The Study On Transgenic FAD8 Of Improving The Plants Cold Resistance In Seedlings Of Jatropha Curcas

Jatropha curcas of the family Euphobiaceae is a deciduous shrub or small tree,it has large output and the seeds contain high oil.the oil components meet the standard of biodiesel,so that is an ideal raw material for the biodiesel.But the Jatropha could be injuryed seriously by the low temperature and the drought.It is important to study on improving it's fatty acid composition and cold resistence through the way of biotechnology.Arabidopsisω-3 fatty acid desaturase FAD8 is an enzyme which catalyze that the dienoic acid rapidly unsaturase to produce trienoic acid in cold condition.,which can increase the content of unsaturased fatty acid in the seeds and plants,and improve the plant oil nutrition and provide plants obviously resistance in cold condition.In our research,set up better regenerated and transformed system,used agrobacteria-medidated method transformed Arabidopsis FAD8 gene into the Jatropha,identified and filtrated the transgenic plant, and studyed fatty acid metabolism and cold-resistance in the transgenic Jatropha.1,Establishment of high efficient regeneration system of Jatropha curcasPeeled the seeds of Jatropha curcas and sterilized them.The explants of 15 days hypocotyls,cotyledon and the petiole of cotyledon from aseptic seedings of Jatropha curcas were used to callus induce and shoots regeneration,which were growed in MS containing 6-BA 3.0mg/L and IBA0.5mg/L medium.The best cotyledon's differentiated medium is MS+6-BA 3 mg/L+ IBA 0.5 mg/L,and the differentiated rate is 82.9%.The best differentiated medium for hypocotyls's bud is MS+6-BA 2 mg/L+IBA 0.2 mg/L,and the differentiate rate is 23.8%.The best petiole of cotyledon's differentiated medium is MS+6-BA 3mg/L+IBA 0.1mg/L,and the differentiated rate is 32.7%.Adding AgNO₃ 1.0mg/L into the regeneration medium of explants could promote the bud differentiation,and inhebit browning.The WPM medium is the best one in rooting of the regeneration buds,and the rooting rate is 29.6%.The differentiated rate of each explants of Jatropha curcas is higher than ever reported,and get the regeneration plants from petiole of cotyledon for the first time.2,Establishment the transformation System of Jatropha curcas Mediated by Agrobacterium tumefaciensUsed agrobacteria-medidated method transformed the FAD8 genes(the plasmid is pSH-FAD8) into the 15 days aged Jatropha curcas hypocotyls,cotyledon and petiole of cotyledon. The explants were submerged into 0.5(OD₆₀₀)Agrobacterium suspension contained 200μg/L AS (hypocotyls for 10 min and cotyledon and petiole of cotyledon for 8min),and placed on co-cultivation medium for 3 days dark in 28℃.Then placed them into the medium of MS+6-BA 3mg/L + IBA 0.5mg/L content cephalosporin 100mg/L for 5 days.The content of kanamycin(Kan) selection pressures were determined as 30mg/L for shoot regeneration of cotyledon and petiole of cotyledon,50mg/L for hypocotyls,the results showed that the extra gene fad8 was integrated into Jatropha curcas genomes successful confirmed by GUS staining,PCR and RT-PCR,and get 27 transgenie plants.In this study,we established more perfect ransformation System of Jatropha curcas than ever,and get the transgenic plants.3,Analysed the fatty acid content in the transgenic and control Jatropha curcas by the reverse phase chromatography,and showed that the sort of the fatty acid in the leaf of the transgenic Jatropha curcas was not changed.4,The test about physiological indicators of cold-resistence showed that the cold-resistance ability of the transgenic Jatropha curcas were higher than the control.Choosed two transgenic plants,and studied the cold-resisted ability:the Chlorophyll Comparison of light damage,CAT,POD,malondialdehyde and proline content all enhanced more in transgenic FAD8 gene Jatropha curcas plants.