论文标题:毛白杨雄株花粉败育细胞学观察和双抗虫基因转化的研究
A Cytological Observation on the Pollen Abortion in Male Populus Tomentosa and Study on Transformation with Two Genes of Insect-resistant
论文作者 孟永红
论文导师 路丙社；杜克久,论文学位 硕士,论文专业 森林培育
论文单位 河北农业大学,点击次数 192,论文页数 54页File Size5014k
2004-04-01论文网 http://www.lw23.com/lunwen_3263257/ 花粉败育;双抗虫基因;转化;抗虫性;毛白杨雄株
pollen abortion; two insect-resistance gene; transformation; insect-resistance; male populus tomentosa
通过基因工程培育抗虫且对环境安全的杨树新品种，已经成为杨树抗虫基因工程研究的主要方面。为避免雌株飞絮，雄株花粉漂移携带外源基因转移等环境问题，本研究确定了以毛白杨败育雄株为双价抗虫基因遗传转化工程体。在对其进行花粉败育形态学和细胞组织学研究基础上，通过农杆菌介导法对其进行双抗虫基因(Bt基因和API基因)的转化，并对筛选获得的卡那霉素抗性植株进行分子生物学检测和抗虫性鉴定。主要研究结果如下： 1) 毛白杨雄株花药外部形态及花粉发育过程连续两年实验数据表明：从花药发育的外部形态观察，在花药发育的过程中没有观察到散粉现象；从花粉发育的细胞组织学观察，其花粉败育的原因可能是绒毡层细胞解体延迟导致小孢子发育受阻，不能形成正常成熟的花粉粒。由此确定该毛白杨雄株为雄性不育。 2) 建立了毛白杨雄株遗传转化再生体系。确定其转化时所用的诱导芽分化培养基为：MS+TDZ0.1mg／L+IAA0.1mg／L附加蔗糖3％、琼脂0.6％；转化芽增殖培养基为：MS+6-BA0.3mg／L+IBA0.1mg／L附加蔗糖3％、琼脂0.6％；生根培养基为：1／2MS+IBA0.5mg／L附加蔗糖1.5％、琼脂0.6％。 3) 通过农杆菌介导法对258个叶片外植体进行双抗虫基因的转化，其中分化数为81个，共得到108个转化芽。将所得的转化芽经卡那霉素抗性筛选，共得到21株卡那抗性植株，将部分卡那抗性苗经PCR检测，阳性率为90％，3株阳性植株经Southern杂交分析，证明外源Bt基因已整合到杨树基因组DNA中。 4) 用转基因毛白杨雄株的幼嫩叶片喂养杨扇舟蛾和舞毒蛾幼虫，虫试结果表明，转基因毛白杨雄株对幼虫的毒性明显大于对照未转基因植株。根据昆虫的总死亡率将转基因植株划分为高抗、中抗、低抗、不抗四个等级，筛选出4个高抗无性系(D-84、D-87、D-94、D-95)、3个中抗无性系(D-74、D-85、D-88)。对测试昆虫的生长、发育进行测定，结果表明：各转基因无性系对测试昆虫的生长发育有明显抑制作用，转基因无性系中昆虫各发育龄期比对照明显延长，昆虫体重增长速率比对照明显减慢。说明Bt基因和API基因在植物细胞内能够有效地表达，其表达产物具有较强的毒性。从而提高了该转基因毛白杨雄株的抗虫性。
It has been a main aspect to cultivate new variety of poplar that is insect-resistant and sureness to enviroment. In order to avoid the pollution that caused by the seed of female poplar and the pollen drifting of male poplar, the research selected new variety of male populus tomentosa as material to be contransformed. On the base of the study of the morphology and histology of pollen abortion , the modified Bacillus thuringiensis (Bt) -endotoxin gene and arrowead proteinase inhibitor (API) gene were contransformed to the male poplar by Agrobacterium tumefaciens. The transgenic plants were selected on the medium containing kan. The resistant plants were tested by molecular biology measures and raising worms experiment. The main results as follow:1) The anther modality and the process of pollen growth of the male poplar were observed for two continual years. The result shows that it is no pollen during the process of anther growth. The reason of pollen abortion probably is tapetum cell delayed disintegration .The sporule can"t growth normally. So the male populus tomentosa was ascertained to be male sterility.2) The transformation regeneration system of the male populus tomentosa was established. The bud differentiation media is MS+TDZ0.1mg/L +IAA0.1mg/L. The bud multiplication media is MS+6-BA0.3mg/L +IBA0.1mg/L .The rooting media is 1/2 MS+IBA0.5mg/L.3) By Agrobacterium tumefaciens, 258 leaf-explant were contransformed with two genes of insect-resistantance. The number of differentiation is 81. The differentiation rate is 31.39%. 108 resistant buds were obtained. The transformed buds were selected on the medium containing kan and obtained 21 resistant plants. The PCR analysis and southern hybridization proved that Bt gene and protease inhibitor have already combined in genome DNA of male poplar.4) Feeding Clostera anachoreta(Fabricius) and Lymantria dispar(Linnaeus) with the young tender leaves of transgenic male white poplar. The result showed: Toxicity of transgenic poplar is significantly higher than contrast. According to the total death rate of Clostera anachoreta(Fabricius) , the transformed plants were devided into 4 grades: high-insect resistance, middling insect-resistance, low insect-resistance and none insect-resistance. Four high insect-resistance clones (D-84 D-87, D-94 D-95) and three middling insect-resistance clones (D-74 D-85 D-88 ) were sieved. In addition, the insect"s growth and development were tested. The result indicated: the transformed clones inhibited the insects" growth and development significantly. The auxeticduration that eating transformed clones was obviously delayed contrasting with control. The growth rate of larva"s weight that fetched the transformed clones was obviously slower than CK. That means Bt gene and Api gene can express effectively in the plant"s cell and give off strong toxicity. Meanwhile, it proved these 2 genes have different insecticidal mechanism which conspire to poison function. So the insect-resistance of transgenic male poplar was improved.
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