【作者】 张廷婷；

【导师】 温孚江；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 植物生长发育经常遇到的逆境以干旱、盐碱、虫害等最为严重,在干旱、盐碱等逆境胁迫下会导致植物的渗透胁迫和氧化胁迫伤害,使植物细胞膜的结构和功能遭到破坏,以致光合作用和信号转导作用大大降低。如果通过基因工程的手段将植物细胞膜上起到重要信号传递作用的磷脂酶Dα(Phospholipase Dα,PLDα)基因导入植物,发挥抗氧化、渗透调节功能,将能有效的提高植物抗旱、耐盐等抗逆能力。毛白杨是我国特有的乡土树种,是我国北方地区重要的造林和平原绿化树种。随着杨树人工林面积的不断扩大,虫害问题越来越突出,危害严重,利用基因工程技术培育抗虫杨树新树种,是防治害虫的有效手段。本研究将正、反义PLDα基因分别转入毛白杨,进行了基因功能的鉴定。在此基础上,选取超表达PLDα基因耐盐、抗旱性较强的毛白杨优良株系,导入具有抗虫功能的Bt毒蛋白基因,培育获得了既耐盐、抗旱又抗虫的多抗毛白杨新品种。转基因植株的获得为探讨植物抗逆的生理和分子机制以及抗性转基因杨树的实际应用打下良好的基础。主要实验结果如下:1.正、反义PLDα基因转化毛白杨及基因功能的鉴定(1)利用农杆菌介导的叶圆盘转化法分别将正、反义PLDα基因转入毛白杨。在含有卡那霉素的培养基上筛选,获得转正义PLDα基因的抗性植株45个株系,转反义PLDα基因的抗性植株49个株系。(2)微量提取植物总DNA进行PCR检测,结果显示,在含有卡那霉素培养基上筛选的抗性植株(除转反义基因1株外)均扩增出约780bp大小的目的带,初步证明卡那霉素抗性植株为转基因植株。(3)大量提取转正、反义PLDα基因植株的总DNA分别用限制型内切酶XbaI和BamHI酶切,Southern blot分析结果显示,PCR检测结果呈阳性的毛白杨植株均检测到信号,证明目的基因已经整合到杨树的染色体中,并且有1～5个拷贝数。(4)Northern blot分析结果显示,各转正义PLDα基因植株均检测到明显的杂交信号,转反义PLDα基因植株杂交信号弱于野生型,表明正义PLDα基因在转录水平进行了有效的超表达,反义基因抑制了植物内源PLDα基因的表达。(5)试管苗的耐盐抗旱性实验表明,在含有不同浓度的NaCl(0,68,102,136,172mmol/L)和甘露醇(0,150,200,250,300mmol/L)的培养基上,转正义PLDα基因杨树的生根率和平均根长都明显高于野生型和转反义PLDα基因的植株,表明转正义PLDα基因杨树的耐盐、抗旱能力较强。(6)干旱或盐胁迫处理条件下,不同转基因植株的Northern blot分析结果显示:经过干旱或盐胁迫处理的野生型和转正义PLDα基因植株的信号都强于正常浇水的植株;在正常浇水的转反义PLDα基因植株中检测不到信号,而干旱或盐处理植株中都检测到信号。表明干旱或盐胁迫可以诱导植物PLDα基因的表达,或者使PLDα基因的表达量有不同程度的提高,也说明转反义PLDα基因并没有完全抑制内源PLDα的转录。(7)在20% PEG6000模拟干旱条件下,转正义PLDα基因杨树的质膜损伤程度小于野生型,而转反义PLDα基因杨树的质膜损伤程度大于野生型;同时在200mmol/L NaCl模拟盐害条件下,也得出了类似上述结果。表明转正义PLDα基因提高了植物的抗旱和耐盐性,而转反义PLDα基因反而降低了植物的耐盐、抗旱能力。(8)杨树叶片细胞形态学分析得知,正常条件下细胞膜系统完整,细胞膜,线粒体膜,叶绿体膜,叶绿体基粒片层等膜系统和结构都比较规则。经过200mmol/LNaCl或20%PEG6000处理以后,转基因和野生型植株细胞膜系统都有不同程度的破坏;野生型植株细胞膜受到破坏,叶绿体变圆,外膜受到损伤,线粒体破坏严重,膜不完整;转正义PLDα基因植株细胞受到的破坏较轻,线粒体外膜有点损伤,细胞膜、叶绿体膜和液泡膜还比较完整;而转反义PLDα基因植株细胞膜系统破坏最严重,叶绿体变圆,基粒片层错乱,叶绿体外膜、细胞膜和液泡膜都受到严重的损伤。由此推测,转正义PLDα基因植株中PLDα基因的超表达,对渗透胁迫条件下植物细胞膜系统有保护作用。2.耐盐、抗旱、抗虫多抗转基因毛白杨的获得(1)构建了包含Bt毒蛋白基因的植物表达载体pBT1946,内含有抗除草剂(bar)基因,作为选择性标记基因。叶片分化实验确定适宜的筛选压力为PPT浓度0.4mg/L。(2)选取具有较强耐盐、抗旱能力的转正义PLDα基因毛白杨9号株系(S9)作为受体材料,利用农杆菌介导法导入Bt毒蛋白基因(cry1Ab),在含有除草剂(PPT)的培养基上筛选获得70个抗性株系。(3)微量提取植物总DNA进行PCR检测,结果显示,在含有除草剂培养基上筛选获得的抗性植株,有50个株系扩增出约500bp大小的目的带,初步表明外源基因已整合到这些植株的染色体基因组中。(4)大量提取转Bt毒蛋白基因植株的总DNA,用限制型内切酶HindⅢ酶切。Southern blot分析结果显示,PCR检测结果呈阳性的毛白杨植株均检测到信号,证明目的基因已经整合到毛白杨S9的染色体中,并且存在1～5个拷贝数。(5)Northern blot分析结果显示,各转基因植株均检测到明显的杂交信号,而非转基因植株无任何杂交信号,表明Bt毒蛋白(cry1Ab)基因在转录水平得到了有效表达。(6)ELISA检测结果表明,Bt毒蛋白基因在翻译水平进行有效的表达,植物中Bt蛋白的含量为725.11ng/g～866.43ng/g。(7)饲虫实验结果表明,盆栽苗幼嫩叶片饲喂舞毒蛾幼虫30d后,各个转基因株系幼虫死亡率均在90%以上,最高达到98.9%;而非转基因植株幼虫死亡率仅为10%。证明转Bt毒蛋白基因杨树具有较强的抗虫能力。更多还原

【Abstract】 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.更多还原