Constuction Of ICE1 Gene Plant Expression Vector And Research Of Transformation Into Tomato

Tomato named Lycopersicum esculentum Mill in Latin,an annual herbage plant in the solanaceae or nightshade family, becomes a world-wide vegetable because of its rich nutrition and delicious taste now. Low temperature is the key factor that limits the geographical distribution, quality and yield of tomatoes. The topic of how to improve the cold-resistance of tomatoes has always been brought into focused that people attach importance to. Recently new approaches have been achieved by speedily developing plant genetic engineering technique,the stress tolerance by transforming foreign genes has become dominant methods for improving modern plants breed. Construction of plant expression vector was realize foreign gene in recipient cell stable exist, and may inherited to next generation ,also the key to induce target gene expression and play a role .This research cloned cold tolerance gene ICE1, constructed the plant expressing vector, ICE1 gene into tomato mediated by Agrobacterium tumefaciens. Functional analysis demonstrated its crucial role the responses of plant to cold stress. Research results are mainly as follows:(1)The experiments digested tomato ICE1 gene which was cloned to PMD18-T vector with doubleEnzymes NCOI andBSTEII contain 1500bp.expression vector of tomato ICE1gene was successfully constructed after linking the1500bp fragment and pCAMBIA3301vector which was digested by NCOI and BSTEII using T4DNA ligase enzymes.The result showed that ICE1 successfully linked to Plant expression vector pCAMBIA3301.(2)The explants were cotyledon,culture medium supplied with different combination.This was the best bud induction medium:MS+2mg/L6-BA+0.2mg/L IAA+3% sucrose+ 0.8%Agarose,PH5.8(3)Study the factors that effect tomato transformation.An efficient gene transformation system was established,and the optimum conditions for the transformation as follows:Cotyledons were pre-culture 48h on bud induction medium:Agrobacterium were diluted,in which explants were soaked for 8-10 minutes.Then the explants were co-cultured for 36h. After that,the explants were cultured on select medium.When shoots were more than 1cm high,they were cut and transferred on medium(1/2MS+0.5mg/L IAA+20mg/L Kan+300mg/L Carb)for rooting.(4)we got 5 Kan-resistance transgenic plants, the PCR and RT-PCR result showed that 3 regenerated pants of them had amplified idea DNA band, positive plant rate was 60%,The results proved that ICE1 gene have been transformation into tomato.(5)The transgenic tomato were treated with low temperature(4℃)stress for 72h,the content of MDA were apparently lowered,the content of Pro and the activity of CAT and POD were significant higher. compared with non-transgenic tomato.