Construction Of Expression Vector And Genetic Transformation In Phalaenopsis Of Phalaenopsis Floral Meristem Identity Gene LFY

ABSTRACT: LFY-floral meristem identity gene-is widely expressed in vegetative organs and vegetative organs in plants.LFY is closely related to the gene that regulate and control plant flowering time.Four ways that regulate flowering time are all expressed and integrated in the upstream region of LFY. The expression of LFY gene will promoter after floral induction.LFY is a feature gene that controlled inflorescence meristerm tissue by studying the model plant Arabidopsis deeply.LFY gene promote the formation and control the attribute of flower meristem.LFY can maintain the normal function of flower meristem,initiate flowering,prevent the reversion of flower meristem.LFY is firstly expressed among flower meristem identity genes.LFY gene encoding a development on-off,its activity is enough to change all rachis to single flower bud,can promote the blooming.LFY gene can't form flower until its expression is enough and its over-expression have no effect on flowering model but improve the flower formation. With the energetic development of biotechnology people begin to study the using of LFY by transgenic technology, over-expression of LFY gene make flowering early in plants such as poplar,tobacco,chrysanthemum and rice. In this experiment, we construct a expression vector pRI101-LFY based on the previous studies and transform it into Phalaenopsis using Agrobacterium-mediated method. Then identificating the transgenic plants to study the transformation and expression of LFY gene in Phalaenopsis,then we can get the early-flowering cultivar of Phalaenopsis if LFY gene was over-expressed. Our results of this research were as follows:(1) Acquiring and cloning of LFY in PhalaenopsisAccording to the reported LFY cDNA in NCBI, RT-PCR were adopted to gain LFY gene in Phalaenopsis.The LFY gene was connected to the cloning vector pMD19-T and transferred into E.coli DH5α.Then the recombinant plasmid pMD19-T LFY structure was verified by PCR,double-enzyme cleavage and gene sequence analysis. (2) Construction and identification of the expression vector pRI101-LFYUsing gene recombination technique to clone the LFY gene to expression vector pRI101 and transfer pRI101-LFY into E.coli DH5α.Identified the recombinant plasmid via PCR and double-enzyme cleavage.(3) Transferring pRI101-LFY to Agrobacterium tumefaciensThe recombination plasmid pRI101-LFY was introduced into Agrobacterium tumefaciens EHA105 by freezing and thawing. The positive clone was screened with Rif and Kana. Through plasmid PCR and double-enzyme cleavage identification, it is proved that pRI101-LFY was transferred to Agrobacterium tumefaciens EHA105.(4)Construction of Phalaenopsis regeneration systemPhalaenopsis protocorm were used as explants and MS as basic culture medium. The medium 1/4 MS+KT 0.5mg/l+AC 1g/l+peptone 1g/l+coconut juice 50% was selected for seeding; 1/2 MS+NAA 5mg/l+IBA 5mg/l+AC 0.5%+potato 50g/l for sprout; 1/2 MS+6-BA10mg/L+coconut juice 20g/l for inducing the differentiation of adventitious shoots; 1/2 MS+6-BA 0.3mg/l for rooting cultivation.(5) Transformant selection and plant regenerationAfter be precultured for 3-4 days, the explants were dipped in suspension of Agrobacterium tumefaciens for 5-15 minutes and cultured for 2-3 days. Then transfer them to the medium that containing MLPN and Kar for differentiation and selection.The transformants transferred into foreign genes would be germinated and took root. Thus the transgenic plants were obtained.(6)PCR test of transgenic PhalaenopsisTransgenic Phalaenopsis was performed with PCR reaction amplification using the genomic DNA as a template.The results showed that LFY has been transformed into Phalaenopsis and the transformation rate is 25%.