The Construction Of Multi-gene Plant Transformation Vector And Genetic Transformation Of Tobacco

The two most important factors which always block agroforestry’s production were insect pests and soil salinization.The development of plant genetic engineering provides a way to solve the two problems effectively.In order to get plant transformation vectors which can efficiently insect-resistant and both insect-resistant and salt-tolerant.Choose escherichia coli DH10B,agrobacterium EHA105 and tobacco as the main test materials.Different structure of plant transformation vectors were constructed by changing sequence of Bt gene ORF,changing the direction of promoters,joining the middle partition at both ends of the first Bt gene,changing the direction of selection marker gene and adding salt resistant genes.Use agrobacterium-mediated method to conduct genetic transformation.Identify each transfor-mation strain by PCR.Study the m RNA transcription abundance by fluorescence quantitative PCR.Identify the Bt toxalbumin expression quantity and insect resistance by ELISA method and feeding-insect test.Use salt stress method to detect transformation strain’s salt resistance.We get the following results:1.In this experiment ten different structure of plant transformation vectors were constructed.Among them N14,N15,N17,N20 and N22 contain Cry1Ac and Cry3A two insect-resistant genes at the same time;B2 contain Cry1Ac and gus two exogenous genes;N23,N24,N25 and N26 were got on the basis of existing vector N5 and added mtl D,BADH,mtl D+BADH and mtl D+BADH+Sac B salt-tolerant genes separately.Genetic transformation of tobacco was conducted using the above 11 vectors.Made the PCR detection.In the end we received 76 transgenic lines.2.Two vectors which the order of Bt gene is different.The average expression quantity of Cry1Ac toxalbumin of N15（Cry1Ac gene in the downstream）was 6.69μg·g^-1which 5.3times higher than that of its average expression quantity of Cry3A toxalbumin(1.26μg·g^-1).The Cry3A toxalbumin average expression quantity of N17（Cry3A gene in the downstream）was 6.69μg·g^-1which less than Cry1Ac toxalbumin average expression quantity(4.05μg·g^-1).According to transcription abundance of Cry1Ac and Cry3A genes,vectors N15 and N17 have no significant difference.It can be seen that it has no influence to the transcription and translation’s high or low of the order of Bt gene.3.Two vectors which the direction of Cry3A gene promoters is different.The Cry3A toxalbumin average expression quantity of vector N20 was 2.67μg·g^-1,which 1.5 times higher than N5(1.81μg·g^-1).The Cry1Ac toxalbumin average expression quantity of vector N5 was 12.48μg·g^-1,which 1.7 times higher than N20(7.31μg·g^-1).Cry3A gene average transcription abundance of N20（7.36×10⁷）was 1.7 times higher than N5（4.31×10⁷）.It can be seen that changing the direction of Cry3A gene promoter can improve the Cry3A gene’s expression quantity and stability.But it reduced the amount of Cry1Ac gene’s expression.4.Join the middle partition at both ends of Cry3A gene of N14.Both the Cry1Ac and Cry3A toxalbumin average expression quantity of vector N14 were lower than N5.The Cry3A toxalbumin average expression quantity of vector N5(1.81μg·g^-1was 6 times higher than N14(0.30μg·g^-1).Compared with N20,N22 has one more middle partition 144.The Cry1Ac toxalbumin average expression quantity of vector N22(4.19μg·g^-1)lower than N20(7.31μg·g^-1)significantly.The Cry3A toxalbumin average expression quantity have no significant difference.The size of the Bt gene average transcription abundance of N14 and N5was opposite.The Cry1Ac gene average transcription abundance of N20 and N22 is not reach significant difference.The Cry3A gene average transcription abundance of N20（7.36×10⁷）was higher than N22（3.28×10⁷）.It can be seen that joining the middle partition reduce the Bt gene toxalbumin average expression quantity at a certain extent,but has no specific rules for its transcription.5.The direction of selection marker gene npt II of B2 was opposite with other 6 vectors.The Cry1Ac toxalbumin average expression quantity of vector B2(0.26μg·g^-1)was lower than other vectors significantly.The Cry1Ac gene average transcription abundance of B2（3.83×10⁵）was lower than other vectors significantly.It can be seen that changing the direction of npt II gene may inhibit the Cry1Ac gene expression seriously,even lead to no expression.6.Feeding-insect test showed that each strain of transgenic tobacco can produced resistance against lepidoptera pest Helicoverpa armigera and Coleoptera Epilachna vigintioctopunctata.The insecticidal efficacy of each strain of transgenic tobacco was not consistent with toxalbumin expression.7.Deal with salt stress for 36 days.The influence of plant height,chlorophyll content,relative electric conductivity,photosynthesis and chlorophyll fluorescence parameters between tobacco transformation strains and non-gmo contrast have no significant difference.The results showed that transferred mtl D,BADH,mtl D+BADH and BADH+mtl D+Sac B salt-tolerant genes to tobacco cannot significantly improve its salt tolerance.8.Added single salt-tolerant gene mtl D and BADH limit the Bt gene toxalbumin expression.The Cry1Ac and Cry3A toxalbumin expressed of vector N25 which have two salt-tolerant genes mtl D and BADH,but its expression quantity was low.The vector N26 has three salt-tolerant genes mtl D,BADH and Sac B.The Cry1Ac toxalbumin was not expressed,but its Cry3A toxalbumin has low expression.