Cloning And Functional Studies Of Tea Tree Flower Development Genes CsFUL And CsAGL6

Tea plant(Camellia sinensis(L.)O.Kuntze)is a kind of economic crop for leaves consuming,tea flower bud and leaf bud was together born in axils.It will take about 15-16 months from flower bud differentiation to seed maturation,which consumes a great deal of nutrient and will inevitably disturb leaf developent.Therefore,shortening the reproductive cycle and promotion vegetative growth is one of the methods to solve the major problems facing the current tea production.Flower development requires a series of genes.According to the stage of plant flowering process,genes were divided into three groups:flowering time-related gene,flower meristem identify gene and flower organ identify gene.API,FUL and AGL6 are both the floral meristem identity genes and floral organ identity genes.Flower development early,they can promote the differentiation of inflorescence meristem to floral meristem,at the later stage of flower development,they regulate the development of different floral organs.It is necessary to clone and study the function of the CsAPI,CsFUL and CsAGL6 genes,then elucidate the molecular regulation mechanism of tea plant flower transition.This study will provide theoretical basis for the further use of molecular biological methods to shorten the flowering time of tea plant.In this study,the genes CsFUL and CsAGL6 were successfully obtained from the differentiating flower buds of the representative of Shaanxi province tea plant "Camellia sinesis cv.Ziyangzhong"by homology cloning and RT-PCR.Bioinformatics prediction and phylogenetic analysis were employed for those two genes.Then functional analysis of them from three aspects.First,the function of CsAP1,CsFUL and CsAGL6 genes was analyzed by 35s promoter ectopic expression of Arabidopsis thaliana.Second,real-time quantitative PCR(RT-qPCR)analysis genes expression patterns.Third,the use of subcellular localization,yeast one-hybrid and yeast two-hybrid technology to determine the interaction of three proteins.The main conclusions are:1.We isolated two cDNA sequences with 735 bp and 726 bp open reading frame by using the homology cloning and RT-PCR from the differentiating flower buds "Camellia sinesis cv.Ziyang zhong".They were homologous to the FUL and AGL6 gene of Camellia japonica,Actinidia chinensis,Ipomoea nil,Arabidopsis thaliana and other species by BLASTN alignment,and contained the conserved MADS domains,K domain and characteristic motif.Therefore,they were named after CsFUL and CsAGL6,respectively.Then CsFUL and CsAGL6 were submitted to GenBank under the accession number of KU862280 and KU862281,respectively.2.Bioinformatics analysis showed that CsFUL and CsAGL6 were APl/FUL-like family and AGL6-like family respectively and encoded a MIKC-type MADS-box transcription factor.The two transcription factors had similar conserved motifs,CsFUL protein had MPPWMI motif;protein CsAGL6 had two motifs DNETVLQIGY(AGL6-I motif)and GWVL(AGL6-II motif),which promoted the family protein to form a similar high-level structure that confer a specific biological function.3.CsAP1 gene was expressed in sepals and petals,CsFUL gene was higher expression in flower buds and fruits,CsAGL6 gene was expressed in four floral organs and high expression in sepals,petals and carpels.CsAP1conformed to the expression pattern of typical class A,while CsFUL had its unique function,it regulated the differentiation of flower primordia and the development of bud leave and fruit.The CsAGL6 was involved in the regulation of four floral organs development.In addition,the three genes were highly expressed in fruit,suggesting that these genes might jointly control the fruit development.4.Phenotypic analysis of transgenic Arabidopsis showed that 35s::CsAP1,35s::CsFUL and 35s::CsAGL6 transgenic plants had early one week flowering.The early stage of reproductive growth led to transgenic plants dwarfism and leaves small.Transgenic Arabidopsis overexpressing CsAP1 had apical inflorescence converted to a terminal flower that were composed of two flowers,the petals from 4 to 5,lateral inflorescence converted to a single flower.It indicated that the gene CsAP1 controlled sepals and petals development and promoted the differentiation of inflorescence meristem to flower meristem.35s::CsFUL transgenic Arabidopsis showed rosette leaves and cauline leaves were curly,indicating that CsFUL gene had a certain effect on leaf development.35s::CsAGL6 transgenic Arabidopsis were short and small,in addition to early flowering,had not other phenotypic changes.5.Subcellular localization,yeast one-hybrid and yeast two-hybrid experiments showed that protein CsAP1,CsFUL and CsAGL6 had nuclear localization ability and no transcriptional activation activity.Three protein formed heterodimers each other.Those results indicated CsAP1,CsFUL and CsAGL6 might produce transcriptional activation activity with the help of other proteins,then combined with the target gene to regulate flowering transition and flower development in tea plant.