BASIC RESARCH FOR IMPROVEMENT OF COLD AND DESEASE RESISTANCE OF PERRENIAL WEED PLANTS BY GENETIC ENGENEERING

这项研究的目标是通过基因工程产生能够提高对冰冻和疾病的耐受性的杂草。从具有较强抗寒性的冬小麦中分离出与抗冻蛋白和抗真菌蛋白具有相似性的抗冻蛋白和抗真菌蛋白基因IS-3，作为产生转基因的候选基因。转基因小麦细胞中产生的AS-3蛋白有效地抑制了雪霉菌(Microdochium Nivale)菌丝的生长。此外，表达is-3蛋白的转基因植株比对照植株对尖孢镰刀菌表现出更好的抗性。我们还从桑树中分离到编码脱水蛋白和第三组LEA的基因，并从苔藓Physcomitrella patents中分离到几个ABA和冷响应基因。为了生产转基因紫花苜蓿植株，我们使用了拟南芥CBF3基因，该基因编码一个转录因子，在许多冷诱导基因的上调中发挥关键作用。利用携带CBF3基因过表达载体的根癌农杆菌接种紫花苜蓿(Medicago sativa var.)Regen-sy)。筛选出了卡那霉素抗性愈伤组织，并通过进一步培养获得了再生芽。成功地获得了9株转基因植株。这些转基因植物显示出正常生长，与非转基因植物没有区别，这表明它们的体细胞突变较少。这些转基因植物被用于冷冻测试。

The goal of this research was to generate weeds that has increased tolerance to freezing and disease by genetic engineering. Experiments were focused on (1) isolation of genes that can confer plants with tolerance to freezing and disease, and (2) production of transgenic alfalfa plants by using the isolated genes.As a candidate gene for production of transgenics, WAS-3 gene, which has similarity to antifreeze proteins and antifungal proteins was isolated from winter wheat that has high degree of winter-stress resistance. The WAS-3 protein produced in transgenic wheat cells efficiently blocked growth of hyphae of the snow mold, Microdochium nivale. In addition, transgenic plants expressing the WAS-3 protein exhibited a better resistance to Fusarium oxysporum than control non-transgenic plants. We also isolated genes encoding dehydrins and group III LEA from mulberry, which exhibits extremely high freezing tolerance in winter, and several ABA and cold-responsive genes from the moss Physcomitrella patens.For production of transgenic alfalfa plants, we used Arabidopsis CBF3, a gene encoding a transcription factor that plays a critical role in the up-regulation of a number of cold-inducible genes. Agrobacterium tumefaciens carrying a construct for overexpression of the CBF3 gene was used for inoculation of leaf discs of alfalfa (Medicago sativa var. Regen-SY). Kanamycin-resistant calli were selected and shoots were regenerated by further cultivation. Nine transgenic plants were successfully regenerated. These transgenic plants showed normal growth indistinguishable from non-transgenics, suggesting they have less somatic mutations. These transgenic plants were being used for the freezing tests.