Stability Identification And Preliminary Safety Evaluation Of Glyphosate-resistance Transgenic Rapeseed

Rapeseed is an important economic crop in China.Weeds cause serious damage to rapeseed yield and quality.Breeding glyphosate-resistant transgenic rapeseed is one of the effective ways to solve the weed damage.Previously we transferred a novel glyphosate-resistant gene I.variabilis-EPSPS*to the Brassica napus J9707 to obtain a batch of transgenic rapeseeds.Preliminary glyphosate tolerance analysis and expression analysis of transgenic materials had been carried out.On that basis,we selected the single-copy transgenic lines JH-14,JH-125 and JHX-6,identified the genetic stability and evaluated the environmental safety and edible safety of transgenic lines,provided support for the industrialization of glyphosate-resistant transgenic rapeseed.The major results are as follows:1.The integration stability analysis of the target gene proved that each functional element of the T-DNA insertion was stably integrated into the rapeseed genome,and the T-DNA insertion position of each transgenic line was stable across different generations.The expression stability of the target gene showed that the target gene and its expression products were stably expressed across different generations and has similar tissue-specific expression,which was expressed highest in leaves and lowest in seed.2.The glyphosate tolerance analysis showed that the transgenic plants exhibited a high glyphosate tolerance in T₃ and T₄ generation,which can tolerate four times the glyphosate dose used in production.However,after spraying 6-fold glyphosate,plant growth and development of transgenic rapeseed were greatly affected,the height of each transgenic plants decreased,the leaf turned yellow and the pollen abortion occurred.3.We conducted a preliminary environmental safety assessment of transgenic lines.Survival competitiveness analysis showed that the agronomic traits of the transgenic lines and the recipient control were no significant difference.Non-target herbicide tolerance analysis showed that there were no significant difference in the tolerance of the transgenic plants and the recipient control to non-target herbicides.Compared with the recipient control,the transgenic rapeseed had no competitive advantage in survival and had a lower risk of becoming weeds.4.We analyzed the preliminary edible safety of transgenic materials and detected the content of newly expressed protein in edible and feed parts of transgenic plants by ELISA method.The results indicated that the content of newly expressed protein was highest in leaves(5.63×10^-2～10.36×10^-2μg/g fresh weight),and the content in seeds was very low(0.11×10^-2～0.74×10^-2μg/g fresh weight),while no newly expressed protein was detected in non-transgenic materials.Bioinformatics analysis results showed that the allergenic risk,anti-nutritional risk and potential toxicity of the newly expressed protein were all at low levels.In conclusion,the I.variabilis-EPSPS*gene was stably inherited and expressed across different generations of transgenic rapeseed.Compared with the recipient control,the transgenic rapeseed showed excellent glyphosate resistance,had no survival competitive advantage and lower edible risk.In this study,the safety assessment of excellent single-copy transgenic lines JH-14,JH-125 and JHX-6 in the environmental release stage had been completed and successfully approved,approval number is Nongjian Approval（2020）No.182,which laid a foundation for the cultivation of glyphosate-resistant rapeseed varieties in China.