Characterization And Functional Analysis Of Genes Associated With Leaf Curvature In Non-heading Chinese Cabbage(Brassica Rapa Ssp.chinensis)

Non-heading Chinese cabbage(Brassica rapa ssp.chinensis;NHCC),belongs to Brassica genus of Cruciferae,is one of the most important leafy vegetables,which originated in China and widely cultivated in the middle-lower reaches of the Yangtze river.NHCC has abundant genetic resources and varieties,forming different morphological structures.NHCC leaves are the main edible and important organs for many physiological functions,including photosynthesis,transpiration and respiration.Therefore,leaf morphology is one of the most important agronomic traits in NHCC.Generally,the flatten leaves can maximize photosynthetic efficiency.The study on the genes related to leaf curvature will be helpful to elucidate the molecular mechanism of leaf shape formation,and provide a theoretical basis for the breeding of Non-heading Chinese Cabbage varieties with high quality,high yield and good appearance via molecular genetic breeding mechanism.In this study,we isolated and cloned three leaf curvature genes(BcMYB101,BcKAN2,and BcYABBY3)from Non-heading Chinese Cabbage cultivar'suzhouqing'.The expression profiles in different tissues and the response patterns to diverse stresses were analyzed using qPCR.The transgenic Arabidopsis lines and virus-induced gene silencing(VIGS)plants in Non-heading Chinese Cabbage were generated for investigating the function of three genes.Furthermore,yeast two-hybrid system was used for screening the potential interacting proteins.The main research contents and results for this study were listed as follows:1.The cDNA sequence of BcMYB101 was cloned from Non-heading Chinese Cabbage(Brassica rapa ssp.chinensis)cultivar 'suzhouqing'.The putative BcMYB101 protein contains high conserved R2R3-MYB domain and GAMYB-specific motif(Box1,Box2,and Box3),and have high identity with their Arabidopsis ortholog.qPCR analysis displayed that BcMYB101 had higher expression level in leaves,roots,and floral organs,and responded to GA and ABA treatments.Subcellular localization assay revealed that BcMYB101 protein localized to the nucleus.Meantime,one potential interacting protein,BcTCH4,was screened and identified by yeast library and yeast two-hybrid assay.BcMYB101 overexpression plants exhibited down-curly cauline leaves and generated more leaves than wild type in Arabidopsis.While upward rolled leaves were found in BcMYB101-silencing plants in Non-heading Chinese Cabbage.These results indicated that BcMYB101 involved in the regulation of leaf curvature.2.We isolated BcKAN2 from Non-heading Chinese Cabbage cultivar 'suzhouqing'.Sequence analysis showed that BcKAN2 protein contains conserved Myb-DNA binding domain and KANADI-specific motif,shared high similarity with Brassica oleracea orthologs.Subcellular localization and qPCR assays indicated that BcKAN2 encoded a nuclear localization protein and relatively have higher transcript levels in stem tissues.Overexpression of BcKAN2 in Arabidopsis showed leaf serration and curl downward.Meantime,leaves of BcKAN2-silencing plants displayed upward curling phenotype in Non-heading Chinese Cabbage.To further investigate the functional mechanism of BcKAN2,yeast two-hybrid library and online website STRING were used to screen and predict the underlying interacting proteins.Four proteins(BcRVE1,BcAFO4,BcYAB3,and BcARF3a)were shown to interacted with BcKAN2 via yeast two-hybrid,colocalization,and luciferase complementation imaging assay(LCI).The results suggested that BcKAN2 may participate in the control of leaf curvature by acting with adaxial determinants.3.In this study,twelve YABBY-like genes were identified from Non-heading Chinese Cabbage,and their proteins have high identity with Arabidopsis orthologs.Subcellular localization exhibited that BcYABBYlb,BcYABBY2c and BcYABBY3 proteins were mainly localized in the nucleus,consistent with the online prediction.qPCR analysis revealed that BcYABBY genes were rarely expressed in roots,and BcYABBY4a,BcYABBY4b and BcCRC were restricted in floral tissues,BcYABBY1/2/3/5 have high transcription abundance in leaf and floral organs.Meantime,expression analysis of different stress treatments indicated that the expression profile of BcYABBY genes was significantly different in response to different stress treatments.However,YABBY genes in the same subgroup have similar expression patterns,suggesting that these genes may be functional redundancy.We furtherly investigated the molecular function of BcYABBY3 due to BcYABBY3 have an interation relationship with BcKAN2.BcYABBY3-overexpression Arabidopsis plants were generated by Agrobacterium-mediated floral-dip transformation,and significantly down-rolled and narrower leaves,increased leaf number,and delayed flowering phenotypes were found in transgenic plants.In addition,BcYABBY3-silencing plants generated by virus-induced gene silencing(VIGS)system,showed up-rolled leaf phenotype.In conclusion,these results demonstrated that BcYABBY3 play a role in the regulation of leaf shape.