| Herbicide-resistant transgenic rape is one of the world’s four major transgenic crops.Although transgenic oilseed rape has not yet been commercially grown in China.However,a large amount of herbicide-resistant transgenic oilseed rape is imported every year for the processing of edible oil.Seeds may be scattered during transportation,processing and production to form volunteer seedlings.Once the resistance gene of the volunteer seedlings flows via pollen and introgress into the relative weeds,it may cause adverse effects on weed control and ecological environment.Therefore,it is of practical significance to evaluate the ecological risk of herbicide resistance transgenic rape.It was found that plants carrying and expressing the resistance gene(resistance-expression)(E),plants carrying the resistance gene,but not expressing resistance(resistance-silence)(S),and plants without transgene(noresistance-no-expression)(The first generation of resistance segregation)were segregated from the resistant backcross generation between glufosinate-resistant transgenic Brassica napus(transgene pat located on C chromosome)and wild Brassica juncea;and then it was found that in the first generation of resistance segregation,the above three types of plants were segregated from the self-progeny of resistance-silence plants(The second generation of resistance segregation),and the resistance-silence plants of the pat gene promoter sequence were methylated,which indicates that gene silencing is closely related to the methylation of the gene promoter.On the basis of previous research,we mainly studied on the expressing and silencing laws of resistance-expression and resistance-silence plants identified in the second generation of resistance segregation in continuous two self-progeny;analysis of the correlation between DNA methylation and resistance gene silencing;and the fitness of the resistance-expression and resistance-silence backcross generation of resistance-silence plants.We aimed to clarify the transmission and expression of resistance genes in the backcross generation of transgenic transgenic Brassica napus rape and wild Brassica juncea,to clarify the mechanism of resistance gene silencing,analyze the resistance gene expressing and silencing backcross generation adaptability to the environment and potential ecological effects,provide a basis for the safety assessment of resistance gene infiltration into weeds.The research results are as follow:1.Expressing and silencing of glufosinate-resistant genes in backcross generation between transgenic oilseed rape and wild B.juncea1.1 Expressing and silencing pattern of resistance genes identified in the first resistance segregation generation of self-progeny of S plants:In the first resistance segregation generations,self-progenies of S plants BC1mF5 and BC1pF5,were used as materials(m and p represent wild B.juncea as maternal plants and paternal plants).Molecular identification and applying glufosinate on leaf were used to screen whether the plants carried and expreessed the glufosinate-resistant pat gene.The results showed that:Three types of plants were segregated from the backcross generation,namely resistance-expression,resistance-silence,no-resistant-no-expression.The average proportion of plants expressing resistance and no-expressing resistance segregated from selfprogeny of S was 68%and 28.34%,respectively,recorded as S-E and S-S,the above two kinds belonged to the second resistance segregation generation.1.2 Expressing and silencing pattern of resistance genes identified in the second resistance segregation generation of self-progeny of S-E and S-S plants:In the second resistance segregation generations,self-progenies of S-E and S-S BC1mF7,BC1pF7,BC1mF9,BC1pF9,BC2mF4,BC2pF4,BC3mF5 and BC3pF5,were used as materials.Use the same method as 1.1,to identified the expressing and silencing of resistance genes in these materials.The results showed that:Three types of plants were segregated from the above backcross generation,namely resistance-expression,resistancesilence.no-resistant-no-ex pression.The average proportion of plants expressing resistance and no-expressing resistance segregated from self-progeny of S-E plants was 59.83%and 33.17%,respectively,recorded as S-E-E and S-E-S;The average proportion of plants expressing resistance and no-expressing resistance segregated from self-progeny of S-S plants was 59.09%and 31.25%.respectively,recorded as S-S-E and S-S-S,the above four kinds belonged to the third resistance segregation generation.1.3 Expressing and silencing pattern of resistance genes identified in the second resistance segregation generation of self-progeny of S-E-E,S-E-S,S-S-E and S-S-S plants:In the third resistance segregation generations,self-progenies of S-E-E、S-E-S、S-S-E and S-S-S BC2mF5,BC2pF5,BC3mF6 and BC3pF6,were used as materials.Use the same method as 1.1,to identified the expressing and silencing of resistance genes in these materials.The results showed that:Three types of plants were segregated from the above backcross generation,namely resistance-expression,resistance-silence,no-resistant-no-expression.The average proportion of plants expressing resistance and no-expressing resistance segregated from self-progeny of S-E-E plants was 49.33%and 37.83%,respectively,recorded as S-EE-E and S-E-E-S;The average proportion of plants expressing resistance and no-expressing resistance segregated from self-progeny of S-E-S plants was 45.33%and 43.17%,respectively,recorded as S-E-S-E and S-E-S-S;The average proportion of plants expressing resistance and no-expressing resistance segregated from self-progeny of S-S-E plants was 51.83%and 35.67%,respectively,recorded as S-S-E-E and S-S-E-S;The average proportion of plants expressing resistance and no-expressing resistance segregated from self-progeny of S-S-S plants was 50.5%and 39%,respectively,recorded