Molecular Cloning,Expression Analysis And Its Transformation Of ACS Gene And ACO Gene From Narcissus Tazetta Var.'Yunxiang'

'Yunxiang'(Narcissus tazetta var.'Yunxiang')is a new breed of Narcissus selected and bred by Institute of Genetics&Breeding in Horticultural Plants,Fujian Agriculture and Forestry University,April 1,2012 by Fujian provincial crop variety Approval Committee,compared with other species of Narcissus,'Yunxiang' has more flowers,longer flowering period,strong aroma,scape stand up,can be used as cut flowers.But cut flowers have high requirements for storage and flowering.This study will isolate ACS and ACO gene from 'Yunxiang',analyze its expression and vector construction,and transform tobacco through agrobacterium mediated transformation,and obtain transgenic plants.This will provide experimental basis for further identification of the gene function and subsequent transformation of Narcissus.The aim is to get a longer storage life of 'Yunxiang' through genetic engineering to regulate the ethylene metabolic pathway,so as to expand the use of Narcissus and make it more suitable for fresh cut flowers.During this study,we have achieved the following results:1.Aimed to study the characteristics and functions of ACS gene,in the present study,a 1-aminocyclopropane-l-carboxylate synthetase gene named NtACSl(Genbank KX082936)was cloned based on the RNA-Seq database from the flower of 'Yunxiang' using RT-PCR method.The length of the open reading frame(ORF)of ACS is 552 bp,encoding 183 amino acids coupled with a molecular weight of 20.6 kD and theoretical isoelectric point of 6.30.qRT-PCR analysis showed that the relative expression levels of NtACSl both in perianthes and coronas are decreased gradually along with the aging of flower,moreover,the expression data of NtACSl gene were consistent with those obtained by RNA-Seq,implied that the NtACS1 protein as an ACC synthetase might play a role in the catalytic system 1 of ethylene biosynthesis.Furthermore,sense plant expression vectors of NtACS1 were successfully constructed with agrobacterium mediated transformation,and 6 positive transgenic tobacco plants were ultimately obtained.2.ACC oxidase(ACO)is a key enzyme in the biosynthesis pathway of ethylene.In order to explore the characteristics and function of the ACO gene in Narcissus,the following work has been done in this study:Petals of Narcissus tazetta var.'Yunxiang' were used as experimental materials,a full-length 1 162 bp sequence was obtained by RT-PCR and named as NtACO1(GenBank KX082935).The open reading frame(ORF)length of NtAC01 is 939 bp,which encodes 312 amino acids.Amino acid domain analysis showed that this gene has DIOX_N and 20G-Fe ?_Oxy domains and was identified as a member of the ACO protein family.Molecular phylogenetic tree analysis showed that ACO gene of'Yunxiang' was closely related to that of Narcissus tazetta var.chinensis.qRT-PCR results showed that the expression of NtACO1 increased gradually with Narcissus flower development and senescence,and the expression level in the petals were higher than that in contemporaneous coronas,indicating that the NtACO1 gene may be involved in the flower development and senescence of 'Yunxiang'.We obtained several NtACO1 overexpression transgenic tobacco plants using the Agrobacterium mediated.Compared with wild type,the vegetative growth stage of transgenic tobaccos was reduced,as well as the transgenic tobaccos showed early flowering and less leaves.3.In this research,a gene named NtACOY2(GenBank KX214535)encoding a new ACO(ACC Oxidase)protein was isolated using RT-PCR from 'Yunxiang'(Narcissus tazetta var.'Yunxiang').NtACOY2 consisted of an ORF(open reading frame)with a length of 936 bp,and encoded a 35.83 kD protein with 311 amino acids.Bioinformatics analysis indicated that NtACOY2 contained a typical DIOX_N and 20G-Fe ?_Oxy binding domain.Evolutionary analysis indicated that the amino acid sequence encoded by NtACOY2 was highly conserved.Real-time fluorescence quantitative PCR results revealed that the expression of NtACOY2 in the petal and deputy crown is increasing with the senescence of 'Yunxiang'.The expression of petals in each period was higher than that in the secondary crown,and the highest expression in petals at bud stage.As a result,NtACOY2 may functions as a ACC oxidase gene involved in the development of 'Yunxiang' and related to flower senescence.The expression vector of NtACOY2 was successfully constructed by PCR and identified by restriction enzyme digestion reactions.And the sense plant express vector of NtACOY2 was used to transform the tobacco through Agrobacterium-mediated procedure.PCR detection showed a total of 15 transformed plants were amplified fragments with the same size as the positive controls.The result of RT-PCR identification showed that 12 transgenic tobacco plants were obtained.2 randomly selected transgenic plants(Z1,Z2)and wild type(WT)exactly the same condition of transplanting under greenhouse cultivation,the results of Zl,Z2 were earlier than WT 8 D and 7 d flowering,and the number of transgenic tobacco flowers were more than the wild type,showed that NtACOY2 could normally expressed at the transcriptional level.