Suppressing The Expression Of DgLsL Gene Effectively By Construction Of RNAi Expression Vector And Genetic Transformation Of Chrysanthemum

Chrysanthemum （Chrysanthemum×morifolium Ramat.） is a traditional famous flower in chinawith a long history of cultivation. It is also one of the most important commercial flower types in theworld. Chrysanthemum has various types, and can be easily propagated with strong vitality. Differenttypes of chrysanthemum, such as artistic chrysanthemum, ground-cover chrysanthemum, cutchrysanthemum and potted chrysanthemum, contribute to the broad applications for flower beds, parks,gardens, and indoor decoration.Chrysanthemum has complex genetic background with a high degree of gene heterozygosity of itsgene, which leads to the difficulty of the genetic breeding of chrysanthemum. The development of plantgenetic engineering provides a powerful tool to improve the chrysanthemum quality for the traits. Thecombination of molecular breeding and conventional breeding are expectable to accelerate theinnovation of germplasm resources and therefore shorten the breeding cycle.The breeding of branchless varieties has an important practical value for standard cutchrysanthemum. As it keeps only one inflorescence at a main branch in production, all of the lateralbranches had to be removed manually, which has heavy workload, and greatly increased the labor costsand management difficulties. It is one of the important goals of cut chrysanthemum breeding to cultivateless or no lateral branches of standard cut chrysanthemum variety.DgLsL （Dendranthema grandiflorum Kitamura Lateral suppressor-like gene, GenBank no.AB206874） gene is cloned in chrysanthemum, and it’s homologic with tomato Ls （lateral suppressor）and arabidopsis LAS （lateral suppressor gene in Arabidopsis thaliana） gene. DgLsL gene is related tochrysanthemum’s branch development. It was confirmed that lateral branch occurrence can be inhibitedin transgenic plants with antisense DNA. RNAi is induced by the degradation of homologous mRNA,which derived by double-stranded RNA complexes. It’s a post-transcriptional gene silencing technologywith higher efficiency of gene silencing compared with antisense technology.In this paper, the DgLsL gene was cloned from cut chrysanthemum ‘Jinba’, and the plant antisenseand RNAi expression vector were constructed, then they were introduced into ‘Jinba’ genome byAgrobacterium-mediated transformation. The main results are followed:1. The construction of plant antisense and RNAi expression vector. DgLsL gene, which was relatedto chrysanthemum’s branch development, was cloned from cut chrysanthemum ‘Jinba’. The genefragments obtained by PCR were sequenced and99.4%identity to DgLsL gene in GenBank. The genefragment of the gene was inserted into the plant expression vector pBI121and pFGC5941to construct theantisense vector pBI121-P0, antisense vector pBI121-P2and RNAi vector pBI121-R2. The correctinsertion of the exogenous gene in the vectors was confirmed by PCR, enzyme and sequencing.2. Establishment of efficient regeneration system from leaf disks of cut chrysanthemum ‘Jinba’and‘Youxiang’ and the study on the optimal concentration of antibiotic. The regeneration rate of leaf disks of‘Jinba’ was highest in MS+0.5mg L^-1NAA+0.5mg L^-16-BA, reaching9.3shoots per explant;‘Youxiang’ has3.2shoots per explant in media MS+1.0mg L^-1NAA+0.5mg L^-16-BA. The best antibacterial concentration to control the growth of Agrobacterium for carbenicillin is400mg L^-1. Theconcentration of kanamycin as the screening marker for Leaf discs of ‘Jinba’ and ‘Youxiang’ is70mg L^-1and50mg L^-1, respectively, while120mg L^-1and100mg L^-1of kanamycin is appropriate for rootingselecting.3. Genetic transformation of ‘Jinba’ mediated by Agrobacterium tumefaciens. The kan-resistantplantlets were obtained after the leaf disks of ‘Jinba’ infected by Agrobacterium EHA105with Plantexpression vector pBI121-P0, pBI121-P2and pBI121-R2.18putative transgenic plants were obtainedafter the infection of EHA105with pBI121-R2, and12were confirmed by PCR, which preliminarilyverified the RNAi region had been integrated into the genome.