The Genetic Transformation Of Plant Flowering Pathway Key Genes AtLEAFY And Screening Of Early Flowering Lines For Chrysanthemum Morifolium

Chrysanthemum morifolium is belong to the genus Chrysanthemum in the compose family,which are the perennial herbaceous perennial plant.The plant-type is low and strong,has features such as cold,combat a drought,combat poor,stand up insect pest,flowering prosperous and extensive management.So it has an important role in Landscaping.Chrysanthemum is a typical short-day plant,dependent on Photoperiod.Flowering time is mostly concentrated in September to October months,usually continued 40-50 days.In the cold and high latitudes Northeast,Chrysanthemum could be normally growth and reproduction.But flowering encountered frosts,resulting ornamental significantly decreased and dramatically reduce viewing period.In order to enjoy the sight and meet the market for the early flowering of Chrysanthemum requirements.Pour exogenous gene into others by genetic engineering technology.The exogenous gene over expressed to achieve the purpose of changing the flowering time.Thus,quick access to early flowering of new transgenic varieties of Chrysanthemum has important economic significance and promotional value.Experiment to change Chrysanthemum 'flame' flowering time as a starting point,according to the LEAFY gene which has been reported not only control the transition inflorescence meristem to floral meristem,but also has the role of regulation of flowering time.The use of transgenic technology operations,the screening system of mannose to obtain resistant plants,and associated the assay of molecular biology experiment.It aims to get exposed to early flowering Chrysanthemum'flame' early flowering lines,which can lay a good foundation for further study and discussion chrysanthemum flower induced molecular mechanism.The overall results are summarized as follows:1.Clone and construction of expression vector.Clone LEAFY gene from Arabidopsis thaliana(L.).It is a full-length gene for 1275 bp.The gene were obtained with the database published Arabidopsis LEAFY gene sequence homology was 100%.The use of Gateway technology to construction of over-expression vector.After verification test of PCR and restriction,successfully put AtL EAFY gene into the expression vector,and by repeated freeze-thaw method transferred into EHA105 strain2.Genetic transformation of Chrysanthemum 'flame' and obtain resistance plants.By Agrobacterium-mediated leaf disc method infested Chrysanthemum 'flame' blades genetic transformation total of 5650 explants.Use biosafety phosphomannose isomerase(PMI)marker gene of the vector,in a medium containing mannose to filter.After pre-culture,co-culture,the culture pre-screening,post-screening culture,rooting stage screening cultivation,obtained obtaining resistant 218 plants.3.Molecular biological identification of transgenic plants.Resistant plants are detected by biological molecular such as PCR,GUS staining,western blot methods.4.Transgenic plants phenotype identification.The transgenic plants and wild-type plants were acclimated,and transplanting into the land at the same time.Observation of transgenic plants of budding time,the bud stage,the first flower time,and full blooming time were compared with the wild type in order to obtain early flowering lines.Transgenic plants flowering 6 to 14 days in advance,get nine early flowering strains flower lines and two later lines.