Studies On Transgenic B. Napus With Antisense Myrosinase Genes

Rapeseed is one of the most important oil crops worldwide. Among the threemajor types of oilseed Brassica, B. napus have the largest acreage. As of itsimportance, the acreage of rapeseed is still increasing. China is the biggest country forrapeseed production, which accounts for one third of globle production as well asplanting area. Along with the improvement of life quality and a huge demand ofindustry oil, there is still a great potential for rapeseed production in China, whichpromotes the quality improvement.Plant genetic engineering refers to transfering target DNA into a host cell andexpressing it in order to get the transgenic plant. Major methods for genetransformation include Agrobacterium mediated, PEG untake, electroporation,particle gun, microinjection, laser microbeam, pollen-tube pathway, ect.. Genetransformation mediated by Agrobacterium is widely used in Brassica breeding.Genetic Engineering can be applied for quality improvement, disease or insectresistance and herbicide torelence in rapeseed.The secondary metabolites called glucosinolates exist in many Brassicaceaespecies. Upon pest attack or mechanical damage, cellular breakdown exposes thestored myrosinases to degrade glucosinolates. The glucosinolates degradationproducts are thiocyanate, isothiocyanate or nitriles which are involved in defensesystem against insects and phytopathogens and potentially in sulphur and nitrogenmetabolism and growth regulation. However, the myrosinases-glucosinolates defensesystem might not be useful to some specialist insects which depend on theglucosinolates or degradation products to recognize plant. In this paper, myrosinasesgene are inhibited by antisense technology in B. napus, thus the level of myrosinasescontained in transgenic plants down regulated. Furthermore, the transgenic plants maybe resistant to some specialist insectsGene sequence coding for myrosinases in Arabidopsis (TGG) are highlyhomologous to that of B. napus. In this study, we transformed the antisense TGGexpressing vector pBasTG in to rapeseed, wichi is under the control of a superpromoters.. The main results are as follows:1. By testing the regeneration abilities of four B.napus varieties, "zhongshuang6" is choosed as the transformation plant.2. Screening the Agrobacterium of GV3101,LBA4404,EHA105,AGL1,C58,GV3101 is choosed as the transformation bacterium. 3. The cotyledon transformation system is primarily established. The explantsshould be cultivated for 5 or 6 days. The OD600 of GV3101 should be 0.08-0.48. Thecotyledons should be infected by GV3101 for 5-10 min. Then the cotyledons areco-cultured in M1 for 3d; later lay-culture in M2 for 5-6d. After that the cotyledonsgrow in M3 until the green roots appear. AS, 2, 4-D, 6-BA, AgNO₃ are needed intissue culture. 8mg/L of Spect would be proper. This transformation system is simpleand convenient.4. By PCR and RT-PCR testing, 2 strains contain the target gene fragment.5. By testing the resistance of the transgenic plants to Plutella xylostella andaphid, there is no obvious difference between transgenic plants and wild plants.