as S-S-S-E and S-S-S-S,the above eight kinds belonged to the fourth resistance segregation generation.In summary,resistance gene silencing has occurred in self-progeny of four consecutive backcross generations between wild B.juncea and transgenic glufosinate-resistant oilseed rape,and silencing resistance gene in some backcross generations can be restored.2.Resistance gene methylation detection in the backcross generation of glufosinateresistant transgenic rape and wild B.junceaIn the second resistance segregation generation,in E-E and S-E plants,CaMV 35S promoter region was not DNA methylated;There are 3-6 methylation sites in the pat region of the resistance gene.In E-S and S-S plants,there are 3-6 methylation sites in the CaMV 35S region of the resistance gene;There are 3-7 methylation sites in the pat region of the resistance gene.In the third resistance segregation generation,in S-E-E and S-S-E plants,CaMV 35S promoter region was not DNA methylated;There are 4-6 methylation sites in the pat region of the resistance gene.In S-E-S and S-S-S plants,there are 4-6 methylation sites in the CaMV 35S region of the resistance gene;There are 3-7 methylation sites in the pat region of the resistance gene.In the fourth resistance segregation generation,in S-E-E-E,S-E-S-E,S-S-E-E,S-S-S-E plants,CaMV35S promoter region was not DNA methylated;There are 4-6 methylation sites in the pat region of the resistance gene.In S-E-E-S,S-E-S-S,S-S-E-S,S-S-S-S plants,there are 4-6 methylation sites in the CaMV 35S region of the resistance gene;There are 3-7 methylation sites in the pat region of the resistance gene.The methylation sites of pat gene region and CaMV 35S promoter region of resistanceexpression plants and resistance-silence plants identified by the second,third,and fourth generation resistance segregation were determined.It was found that methylation of the promoter region was one of the causes of gene silencing.3.Verification of the relationship between methylation and resistance gene silencing in the backcross generation between glufosinate-resistant transgenic rape and wild B.junceaSelf-progeny of S plants in the first resistance segregation generation,BC1mF5 and BC1pF5,self-progeny of S-E and S-S plants in the second resistance segregation generation,BC1mF6 and BC1pF6,were used as materials.Each backcross generation treated 100 seeds with 5-azaC at 250μmol/L,and 100 seeds without treatment as control group.The treated seeds were sown and cultivated to the seedling stage.Molecular identification was used to screen whether the plants carried the glufosinate-resistant pat gene.The results showed that:two types of plants were selected:carrying the resistance gene and without the resistance gene,and the number of plants carrying the resistance gene in the control group and the 5azaC treatment group of each backcross was equivalent.After spraying and screening 1111 times dilution of 18%glufosinate solution(water used 0.06 L/m2),The number of surviving plants in the treatment group was 26～37 more than that in the control group.Therefore,it is speculated that the DNA methylation level of seeds treated with 5-azaC decreased during growth and development,resulting in the restored of resistance gene expression and the increase in the number of plants expression resistance.It was initially confirmed that there is a correlation between resistance gene silencing and DNA methylation.Using the above mentioned control group as material,the ROS1 and DME genes were cloned.DNA sequences of 4612bp and 5451 bp were obtained.Fluorescence quantification method was used to detect relative expression ROS1 and DME in resistance-expression and resistance-silence plants before spraying 1111 times dilution of 18%glufosinate-soluble solution(water used 0.06 L/m2)0h after 6h and 12h.The results showed that:after spraying,the expression of ROS1 and DME genes increased first and then decreased,and the expression level was highest at 6 hours,and the expression level of resistance-expression plants was higher than that of resistance-silence plants,higher 22.29%-30.61%and 14.62%27.34%,respectively.There was no significant difference in the relative expression of ROS1 and DME between 0h before spraying and 12h after spraying.The above results indicated that ROS1 and DME were involved in the regulation of resistance gene expression.4.Fitness analysis of the backcross generation between glufosinate-resistant transgenic oilseed rape and wild B.juncea4.1 Fitness of resistance-expression and resistance-silence offspring identified in the second resistance segregation generationS-E and S-S of BC1mF5 and BC1pF5,self-progeny of S in the first resistance segregation generation,were used as materials.Vegetative and reproductive fitness components were measured and used to calculate their composite fitness under greenhouse without selection.Furthermore,the differences of the composite fitness between the backcross generations and wild B.juncea were analyzed.The results showed that:the composite fitness of S-E and S-S BC1mF5 and BC1pF5 was significantly lower than that of wild B.juncea,but the composite fitness of resistance-expression and resistance-silence plants was similar.4.2 Fitness of resistance-expression and resistance-silence offspring identified by the third resistance segregation generationResistance identified by second generation resistance segregation segregation S-E and SS segregated from self-progeny of S-E-E,S-E-S,S-S-E and S-S-S BC1mF9,BC1pF9,BC2mF4,BC2pF4,BC3mF5 and BC3pF5 as materials.The analysis method is the same as 3.1.The results showed that:the composite fitness of S-E-E,S-E-S and S-S-E,S-S-S BC1mF9,BC1pF9,BC2mF4,BC2pF4,BC3mF5 and BC3pF5 were equivalent that of wild B.juncea,and the composite fitness of resistance-expression and resistance-silence plants was similar. |
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