Study On Transformation Of CBL1 Gene Of Arabidopsis Thaliana In Maize

For a long time,various biological and abiotic stress have an important impact on the distribuion of plant growth,yield and quality.When plants respond to various external stress, Ca²⁺ as a second messenger which can make an important signal transduction in the plant.A variety of stimuli can cause the concentration of intracellular Ca²⁺ instananeous change in plant cells,calcium signal generated,Ca²⁺/CBL is one of the calcium signal system in plant cells,the calcineurin B-like (CBL) protein family are the Ca²⁺ receptor protein,belong to a multi-gene encoded proteins,participate many environment induced signals and the adjustment process of plant growth and development.CBL1 is a member of the CBL protein family,overexpressed in the model plant Arabidopsis thaliana,can be induced in hypokalemia,drought,salt,damage and promote a series of stress which can induce gene expression to resist external stress.By transfer CBL1 gene into maize,can improve the efficient use of potassium ions in the maize, maize can grow propperly in potassium deficiency soil ,also can reduce the amount of potash ferilizer in agricultural production .In this article callus was induced from immature embryos of maize inbred lines Dan598 to establish regeneration system, which provides basis for further genetic transformation by Agrobacterium tumefaciens-mediated transformation(ATMT). The effects of embryo age,media type,concentration of 2,4-D on the callus induction were studied. It was indicated that 16-18 days post-pollination,2,4-dichlorophenoxyacetic acid concentration at 2.0mg/L are the best condition to induct embryogenic callus; set 5 kinds of culture medium such as basis for further genetic transformation.N6,improved NB,NB,MS,MB to observe the effects of callus induction, effect of maize callus induction with improved NB medium is better than other medium.Differentiation medium supplemented 1mg/L KT,0.5mg/L 6-BA and 0.5mg/L NAA can promote the differentiation of green root growth.The CBL1 gene was trasformed into maize immature embryos,maize embryogenic callus,maize shoot tip by Agrobacterium tumefaciens-mediated transformation(ATMT) . Discuss the infective solution concentration,infective time,co-culture time, Timentin antibacterial concentration and PPT screening concentration to influence the immature embryos and embryogenic callus of maize genetic transformation,as well as obtained the best genetic transformational condition:maize inmmature embryos as explant the best genetic transformational condition is infection concentration OD₆₀₀ = 0.8,infection time is 25min,co-cultured for 3 days,Timentin antibacterial concentration of 500mg/L,PPT screening concentration of 8mg/L, the conversion rate was Dan598 1.3%,H325 1.1% ;maize embryogenic callus as explant the best genetic transformational condition is infection concentration OD₆₀₀ =0.5,infection time is 25 min,co-cultured for 3 days,Timentin antibacterial concentration of 600mg/L,PPT screening concentration of 10mg/L,. the conversion rate was 2.0% .In addition,genetic transformational condition of maize shoot apex the infection concentration OD₆₀₀ =0.8,50kPa pressure infect 10 min, the conversion rate was 2. 2% .The plasmid contain gene CBL1 was inserted ino the maize by pollen tube technology between 8-10 hours after pollinated , plasmid DNA concentration is 500ug/mL and import volume is 200uL, the conversion rate was 0.17%. Obtained the resisant maize according to the screening by Basta. The CBL1 gene was inserted into the maize genom by PCR molecular detection from the resistant maize and seeds previously mentioned.