The Inheritance Law And Ecological Effects Of Exogenous Genes From Transgenic Brassica Napus To Brassica Juncea

With the increased area of cultivating genetically modified（GM）plants worldwide,the ecological risks of GM plants released in the environment have received wide concerns,particularly for potential gene flow from GM crops to their relatives.The position,copy number,and flanking sequence of exogenous genes integrated into the recipient genome will affect the transcription,expression and genetic stability of target genes,and may further affect the fitness of hybrid and backcrossed offspring.In this study,we employed transgenic oilseed rape（Brassica napus）with the Bacillus thuringiensis（Bt）cry1Ac gene,green fluorescent protein（GFP）marker gene and neomycin phosphotransferase gene（NPTII）.We obtained hybrids F₁ and F₂,successive six backcross generations BC₁-BC₆ and BC₁F₁ from transgenic B.napus and its wild relative B.juncea through hand pollination.To study the inheritance law and ecological effects of exogenous genes from transgenic B.napus to B.juncea,we detected the number of gene copy,mRNA transcription and protein expression of exogenous genes by dd PCR,q RT-PCR and ELISA,and the fitness of hybrid and backcross generations.The main results are shown as follow:（1）The number of gene copy was not consistent in transgenic events,with two copies of exogenous gene in transgenic lineages GT1 and GT5 and three copies in GT9.The hemizygous BC₁F₁ had one copy,and homozygous BC₁F₁ had two copies of these genes.There was one copy of Btcry1Ac,GFP,NPTII in hybrid generations F₁ and F₂,and backcross generation from BC₁to BC₆.The Mendelian ratio of the exogenous genes was found in the hybrid and backcross generations,indicating the exogenous genes are on the A chromosome of GT1.（2）The mRNA transcription level of Btcry1Ac and NPTII in transgenic oilseed rape was significantly higher than that in hybrid and backcross generations,and there was no significant difference among hybrid and backcross generations.Homozygous BC₁F₁ showed higher Btcry1Ac and NPTII transcription level than hemizygous BC₁F₁.The transcription level of Btcry1Ac and NPTII increased with the development of plants.Compared to Btcry1Ac,the transcription level of GFP showed a contrary trend,with lower level in GT1 than in hybrid and backcross generations,and the transcription level decreased with the development of plants.There was no significant difference among hybrid and backcross generations.The BtCry1Ac protein concentration in transgenic oilseed rape was significantly higher than in the hybrid and backcross generations in vegetative growth stages,but no significant difference was found between them in reproductive growth stages.No significant difference of BtCry1Ac protein was found among hybrid and backcross generations.Homozygous BC₁F₁ plants showed higher BtCry1Ac protein concentration than the hemizygous BC₁F₁.The BtCry1Ac protein concentration in transgenic parents,hybrid and backcross generations was the highest at the 10-leaf stage.There was a positive correlation between the protein concentration and mRNA transcription of Btcry1Ac for these plant types in each development stage,while there was no relationship between the protein concentration and transcription level for each plant type.These results indicate the gene copy affected the level of mRNA transcription and the concentration of proteins of Btcry1Ac.（3）The vegetative growth fitness of transgenic oilseed rape was not significantly different from its backcross generations,but the reproductive growth fitness was higher than that of hybrid and backcross generations.The fitness of backcrossed plants increased with the generations of backcross.There was no significant difference between the fitness of homozygous BC₁F₁ and hemizygous BC₁F₁,indicating no significant effect of the gene copy of exogenous genes on the fitness of offspring plants.In conclusion,exogenous genes follow the Mendelian ratio in the hybrid and backcrossed generations formed between transgenic B.napus and B.juncea.The copy number,mRNA transcription and protein concentration of exogenous genes are stable in these hybrid and backcross offspring.The copy number of exogenous genes have positive effects on their mRNA transcription and protein concentration for the hybrid and backcross generations,but have no significant effects on plant fitness.Results provide scientific method and dataset for the biosafety assessment and ecological risk management of GM plants released in the environment.