Mapping, Homologous Cloning And Functional Analysis Of Trichome-related Genes In Cucumber

Warty fruit trait is one of the highly valuable external quality traits of cucumber(Cucumis sativus). Histology analysis showed that trichomes and fruit spines aremulti-cellular and non-glandular column-shaped structures. The glabrous1mutant (gl1) is agood material for studying the formation mechanism of the trichomes and fruit spines, whichshowed no trichome on stems, leaves, tendrils, receptacles and ovaries and there were nospine and tumor on cucumber fruit surface. The results of genetic analysis for gl1indicatedthat gl1gene is recessive epistatic to tuberculate gene (Tu). Studying the formationmechanism of trichome can lay the foundation of elucidating the molecular mechanism of thespines and tumors formation on cucumber fruit. In addition, the trichomes and fruit spines aredifferentiated from epidermis cells and the patterning of epidermal cell types in Arabidopsishas become one of the best models for studying the molecular basis of cell specification inplants. It is helpful for enriching the theories of cell differentiation to study the trichomeformation mechanism in cucumber.To mapping glabrous1gene (gl1), map-based cloning was used and we constructed F2segregate populations with ‘Zhuanggua’(Europe greenhouse type) and glabrous1mutant. F2glabrous progenies were used as mapping population. Bioinformatics approach was also usedto identify all of the members of R2R3MYB, bHLH, WD40-repeat and R3single repeat MYBfamily, respectively. Phylogenetic analysis was performed to find putative trichome formationrelated homologous genes in cucumber. To study the functions of trichome formationrelated homologous genes, we performed genetic transformation of Arabidopsis andcucumber, respectively. The results as follows:1.322SSR markers were screened and21polymorphic SSR markers were identified betweenglabrous1mutant and ‘Zhuanggua’. The gl1gene was delimited in the region between SSR21054and SSR117on chromosome3and their physical distance is about3000Kb.Based on primary mapping of gl1gene and re-sequence of gl1mutant,2polymorphic STSmarkers and3polymorphic CAPS markers were developed in the primary mapping region ofgl1gene. Markers analysis was conducted on2400individuals of F2population using thesenew STS and CAPS primers. Then the gl1gene was fine mapped within the region betweenSTS-1and CAPS-1. The physical distance of the STS-1and CAPS-1was311Kb and there are31candidate genes in this region. The results of genes annotation analysis compared toArabidopsis showed that there was not homologous gene related to cell division or epidermiscell fate determination genes. So the mechanism of cucumber epidermis cell division may bedifferent from Arabidopsis.2. Data gene expression profiles of leaves from gl1mutant and ‘Daqingba’ were performedand the results indicated that365genes down-regulated and139genes up-regulated in gl1mutant relative to ‘Daqingba’, respectively. Of these,57genes showed more than20-folddifference. Gene classification based on gene ontology (GO) for differentially expressedgenes in gl1mutant and ‘Daqingba’ showed that the genes related to metabolic, cellular,response to stimulus and cellular biosynthetic processes were abundant in the differentiallyexpressed genes.3. Whole-genome wide analysis of R2R3MYB, bHLH, WD40-repeat (WDR) and R3singlerepeat MYB family was performed using BlastP program, respectively. We identified46R2R3MYB,138bHLH,191WDR and1R3single MYB genes in cucumber genomedatabase, respectively. The homologous genes of Arabidopsis TTG1, MYC1and TRY wasidentified and named as CsTTG1, CsMYC1and CsTRY, respectively. It was worth to notethat there are not epidermis cell fate determination genes in cucumber R2R3MYB family.However, the gene most closely related with Arabidopsis GL1was identified and named asCsGL1L. Sequence analysis indicated that the CDS length of CsGL1L, CsMYC1, CsTTG1andCsTRY is783,1956,1002and249bp and encode260,651,333and82amino acids,respectively.4. To examine the subcellular distribution of the CsGL1L, CsMYC1, CsTTG1and CsTRY protein, CsGL1L, CsMYC1, CsTTG1and CsTRY was fused with GFP，respectively. Confocalimaging showed the four fusion protein localized exclusively in the nuclei of onion (Alliumcepa) epidermal cells.5. Tissue-specific expression of CsGL1L, CsMYC1, CsTTG1and CsTRY were performed andthe results showed that they expressed in different tissues to various degrees. CsGL1L showedhigher expression levels in fruits than any other tissues. CsMYC1showed high expressionlevels in roots, stems, leaves, male flowers and fruits but low levels in tendrils. CsTTG1highly expressed in all of the tested tissues. CsTRY showed higher expression levels in leaves,roots and tendrils and low levels in other tissues.6. To analyze the functions of CsGL1L, CsMYC1, CsTTG1and CsTRY, the over-expressionand RNAi recombinants vectors of these genes were constructed, respectively. The results ofgenetic transformation of Arabidopsis indicated that over-expression CsGL1L in Arabidopsisgl1mutant did not rescue the trichome formation of the gl1mutant. We over-expressedCsGL1L and CsMYC1in Col exhibited significantly increased trichome density to variousdegrees on rosette leaves compared to Col. However, over-expressed CsTTG1in wild typeCol, and the trichome number on rosette leaves of transgenic lines was indistinguishable fromthose in wild-type plants.7. To further study the functions of CsGL1L, CsMYC1, CsTTG1and CsTRY in cucumber, thegenetic transformation of cucumber were performed using Agrobacterium tumefaciens. Todate, we obtained1CsTTG1over-expression line and2CsMYC1over-expression lines. Thephenotype of over-expression CsTTG1line was also analyzed and the transgenic line showedsignificant increase in trichome number and length relative to control, particularly onreceptacles and adaxial surfaces of the leaves. In addition, there are more spines and tumorson the surface of transgenic fruits and more remarkable ribs were also observed.