference in fruit length,C2111 also showed differences in other organs,such as smaller leaves,smaller floral organs,brittle stems,smaller and rounder seeds.The hybrid F₁ from C2111 and wild type was separated,and short-fruit plants are about half,which suggested that C2111 is a heterozygous mutant.In the F₂ population,compared with the heterozygotes,the phenotypes of homozygotes are more severe,with shorter ovary,smaller leaves,and shorter plants height.2.Map-based cloning of SF3The genetic analysis of short fruit traits was conducted in the 9930'C2111 BC₁ and Gy14'C2111 BC₁ populations.The results showed that the separation ratio of short fruit plants to normal fruit plants was consistent with 1:1 by chi-square test.Combining the phenotypic difference between homozygous and heterozygous mutants indicates that the short fruit traits of C2111 is controlled by a semi-dominant gene.Then two BC₁ populations were employed to map SF3,and SF3 was located in the region of chromosome 7,20715883-20734224 by5937 individuals and 19 markers,with a physical distance of 18.3 kb.In this region,only one complete gene Csa V3?7G032910(CsKTN1,encoding microtubule severing protein katanin p60)and the second half of Csa V3?7G032920 gene are included.After comparing the DNA sequences of the two genes,it was found that a base conversion from C^20725952 to T^20725952occurred in the 5th exon of CsKTN1 in the mutant sf3,which resulted in the substitution of S354F on the amino acid sequence of CsKTN1.Except for the SNP,there was no difference in other sequences,therefore,CsKTN1 is the most likely candidate gene for SF3.To verify the candidate gene,a d CAPS marker developed from the non-synonymous mutant SNP was used for linkage analysis in the Gy14'C2111 BC₁ population,and the results showed that the marker was co-segregated with the phenotype of the recombinant individual.The results of site diversity analysis in 132 cucumber germplasms showed that the SNP only exists in mutant.Overexpressing the CsKTN1 gene of wild-type and homozygous mutant can restore the phenotype of Arabidopsis At KTN1 knockout plants,meanwhile,overexpressing the CsKTN1 gene of wild-type and homozygous mutants in wild-type Arabidopsis thaliana,the transgenic plants both showed a similar phenotype to the overexpression of At KTN1.The results suggested that the function of KTN1 in Arabidopsis and cucumber is conserved,and the function of CsKTN1 in sf3 is acquired.CsKTN1 was expressed in all tissues and organs of cucumber,and the transcription level of CsKTN1 was not significantly different between CCMC and sf3.3.Correlation analysis of short fruit formation and hormone content change in the mutantTo explore the relationship between the short fruit of the mutant and hormones,HPLC–ESI–MS/MS was used to determin hormone content in ovary.The results showed that the content of IAA and GA₃ in sf3 ovary was significantly reduced,but the content of ZT,ABA and JA had no significant difference.Exogenous IAA could partially restore the length of the mutant ovaty,while GA₃ treatment had no effect on the length of the mutant ovary.Transcriptome analysis suggested that the decrease of IAA and GA₃ content in sf3 may be due to the down-regulated expression of IAA synthetic gene YUCCA6 and the up-regulated expression of GA inactivating gene gibberellin 2-oxidases(GA2ox),respectively.GUS staining and dual luciferase activity detection revealed that the transcription factor Abscisic acid repressor1(CsABR1)can directly bind to the promoters of YUCCA6 and GA2ox8 to inhibit YUCCA6 and promote the transcription of GA2ox8.It is predicted that Arabidopsis miR169g may target and regulate CsABR1.A possible miR169g sequence was predicted in the cucumber genome,and the q PCR results showed that miR169g was down-regulated about 4times in sf3.The GFP fluorescence observation test proved that miR169g does indeed regulate the CsABR1.4.Screening and verification of CsKTN1 interaction proteinSubcellular localization showed that CsKTN1 was localized in the nucleus and cell membrane.Some CsKTN1 interacting proteins were screened using yeast library.Bi FC were further used to prove that CsKTN1 interacts with ?-tubulin.