Cloning And Functional Analysis Of LFY And MADS-Box Homologous Genes In Platanus Acerifolia And Its Phylogenetic Evolution Analysis

London plane tree（Platanus acerifolia Willd.） is one of the most important landscape trees used for improving city environments.In the ranges of Yellow River and Yangtse River of China and the majority of temperate and subtropical cities all over the world,the tree is extensively planted in roadside,street,courtyard and industrial area due to its many desirable traits,such as fast growth,broad crown,wide adaptability,good tolerance to pruning,and high resistance to environmental pollution,which is called to be "the king of street tree".In some regions,it even becomes the unique tree and non-substitutional by any other plant.However,its pollen and seed hairs disperse widely in the spring,which affect human health.In the past several years,many researchers have carried out a lot of studies on this subject,but have not yet resolved the problem completely.The major objective of this study was breeding male and/or female sterile individuals of London plane tree.For the purpose,an efficient Agrobacterium tumefaciens-mediated transformation system of Platanus acerifolia was optimized and developed firstly by GUS reporter genen.Subsequently,cDNA library of female inflorescence was constructed.On the other hand,several flower development related homologous genes in P.acerifolia,SOC1、LFY and A-,B-,C-,E-class of MADS-box, were isolated by RACE technique,partial genes were expression and functional analyzed by real-time PCR and over-expression in tobacco.The major results as following:1.The pre-treatment of leaf explants with 0.4 M mannitol,an inoculation period of 10 min,a bacterial OD₆₀₀ of 0.8-1.0 and a co-cultivation period of 5 days,were recovery and selection cultured.Twelve putative transformed lines were obtained from 312 explants,eight of them contained the nptⅡT-DNA gene,as confirmed by PCR analysis. Furthermore,Southern blot analysis confirmed that,in at least five of these lines,the transgene was integrated into the plant genome.2.Three RNA extraction protocols,including Trizol Kit（Trizol reagent 1995）, guanidinium thiocyanate and SDS method,were compared with the improved CTAB protocol.A detailed RNA isolation and purification protocol,based on established cetyltrimethyl-ammonium bromide（CTAB） extraction techniques combined with additional purification steps using butanol and the ionic detergent CTAB,which overcomes phenolic compounds in the woody species P.acerifolia,was conducted. Electrophoresis of samples through a 1%（w/v） agarose gel appeared distinct 28S and 18S rRNA bands,indicating good quality of total RNA.The A260/280 ratios were also around 2.0,indicating that the RNA was of adequate purity for further molecular analysis. Furthermore,RNA isolates from female inflorescences were used for the construction of a cDNA library was constructed by SMART（switching mechanism at 5′end of RNA transcript） and LD-PCR（long distance PCR） techniques.The titers of unamplified and amplified cDNA library were 5.3×10⁶ pfu/ml and 8×10⁹ pfu/ml respectively.The rate of recombination was 97%.The length of inserted fragments was 400-2200 bp and the average was 900-1000 bp.To determine the integrality of the cDNAs,a further 100 clones were selected at random from the library and the nucleotide sequences determined. As a result,eighty-seven clones were proved to contain a full length cDNA.3.The full-length of cDNA of LFY,FUL,AP3,AG,SEP1,SEP3 and 3’end of SOC1 homologous genes were obtained by RACE.The seven genes were named PaLFY, PaFUL,PaAP3,PaAG,PaSEP1,PaSEP3 and PaSOC1 respectively.PaLFY is 1419bp in length,and has an ORF of 1122bp which encodes a predicted polypeptide of 374 aa with 5’/3’-UTR of 54 and 213bp.PaFUL is 996bp in length,and has an ORF of 735bp which encodes a predicted polypeptide of 245 aa with 5’/3’-UTR of 128 and 103bp. PaAP3 is 930bp in length,and has an ORF of 678bp which encodes a predicted polypeptide of 226 aa with 5’/3’-UTR of 83 and 139bp.PaSEP1 is 1160bp in length, and has an ORF of 735bp which encodes a predicted polypeptide of 245 aa with 5’/3’-UTR of 163and 251bp.PaSEP3 is 993bp in length,and has an ORF of 720bp which encodes a predicted polypeptide of 226 aa with 5’/3’-UTR of 64 and 179bp.PaSOC1 is 638bp in 3’ end,and has an partial ORF of 546bp which encodes a predicted polypeptide of 182 aa with 3’-UTR of 82bp.PaAG is 838bp in 3’-end,and has a partial ORF of 570 bp which encodes a predicted polypeptide of 190 aa with 3’-UTR of 243bp.PaSOC1 is 638bp in 3’-end,and has a partial ORF of 546 bp which encodes a predicted polypeptide of 182 aa with 3’-UTR of 82bp.4.Expression analysis of PaLFY,PaFUL,PaAP3,PaAG,PaSEP1,PaSEP3 and PaSOC1 were made by RT and Real-time PCR.5.Sense plant expression vectors of PaLFY,PaFUL,PaAP3,PaAG,PaSEP1 and PaSEP3 were construted.Transgenic plants were obtained by the leaf discs method of Agrobacterium-mediated transforomation in tobacco.The nptΠand target genes were both detected in over 85%of the Kanamycin-resistant tobacco according to PCR verification.6.In order to determine the phylogenetic status of P.acerifolia in plants,the conserved MIK or MIKC domain of nucleotide or protein sequences of PaLFY,PaFUL, PaAP3,PaAG,PaSEP1 and PaSEP3 and their respective homologous genes were analyzed by Maximum-likehood（ML）,Mrbeyes and Neighbor-joining（NJ） method to construct ML,Mrbeyes and NJ tree,which indicated that P.acerifolia is a basic eudicot belonging to paleo-species,closed to Proteaceae and Nelumbonaceae of Proteales, Buxales,Trochodendrales,Ranunculales and Sabiaceae in phylogeny.7.Evolutionary force analysis was conducted to test positive selection based on CDS of PaLFY,PaFUL,PaAP3,PaAG,PaSEP1 and PaSEP3 by PAML3.14beta3 software under branch-specific,site-specific and branch-site model.No positive selection was detected by branch-specific or site-specific model.The branch for PaAG、PaSEP1 are evolved under the positive selection withω＞1（ω₂ =26.17 and 277.12,P＜0.01） by branch-site model A.8.We incorporated the closest Arabidopsis ortholog for each gene into the multiple alignments for 5 MADS-box genes,then estimated dates of divergence from a pooled data set including all of these genes.Estimates were based on the method of PAML, assuming a codon model and a molecular clock.This analysis yielded estimates of 134.6 MYA（range 123.4-145.SMYA） for the divergence of Platanus,which was near Oryza sativa（Poales） and Houttuynia cordata（Piperales） of 137.1MYA（rang 125.7-148.5MYA） comparing to Arabidopsis of 120MYA（range 110-130MYA）.