Studies On Transformation Of Descurainia Sophia Trypsin Inhibitor Gene Into Brassica Napus L.

Plant proteinase inhibitors(PIs) have been well established to play a potent defensive role against predators and pathogens.PI genes are of practical advantages including safety, nontoxic or non-deleterious effects on mammals and animal,broad-spectrum resistance to insects.Hence,research on functional PI genes has been much paid attention to enhance plant resistance/protection against pests.Present studies firstly introduced trypsin inhibitor genes from Descurainia sophia (DsTI1 and DsTI2) into Brassica napus L.via Agrobacterium tumefaciens-mediated transformation,then preliminarily explored their functions.Major results are as follows:Firstly,the Plant expression vectors pCAMBIA1301-DsTI1 and pCAMBIA1301-DsTI2 were constructed.The constructs were confirmed by restricted enzyme digestion and transferred into Agrobacterium tumefaciens strain LBA4404 by freeze-thaw method.Secondly,transformation system were established,using cotyledonal petioles of canola line NJ5424.Seeds sterilized were germinated in MS medium containing 4.0 mg/L 6-BA,explants infected were cultured on selection medium supplemented with 3.0 mg/L 6-BA,0.1 mg/L NAA and 12.5 mg/L Hyg.Subsequently,regenerating shoots were elongated on medium with 2.0 mg/L 6-BA,0.1 mg/L NAA and 12.5 mg/L Hyg.Total 41 hygromycin resistant plants were obtained,and 24 of them were positive plants by PCR analysis,which transformed with DsTI1 and DsTI2.Thirdly,transgenic plants had been confirmed by PCR and enzyme activity assay experiments.PCR detection showed that T₁ transgenic families of DsTI genes were conformed to a Mendelian ratio 3:1 except for one family.RT-PCR analysis of T₁ transgenic plants showed that DsTI genes could express.The enzyme activity assay indicated that leaf extracts from transgenic plants exhibited high inhibitory activity towards trypsin and chymotrypsin,revealing that the two DsTI genes functioned as a serine proteinase inhibitor.Artogenia(Pieris rapae L.) larvae reared with transgenic rapeseed leaves,were observed,showing that young insects grew slowly and died,showing high mortality ratio.These facts might indicat that transgenic rapeseed can resist to artogenia.Lastly,to evaluate effects of DsTI genes on agronomic traits of transgenic plants in T₁ generation,traits were observed by randomly sampling.Results showed that there were not siginificant differences between transgenic and wild type plants on most of agronomic traits observed,but there existed significant differences on few traits.For example,in DsTI1-2 family,there were difference on traits silliques density between transgenic and wild type plants,in DsTI1-4 family,trait of significant difference were seed per siliques.