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| Superposition Effect of Cry1Ac Gene in Transgenic Bt Tobacco (Nicotiana tabacum) |
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Abstract Plant transgenic studies have found that multiple copies insertion often lead to decreased expression of exogenous genes. However, when constructing the vector, the same exogenous gene is constructed on the same vector with series connection. Whether it will improve the expression of exogenous genes after transformation of plants has not been reported. In order to improve the expression of exogenous gene in transgenic plants and explore the effect of superposition of the same target gene on gene expression and transgenic plant growth, in this study, the plant transformation vectors carrying single and double Bacillus thuringiensis(Bt) gene Cry1Ac respectively were transformed into tobacco (Nicotiana tabacum) tissue culture seedlings by Agrobacterium (Agrobacterium tumefaciens)-mediated leaf disc method. Then, the transgenic lines were detected by polymerase chain reaction (PCR), fluorescence quantitative PCR (FQ-PCR) (Absolute quantitative) and Bt toxin and insect resistance tests to identify whether or not the exogenous gene was integrated into the tobacco genome and the expression were carried out. At the same time, morphological indexes and physiological and biochemical indexes of the transgenic plants transformed with the two vectors and non-transgenic plants were observed to research the growth of the transgenic plants. The results showed that nine transgenic single Cry1Ac gene lines and 8 transgenic dual Cry1Ac gene lines were obtained by PCR detection, and the target genes were integrated into the tobacco genome. FQ- PCR and toxin detections showed that the Cry1Ac gene transcriptional abundance of the transgenic dual Cry1Ac gene lines was about 2.6 times higher than that of transgenic single Cry1Ac gene lines, the Cry1Ac toxin content of the transgenic dual Cry1Ac gene lines was about 10 times higher than that of transgenic single Cry1Ac gene lines, and the two types of values of the transgenic dual Cry1Ac gene lines were significantly higher than that of transgenic single Cry1Ac gene lines. The result of indoor insect feeding test showed that with the increase of feeding days, the mortality of cotton bollworm (Helicoverpa armigera) fed with transgenic lines leaves increased gradually. There were some differences among different transgenic lines. The lethal rates of the two kinds of transgenic lines against the first instar larvae of Helicoverpa armigera were 100%. When feeding for three days, the larva mortality rate of transgenic dual Cry1Ac gene lines B1, B4, B6, B7 and transgenic single Cry1Ac gene lines A2, A7 reached 100%. Feeding for four days, the larva mortality rate of B3, B5, B8, A1, A3, A4, A5, and A9 reached 100%. At five days, the larva mortality rate of all transgenic lines reached 100%, while the rate of non-transgenic tobacco was less than 10%. Moreover, the lethal rates of the two kinds of transgenic lines against the second instar larvae of Helicoverpa armigera were also 100%. The larva mortality rate of B7 and B6 reached 100% at feeding for five days and six days respectively, while A2 and A5 reached 100% at feeding for eight days. Overall, the lethal time of the transgenic dual Cry1Ac gene lines was shorter than transgenic single Cry1Ac gene lines, and transgenic dual Cry1Ac gene lines had high insect resistance. The morphological and physiological and biochemical indexes detections showed that there were no significant differences in the parameters of ground diameter, net photosynthetic rate (Pn), stomatal conductance (Cond), intercellular CO2 concentration (Ci), transpiration rate (Tr), maximum fluorescence (Fm), chlorophyll a (Chla), and total chlorophyll content (CT) among transgenic dual Cry1Ac gene lines and CK and transgenic single Cry1Ac gene lines. While there were some differences in other parameters. On the whole, the growth of most transgenic lines was not significantly different from that of the control group, and some of the transgenic dual Cry1Ac gene lines showed dwarfing phenomenon. In general, in this study, compared with transgenic single Cry1Ac gene lines, the exogenous gene expression level of transgenic dual Cry1Ac gene lines was improved, and the plant growth and other aspects were not significantly affected. This study will lay the foundation for exploring ways to improve the expression of exogenous genes and to transform other plants.
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Received: 04 May 2017
Published: 10 December 2017
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