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对冷冻和疾病具有更高耐受性的杂草。本研究从具有高度抗寒性的冬小麦中分离出了与抗冻蛋白和抗真菌蛋白相似的WAS-3基因，作为转基因苜蓿的候选基因，并利用该基因进行了转基因苜蓿的研究。在转基因小麦细胞中产生的WAS-3蛋白有效地阻断了雪霉菌Microdochium nivale菌丝的生长。此外，表达WAS-3蛋白的转基因植物表现出比对照非转基因植物更好的对尖孢镰刀菌的抗性。我们还从桑葚中分离出了具有极高抗冻性的ABA和第三组莱亚基因，从藓类立碗藓中分离出了阿坝和冷响应基因。为了获得转基因苜蓿，我们使用了拟南芥CBF 3基因，该基因编码一种转录因子，在一系列冷诱导基因的上调中起着关键作用。将携带CBF 3基因过表达的构建体的根癌农杆菌用于接种苜蓿（Medicago sativa var. Regen-SY）。筛选出卡那霉素抗性愈伤组织，并通过进一步培养再生芽。成功获得9株转基因植株。这些转基因植物表现出与非转基因植物无区别的正常生长，表明它们具有较少的体细胞突变。这些转基因植物被用于冷冻试验。

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.