Resistance Analysis Of Transgenic Populus Tomentosa Plants With The Sense Or Anti-sense Phospholipase Dα And Bt Toxic Protein Genes

The plant growth is affected by many factors, especially drought, salt and insect pest. The plant suffers from osmotic stress and oxidative stress in drought and salt etc. conditions, which causes the damages of membrane structure and function and results in reducing of signal transduction and photosynthesis. The drought resistance and salt tolerance of plant can be increased when the Phospholipase Dα(PLDα) gene on membrane with important signal transduction fulfills antioxidation function and osmotic adjust action after the PLDαis introduced into plants by the way of genetic engineering. Populus tomentosa(P. tomentosa), a native species in China is important tree species for affbrestation and plain greening in northern region of China. The insect pest is more serious with expanding of poplar plantation, which leads more damages. It is an effective method to control insect pest to breed new poplar tree species using genetic engineering technology. Sense PLDαand anti-sense PLDαwere introduced into P. tomentosa respectively in this study and experiments for analysis of plant resistances and gene functional identification were performed. Overexpression of the PLDαgene promoted a higher level of drought and salt tolerance in vivo in transgenic P. tomentosa, the plants with higher drought and salt tolerance were screened out and transformed with anti-insect gene Bt, so as to obtain a new breed of P. tomentosa with high drought and salt tolerance as well as better anti-insect ability. It is new production in application and demonstrated the potential of PLDαto confer osmotic stress tolerance in P. tomentosa. It would be significative to widely planting the high osmotic tolerance and anti-insect trees in the saline soils as well as to investigating the overexpression of PLDαgene in other plants with the aim of improving plant tolerance to multiple environmental stress and further understanding the function mechanism of PLDαgene. The main results are as followed:1.Transformation of P. tomentosa with sense or anti-sense PLDαgene and identification of gene functions (1) The PLDαand anti-PLDαgene were introduced into P. tomentosa respectively by Agrobacterium-tumefaciens-mediated transformation. Plants were selected to survive on the culture medium with kanamycin to obtain 45 resistant plants with PLDαgene and 49 resistant plants with anti-PLDαgene.(2) The result from PCR analysis showed that an about 780bp band corresponding to the size of PLDαgene was observed for the kanamycin-tolerant plants selected on culture medium with kanamycin (except 1 transgenic plant with anti- PLDαgene), which primarily proved that kanamycin-tolerant plants were transgenic plants.(3) Genomic DNA of the plants with PLDαgene or anti- PLDαgene digested by XbaI or BamHI was hybridized with the PLDαprobe. Southern-blot analysis showed that the hybridization signals were specifically detected in the transgenic plants which were positive in PCR but not in the wt plants. It indicated that objective genes had been integrated into plant genome and carried 1 to 5 copies of the foreign genes.(4) Northern-blot analysis revealed the presence of PLDαmRNA in plants of all PLDαtransgenic lines, but the hybridization signals in anti-PLDαtransgenic plants were much weaker than in wild type plants. It indicated PLDαgene had an effective over-expression at transcription level in P. tomentosa and anti-PLDαgene restrained the expression of plant endogenous PLDαgene.(5) Tube seedlings of several transgenic lines and wt plants (control) were evaluated for drought and salt tolerance and gene expression. The results showed that the rhizogenesis rate and the average root-length of trans-PLDαlines were distinctly higher than those of wt and anti-PLDαtransgenic plants on culture medium with NaCl of different concentrations (0, 68, 102, 136, 172mmol/L) and mannitol (0, 150, 200, 250, 300mmol/L) under the same growth conditions. It indicated that PLDαgene transgenic plants exhibited higher drought and salt tolerance.(6) To study the relationship between PLDαexpression and drought or salt stresses in transgenic plants, Northern-blot analysis showed that the signals of PLDαtransgenic and wild type plants with drought or salt treatments were significantly stronger than those well watered corresponding plants. No signal was detected in the well watered plants with anti-PLDαgene, whereas signals were present in drought or salt treatment ones. It indicated that drought or salt stresses may induce or increase PLDαgene expression in different degree, and anti-sense gene had not restrained PLDαat transcription level completely.(7) Under 20% PEG6000, the damnification of plasmalemma in PLDα-s transgenic lines is lower than that in wt plants, whereas the damnification of plasmalemma in PLDα-a transgenic lines is more serious than that in wild type plants. Meanwhile, the same results were achieved with 200mmol/L NaCl treatment. Which further testified that plant drought and salt tolerance were promoted by PLDα-s transformation but reduced by the anti-sense gene.(8) Cytomorphology analysis showed that cell membrane system was of integrity under normal conditions, and the membrane system including cell membrane, mitochondrial membrane, chloroplast membrane and chloroplast membranes etc. is relatively regular. After 200mmol/L NaCl or 20% PEG6000 treatment, the membrane systems of transgenic and wild type plants were damaged in different degree; for the wt plant, the membrane was damaged to be not complete, chloroplast became round, membrane of chloroplast and mitochondrial was seriously damaged; for the PLDαtransgenic plants, the damage was light, mitochondrial membrane was damaged a little and cell membrane, chloroplast membrane and tonoplast were comparatively complete; while the anti-PLDαtransgenic plant cells were damaged most seriously, chloroplast showed round, grana lamella garbled and chloroplast membrane, tonoplast and cell membrane were damaged seriously. It is supposed that overexpression of PLDαgene in transgenic PLDαgene plants could protect the plant membrane system under osmotic stresses.2.Obtaining of transgenic P. tomentosa with high salt and drought-tolerance and insect-resistant(1) The Bt gene was cloned into the pE1946 vector, The resulting plasmid pBT1946, contained bar gene as the plant selective marker and was mobilized to Agrobacterium tumefaciens strain EHA105 for plant transformation. The sprout proliferation experiment was carried out, and PPT concentration was fixed on 0.4mg/L as the appropriate selectivity pressure.(2) High salt and drought tolerance P. tomentosa S9 was screened out from PLDα-s transgenic lines as plant receptor, Bt gene (cry1Ab) was introduced into S9 by Agrobacterium-tumefaciens-mediated transformation. 70 resistant plants with Bt gene were selected to survive on the culture medium with PPT.(3) The result from PCR analysis showed that an about 500bp band corresponding to the size of Bt gene was observed for 50 PPT-tolerant plants selected on culture medium with PPT, which primarily confirmed that Bt gene had been integrated into genome of these plants.(4) Genomic DNA of trans-Bt plants digested by HindⅢwas hybridized with the Bt probe. Southern-blot analysis showed that the hybridization signals were specifically detected in the transgenic plants which were positive in PCR but not in the control plants. It indicated that objective gene had been integrated into S9 P. tomentosa genome and carried 1 to 5 copies of the foreign genes.(5) Northern-blot analysis showed that the hybridization signals were only specifically detected in the transgenic plants but not in the control plants. It indicated that Bt gene took an effective expression at transcription level in S9 P. tomentosa.(6) ELISA showed that Bt toxic protein gene could be efficiently expressed at translation level and the content of Bt protein in plants was 725.11ng/g-866.43ng/g.(7) Insect feeding test showed that after 30 days feeding Lymantria dispar larva with potted plant tender leaves, the mortality rate of Lymantria dispar larva fed with every transgenic line was over 90%, even up to 98.9%; but the mortality rate of larva fed with non-transgenic plants was only 10%. It indicated that the transformation of P. tomentosa with Bt gene enhanced its anti-insect ability.