| Currently, Bt is the most extensive, most potential and most promising insecticidal gene. Many kinds of Bt genes have been successfully imported in tobacco, corn, cotton, poplar and other plants, obtaining a large number of plants with good insect resistance. Due to specific resistance of these Bt genes, plants transformed from single Bt gene are limited in insect resistance. Therefore, many scholars explored to transform multi-Bt genes into plants, to improve insect-resistance of plants and expand the insect-resistance spectrum. However, as a result of higher homology, interference among different Bt genes may affect the expression. In this experiment, Cry1 Ac and Cry3 A were constructed on a vector in different ways, e.g. applying MAR structure, selecting different promoters, applying different enhancer sequences after promoter and changing sequence of exogenous gene ORF;four types of vectors were constructed, genetic transformation of tobacco was conducted, and the series transgenic resistance strains were obtained respectively; PCR identification, ELISA detection and fluorescence quantitative PCR detection and insect resistance test were conducted against the transformation strain of each vector and the controlled plants, so as to obtain the optimal combination of Bt genes and transgenic plants with high expression and broad spectrum of insect-resistance to conduct the genetic transformation of tobacco, and lay a foundation for breeding of insect-resistant transgenic poplar. Conclusions:1. The order of Bt gene in the vector influences its expression. Toxalbumin detection showed average expression quantity of Cry1 Ac toxalbumin in transgenic strains of N5(8.77 ?g·g-1, the ORF of Cry3 A was in upstream, before the Cry1 Ac gene) was 67 times higher than that of N4(0.13 ?g·g-1, the ORF of Cry1 Ac was in upstream, before the Cry3 A gene), showing significant difference; and average expression quantity of Cry3 A toxalbumin in strains of N4(12.70 ?g·g-1) was 1.5 times higher than that of N5(8.21 ?g·g-1), showing significant difference. Bt gene in upstream had lower quantity of expression, it seems to be affected by some certain inhibition. The opposite Bt genes in downstream had higher quantity of expression. The influence is more obvious in Cry1 Ac gene expression. According to transcription of the two Bt genes, though only the three strains with the highest quantity of toxalbumin expression were tested, no significant difference was found in transcription level between strains of N4 and N5, but Cry3 A gene of all strains was two magnitudes higher than Cry1 Ac gene in transcription abundance, which is consistent with the features of the two genes. It follows that Bt gene sequence may only affect the toxalbumin translation, but has smaller influence on gene transcription.2. The difference of Enhancers has obvious influences to the expression Cry1 Ac gene. Toxalbumin detection showed average Cry1 Ac toxalbumin content in strains transformed from N5(8.77?g·g-1, At ADH 5?-UTR is applied before Cry1Ac) was 13 times higher than that transformed from N10(0.67?g·g-1, omega sequence is applied before Cry1Ac), reaching extremely significant difference. However, in Cry1 Ac gene transcription detection, average transcription abundance of all strains transformed from N5 reached 3.15E+5, showing no significant difference with that transformed from N10(3.12E+5). Therefore, At ADH 5?-UTR is more influential in improving Bt toxalbumin translation for Cry1 Ac gene; and in constructing plant transformation vectors, appropriate enhancer plays an important role for improving gene expression level. Application of different genetic sequence before Cry1 Ac also exerted certain influence on Cry3 A. Average Cry3 A toxalbumin content in strains transformed from N5(8.21?g·g-1) was 2 times higher than that transformed from N10(3.46?g·g-1), reaching significant difference, and the content among strains of N10 was significant different.3. MAR structure on both ends of the exogenous gene improve the Bt gene expression level and stability. Toxalbumin detection showed average content of Cry1 Ac toxalbumin in strains transformed from N5(8.77?g·g-1, MAR sequence) was 5 times higher than that from S23(1.63 ?g·g-1, none of MAR sequence), reaching significant difference. Average content of Cry3 Atoxalbumin in strains transformed from N5(8.21?g·g-1) was 1 time higher than that from S23(6.48 ?g·g-1). The quantity of toxalbumin expression differed significantly among strains transformed from S23 vector that contains no MAR structure, especially genetic difference of Cry3 A was the most significant, the variation coefficient reached 49.45%. Based on comparison of transcription detection among three strains with the highest toxalbumin content in vectors N5 and S23, it can be seen that transcription abundance of Cry1 Ac and Cry3 A genes in three strains transformed from N5 was higher than that from vector S23.4. Comprehensive analysis shows that N5 is the optimum combination of the four vectors N4?N5?N10 and S23, namely the ORF of Cry3 A gene before the Cry1 Ac gene, in the upstream, near the selection marker gene npt II, adding enhanced sequence At ADH5'-UTR before Cry1 Ac gene, adding the MAR structure on the both end of exogenous gene.5. In feeding experiment on some strains with high quantity of Bt toxalbumin expression, all the transgenic strains produced resistance against Helicoverpa armigera(Hubner)(lepidoptera pest) and Apriona germari(Hope)(coleoptera pest), but the correlation between insecticidal effect and toxalbumin content was not obvious. According to the results of analysis, when Bt toxalbumin content reaches a certain threshold, insecticidal effect is significant against young larvae and is not obviously related with toxalbumin content.6. Based on the above research,two types of vectors e.g. plant transformation vectors p14A6846Y71(N12) and p70F68Y71(N16) were constructed. Two different length frangment was added to front-back ORF of Cry1 Ac gene in upstream of N12. The promoter of Cry1 Ac gene was FMV in N16. Colony PCR identification was conducted against the each vector to detect Cry1 Ac and Cry3 A, the construction of plant transformation vectorshave been successfully completed.Genetic transformation of tobacco was conducted, PCR identification, ELISA detection were conducted against the transformation strain of each vector. 10 transgenic resistance strains were obtained respectively.7. The MAR structure berore Bt gene in the upstream or the promoter or ORF of gene after Bt gene in upstream of the vector influences its expression.Cry1 Ac toxalbumin detection showed average expression quantity of Cry1 Ac toxalbumin in transgenic strains of N12(2.95?g·g-1) was 18 and 21 times higher than that of N16(0.16 ?g·g-1) and that of N4(0.13 ?g·g-1), showing significant difference. The transgenic strains of a vector show significant difference. |
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