Cloning And Functional Analysis Of The Promoters Of Flower Development Genes PaAP3?PaPI And PaSTK In London Plane Tree And Their Application In Sterility

London plane tree(Platanus acerifolia)is one of the most important trees in our country in the greenization of the city.Due to its many desirabletraits,such as big crown shadow,rapid growth,strong adaptability and ability to purify the air,thus it waswell known as the king of trees.The tree was widely cultivated in the world,which occupies an irreplaceable role in the landscape architecture.However,seed fair after fruit cracking seriously affect the city's environment and people's health.In recent years,many researchers have done lots of work in solving this problem,such as mutation breeding and branches pruning.Howerver,none work can fundamentally solve the problem of seed fair pollution.With the development of technology of plant molecular biology,especially in the use of genetic engineering to create sterile plants,has made great breakthrough and bring new hope for the cultivation of sterile trees.Since the ABC model was announced,the researchers have made a broad analysis on regulatory network of floral organ development,which makes research from the morphological description and physiological level to the molecular regulation.With the cloning of London plane tree flowering related genes,the mechanism of flowering was understood gradually.This work will improve the ultilization of genetic resources to landscape tree breeding.We separated two promoter sequences of class B genes of flower development by using TAIL-PCR from London plane tree.We performed bioinformatics,promoter truncation,GUS staining and expression analysis of the promoters.Afterwards,the toxic gene BARNASE was fused to the promoters and then transformed to tobacco leading to flower abortion.Meanwhile,the genomic and promoter sequences of STK were also isolated and the function was systematically analyzed.Our study will provide more information for fully understanding the flower development mechanism of London plane tree;also lay the foundation for obtaining sterile plants by genetic manipulation.The main results are as follows:1.The promoter sequences of PaAP3(pPaAP3)and PaPI(pPaPI)were isolated by TAIL-PCR according to the known CDS sequences.The promoter region was analyzed by bioinformatics after sequence validation.According to the core promoter sequences,both of them contained some tissue specific targetin sites,such as flowering related MADS genes and stem cell genes WUS.It is speculated that these promoters are likely to have the characteristics of tissue specific expression.2.Different truncated promoters(pPaAP3-p1 to pPaAP3-p8,pPaPI-p1 to pPaPI-p5)fused with GUS genes were transformed into tobacco according to the bioinformatics information.The result of histochmical staining showed that the activity of pPaAP3 promoter was high with 2900 bp length(pPaAP3-p8),but the expression of GUS was detected in all the four whirl flower organs,stem and leaves.In the relative,the pPaPI promoter had high activity in the flower petal,anther and pistil with 2800 bp fragment(pPaPI-p5),a certain level activity in stems and no activity in leaves.The expression of GUS gene was also detected in the transformed plants by semi quantitative PCR method.The results showed that both of the pPaAP3 and pPaPI had the basic function as promoters to activate the expression of genes.However,based on the different expression model of these two genes,the pPaPI promoter was more suitable for the sterile material creation.3.Considerring the result of GUS staining of different truncated promoters,pPaAP3-p8 and pPaPI-p5 were selected to fuse BARNASE gene and then transformed into tobacco.The results showed that only a small mount of positive plant transformed pPaAP3-p8::BARNASE could survive to the period of flowering,and the survivals couldn't form normal buds and the apical meristem was brown withered.Otherwise,the transformed plants of pPaPI-p5::BARNASE had normal vegetative growth and branch number was increased.It is 60 days later compared with the wild control when the abnormal buds were formed.The flower organ only harbors residual sepals and abnormal buds fall off early.RT-PCR results showed that the expression of BARNASE gene in non reproductive organs was less in pPaPI-p5::BARNASE than pPaAP3-p8::BARNASE transgenic plants.These results suggested that the pPaPI promoter may be more suitable for creating sterile plants of London plane tree.4.The MADS-box ABCE like genes except that of D-class genes were isolated and studied before.Therefore,we cloned D-class gene PaSTK by conserved doamain amplification and RACE method.The conserved domain of PaSTK was studied.Alignment of PaSTK with homologs in other species reveals its status in the evolution.In a addition,the expression quantity of PaSTK was deceted in various tissues by qPCR,and the result shows that the expression of PaSTK was greatly upgraduated in female infloresence in April and June.This study laid the foundation for a better understanding of the molecular mechanism of flowering D class genes.5.According to the PaSTK gene information,the promoter sequence(pPaSTK)was isolated by TAIL-PCR.The promoter driven GUS gene was transformed into Arabidopsis thaliana.High expression of GUS gene in seed was detected,and it was weakly expressed in other nutritive tissues and various development stages of flower.The promoter fused BARNASE gene was also transformed into Arabidopsis thaliana.It was found that the transgenic plants developed normally,and and formed normal flower and pod.However,seeds were not found in the pod which means the expression of BARNASE gene was activated in the early stage of ovule development driven by pPaSTK.These results also suggested that the pPaSTK promoter could be utilized for creating sterile plants.