【作者】 王洪利；

【导师】 楼程富；

【作者基本信息】 浙江大学 ， 特种经济动物饲养学， 2002， 博士

【摘要】 桑树虫害是造成桑叶减产、蚕茧减收的主要因素之一。利用植物基因工程技术开展桑树抗虫性分子育种研究是蚕桑生物技术的发展方向。目前，用于植物抗虫工程研究的抗虫基因主要是苏云金杆菌毒蛋白(Bt)基因和蛋白酶抑制剂(Pl)基因，但Bt和大多数的Pl都对鳞翅目昆虫具有毒杀作用，而家蚕是鳞翅目昆虫的一种。因而桑树抗虫基因工程研究不能利用Bt和大多数的Pl基因。而水稻半胱氨酸蛋白酶抑制剂(Oryzacystatin)基因本身是植物天然的抗虫基因，大量的研究认为水稻半胱氨酸蛋白酶抑制剂对鞘翅目害虫有较强的抑制作用，而对鳞翅目昆虫无特异性抑制作用。因此，本研究在优化桑叶片培养再生技术体系的基础上，选择水稻半胱氨酸蛋白酶抑制剂基因作为目的基因，利用农杆菌介导法和基因枪轰击法进行桑树遗传转化研究，获得了初步成功。 1．通过比较植物激素和抗生素对桑树新一之濑试管苗不定芽分化影响的研究，结果显示：6—BA与NAA的比例以20∶1到30∶1都能分化出较多的不定芽；在含果糖(F)较多的分化培养基上，叶片分化后不定芽生长能力较强；各种抗生素只要在适宜的浓度范围内对不定芽的分化能力影响不显著。 2．利用根癌农杆菌介导法进行了水稻半胱氨酸蛋白酶抑制剂(Oryzacysta—tin)基因的遗传转化研究。通过对影响桑叶片培养和遗传转化条件的试验研究，认为桑树受体对OC基因或携带OC基因的载体亲和性较差，因而根癌农杆菌感染后抗性筛选所得到的不定芽分化率很低，其遗传转化效率仅0.5％左右。对得到的少数转基因抗性苗进行PCR检测、DNA点杂交、Southern blotting鉴定证实，首次将水稻半胱氨酸蛋白酶抑制剂基因(OC)导入到桑树组织细胞，并整合进再生植株的基因组中。 3．通过对基因枪轰击参数与桑树遗传转化效率的研究表明，中等大小的桑苗茎尖、冬芽、腋芽及愈伤组织均可作为基因枪法遗传转化的受体。最佳参数以轰击强度为7cm距离、压力为1350psi，最适金粉用量为每枪500ug，在此 浙 江 大 学 博 士 学 位 论 文 条件下轰击受体材料所得到的遗传转化频率最高。 4．本研究首次成功地利用基因枪轰击法将水稻半脱氨酸蛋白酶抑制剂基 因（OC）导入到桑树组织细胞中。经 PCR。DNA点杂交、Soutnern bloting鉴 定以及RNA点杂交、Northern bloting鉴定，不仅证实外源基因已经整合进再 生植株的基因组中，并且己经在mRNA的水平上进行了表达。同时证实RNA 的表达量在不同的转基因植株间有较大差异，即使是同一转基因植株的不同器 官中，RNA的表达量也存在着一定的差异。更多还原

【Abstract】 Insect pests of mulberry are one of the main factors which cause the damage of mulberry leaves and loss of cocoon. It is a future in agricultural practice that applying plant anti-insect genetic engineering controls insect pests of mulberry. Currently, The gene used for plant anti-insect genetic engineering mainly is Bacillus thuringiensis (Bt) insecticidal crystal protein and proteinase inhibitor (PI) genes in Oryza sativa (Called as Oryzacystatin). Bt insecticidal crystal protein and PI is toxic to lepidopterous insects, while silkworm belong to their species. Therefore, Bt insecticidal crystal protein gene and the majorities of PI gene are not used for plant anti-insect genetic engineering. Oryzacystatin gene itself is a natural anti-insect gene, which strongly inhibit coleoptera insects in a lot of reports but not in lepidopterous insects., There are several successful cases applied in plants on the side. Consequently, we selected Oryzacystatin gene as target gene to introduce it into mulberry tissue by Agrobacterium-mediated and biolistic methods and primarily achieved goal.1. the results,which compared effect of phytohormone and antibiotic on the differentiation of adventitious buds in test-tube seedling of Morus alba, show that the concentration ratio of 6-BA to NAA from 20:1 to 30:1 promotes the differentiation of adventitious buds; the cultures containing abundant fructose promote ability of adventitious buds to grow strongly after leaves differentiated; the effect of a variety of antibiotic on the differentiation of adventitious buds within a suitable concentration is not prominent.2 genetic transformation system was improved and perfected in studying on Oryzacystatin gene transformed into mulberry by Agrobacterium-mediated method. The experiment of the effect on tissue culture of mulberry and transformation conditions demonstrates that affinity of mulberry receivers to OC gene or vectors containing OC gene is poor, transformation efficiency of adventitious buds which was infected by Agrobacterium on selected cultures is lower, about 5%., It was confirmed firstly that the OC gene in a few transgenic plants was transformed into the tissue cells of mulberry and integrated into genome of regenerative plant by PCR identification, DNA blot hybridization and southern blotting hybridization.3 the results researching bombarded parameter of boilistic method and transformation efficiency display that peak transformation efficiency occurred when mulberry explants selected from tip of young stems, buds in the winter, axillary buds and calli of middle size were bombarded with 500 gold particles coated with OC gene each times under the pressure of 135 psi at 7 cm distance.4. The result which Oryzacystatin gene was introduced into cell tissue of mulberry by biolistic method was confirmed by PCR identification, DNA blot hybridization and southern blotting hybridization as well as RNA blot hybridization, northern blotting hybridization. At the same time, it is certain that not only have foreign genes been incorporated into the genome, but also have expressed at RNA level. We still found that the amount of RNA expressed among different transgenic plants is different, even in different organs of a transgenic plant.更多还原