Studies On Physiological And Biochemical Characteristics Of Transgenic Populus Tomentosa Carr. And Construction Of Multiple Gene Expression Vector

To reveal the effect of introduced rolB-pttGA20ox double genes on the root physiology, photosynthetic characteristics, stress resistance and growth of transgenic Chinese white poplar (Populus tomentosa Carr.), four Chinese white poplar lines with the indroduction of rolB-pttGA20ox double genes and a control strain PT-16 were invesgitated. To further improve the insect resistance and salt tolerance of Chinese white poplar, Bacillus thuringiensis toxin gene (BtCryIAc), betaine aldehyde dehydrogenase gene (BADH), GA20 oxidase gene (pttGA20ox) and the rolB gene were constructed in one plant expression vector pYL1305 through a set of multi-genes expression vectors based on Cre/loxP recombination system. Furthermore, the multi-gene vector was transformed into tobacco by agrobacterium mediated procedure. The main results of the research are as follows:1) The difference of endogenous IAA, GA3, ZR and the ABA content changes between transgenic Chinese white poplar with rolB-pttGA20ox and the control was significant during the rooting process, and transgenic Chinese white poplar had better rooting ability as shown in the indexes investigated. In the transgenic lines, the the range of ecological adaptation of light was increased in different degrees. The net photosynthetic rate was significantly correlated with the basal stems, but was of no significant correlation with the height. The height of potted transgenic lines varied greatly and the height of RG-1, RG-2, and RG-3 was significantly higher than that of RG-4 and the control PT-16. Whereas the stem difference was not significant during the potted period. After the lines being moved to farmland, the result was just opposite, with RG-4 being the highest, which reached 231.25cm with the average stem of 13.91mm. The SOD activity and MDA content in different lines indicated that RG-4 has the strongest resistance to adversity, however, the difference was not significant when compared with PT-16.The introduction of rolB-pttGA20ox double genes may have changed the physiological metabolic pathways of Chinese white poplar. Meanwhile the transgenic lines were significantly different in photosynthetic characteristics, growth, stress resistance, etc., which laid a foundation for the selection of high-quality transgenic plants.2) pYL1305-Bt was constructed by replacing the gus gene of pYL1305 vector with the Bt gene obtained through PCR. The CaMV35S-BADH-nos expression cassette was amplfied and inserted into pYL 1305-Bt, resulted in the vector pYL 1305-Bt-BADH. The CaMV35S-GA20ox-nos and GH3-rolB-nos expression cassettes were insered into the supplying vector pYL-d1, pYL-d2, respectively; then the multi-gene vector was obtained by two rounds of reconstruction, which was transformed into EHA105.3) The multi-gene vector was transformed into tobacco through Agrobacterium-mediated leaf disc transformation. The resistant plantlets got were screened by the culture on the medium with 25mg/L of Hyg. PCR analysis and Southern-blot detection to the resistance plantlets selected by antibiotics indicated that the genes had inserted into tobacco genome. Further RT-PCR test showed that all the target genes were transcribed in the transgenic plants, and that the expression of genes in line T2 was the highest. It was also showed that multigene is truly inserted tandemly.4) The IAA levels in the roots, the stems and the leaves of transgenic tobacco plants were 1.90 fold,1.92 fold, and 1.17 fold as high as those of the control, respectively. The GA3 content of transformed plants in all parts was higher than those in the control plants, and that in the stems was the highest while that in the leaves was the lowest. ABA contents in the roots and the stems of transformed plants were slightly lower than those in the control ones, whereas in the leaves, they were higher in the transgenic plants than those in the control plants. IAA/ABA ratios of all parts in transformed plants were higher than those in the control ones, with that in roots being the highest, which was 1.82 times as large as that of the control plants.5) Under 300mM NaCl.treatment, the gowth of control plants was restrained. The leaves turned yellow and it could not take roots. Whereas the growth of transgenic plants was normal with green leaves and the roots in good conditions. The SOD activity of transgenic plants was higher than that of the control plants when stressed by different concentration of NaCl for long duration, but MDA content changed slightly. While the MDA content of control plants decreased sharply with the increasing concentration of NaCl.6) The Bt toxic protein was detected in both stems and leaves of the transgenic plants, with that of T2 plants being the highest, of which the Bt toxic protein in the leaves and stems were 585.53 ng/g.FW and 573.66 ng/g.FW respectively. While in the control plants, no Bt toxic protein was detected. A one-week feeding experiments showed that the corrected death rate of 2-year-old tussock moth was 78.05%, and the death rate of 3-year-old tobacco cutworm was 30%, which restrained the growth of larvae obviously.7) The transgenic tobacco plants grew obviously after being transplanted. The stems were longer, which showed the characteristic of the pttGA20ox genes expression. The basal stem was also bigger, and the leaves were dark green and the plants were strong. After 4 weeks, the average height was 19.28cm, which was 1.61 times as high as that of the control plants; while the basal stem was 5.34mm, which was 1.37 times as big as that of the control plants.In this study, the multiple genes could be expressed normaly in tobacco without resulting in gene silence, which showed that the construction of vector which has a certain number of homologous promoter and terminator was feasible. But with the increasing of the number of the genes introduced, the replacing of different promoters and the employment of MAR should be considered to avoid gene silence. The successful construction of single multi-gene vector and the normal expression of the genes will lay an important foundation for further improvements of Chinese white poplar and other trees.