Studies On Transferring Turnip Mosaic Virus Coat Protein Genes Into Chinese Cabbage

The Chinese cabbage is a kind of popular vegetable.But the Chinese cabbage disease hasn't been solved till now. introduced infected by the turnip mosaic virus.There are many useful objective genes can be used in vegetable genetic transformation,but the research work of Chinese cabbage genetic transformation is little.The reaserch on transforming Chinese cabbage using Agrobacterium-med\ated method hasn't been report at home and abroad.ln the test ,TuMV-CP gene was transferred into Chinese cabbage(Brassica campestris L.ssp.pekinensis) via Agrobacterium -mediated method.A high effective regeneration and genetic transformatin system has been established,The detection by the method of molecular biology, has proved that the regenerative plants are transgenic plants and the target genes have been expressed transgenic plants partly.Meanwhile transgenic progenies were traced and investigated so that heredity,stability and expression of target gene were researched.the virus resistant,stable plants were expected to obtain so that theoretical base can be established for Chinese cabbage breeding by gene engineering.Conclusion of this experiment are as following:1. A high effective regeneration system has been established.When five-day cotyledon of ErNiuXin cabbage,was placed on the polarization medium(MS+5mg/L6-BA+0.5mg/LNAA+5mg/LAgNO3).the number of shoot Differentia -tion is best of all;which was placed on rootage medium(MSi/2+0.2mg/L IBA),the rootage effect is very fine,and gains more regeneration plants.2. The genetic transformatin system has been established.The suitable antibiotics and their concentration were studied for the selecting of transformants. The suitable concentration of the filter, Kanamicin is lOmg/L; The suitable concentration of Cefazolium natriun, the most effective antibiotics,is 300~500mg/L.The genotype is a main factor in the genetic transformation via agrobacteriwn -mediated.The results of orthogonal experiments of the factors which affect mainly the transformation illuminates that the ErNiuXin genotype +bacterium 15 X +explant+dip-dye is the best one .The cocultivation time is 28 days.The experiment shows that:The ErNiuXin cotyledon regeneration frequency is very high, and that the Dongnong901 hypocotyl regeneration frequency is very high.The influence of different strain. The ability of invasion of R1000 was much better than EHA105 when comparing the number of resistant buds.But,the number of transformationpanlt ,R 1000 was lower than EHA105.3. Chinese cabbage resistant virus transgenic plant have been obtained.Transgenic T0 progeny were checked by PCR and PCR-Southern methods,The results prove the existence of target genes in transformation plants. 15 masculine plants, but RT-PCR check,only!2 plants express. The phenomenon of gene silence existed in the part of transgenic plants. The segregation proportion of target genes in transgenic Tl progeny is 3:1 .accordant with the law of Mendelian segregation in a single loci.4. Meet virus experimentThe virus inoculation experiment suggests that the disease incidence and disease index of transgenic Tl progeny were lower than those of CK.Name:Ma WeiMajonCrop Genetics and Breeding Tutor:Prof.ChuiChongShi...