Cloning And Functional Analysis Of The Second Intron Of Agamous Gene In Landon Plane

The London plane(Platanus acerifolia Willd)is one of the widely cultivated street trees.It’s owns many excellent growth characteristics,such as simple to cultivate,grows fast,graceful shape,thick crown,innate disease-and insect-resisitant,and have strong adaptability to city environment.However,when it comes to the late spring and early summer in every year,the London plane could harm the environment and endanger public health that its dispersion of seeds and release of seedhair,tomentum from leaves and the pollen,which would not only pollute the environment but also may result in serious pollinosis to affect human’s normal life.This negative trait reduce the value of its garden application value.The major methods used in this thesis is to obtain the complete sterile London plane by floral organ-specific intron strategy.We isolated the second intron of homologous AG genes in London plane by TAIL-PCR.And then we constructed the b-glucuronidase(GUS)fusion vectors and cytotoxic gene sterile vectors which drived by the intron.Finally,we analyzed the activation of the GUS reporter gene by enhancers contained within the AGAMOUS second intron(Plac AGI).Following is the major results:1.A 4644 bp length the second intron of AG homologous gene Plac AG in Landon plane was cloned,and was named as Plac AGI.Putative cis-elements analysis through bioinformatics showed that Plac AGI owned abundant tissue specificity cis-elements.2.The f Plac AGI::GUS,r Plac AGI::GUS,f Plac AGI-mi35S::GUS,r Plac AGI-mi35S::GUS was constructed and transformed into arabidopsis and tobacco.The function of Plac AGI was analysed through the Histochemical GUS staining of transgenic tobacco and transgenic arabidopsis.The result displayed that GUS expression specifically in carpel and stamen.3.The sterile vectors f Plac AGI::Barnase-mi35S-Barstar,r Plac AGI::Barnase-mi35S-Barstar,f Plac AGI-mi35S::Barnase-mi35S-Barstar,r Plac AGI-mi35S::Barnase-mi35S-Barstar were constructed and transformed into tobacco.We did get transgenic tobacco.As we expected,this highly specific intron(Plac AGI)can also direct the expression of the Barnase cytotoxic gene exclusively in carpels,stamens and petals,resulting in complete sterility through the precise ablation of targeted floral organs.The orientation of the Plac AGI enhancers significantly influenced the effectiveness of the chimeric promoters.Forward-oriented were more effcient to the reverse-oriented promoters in terms of floral organ ablation efficiency.The result showed that Plac AGI was not only have tissue specificity,but also have promoter characteristics.