Transformation Of Bt Cry1Ia8/cry1C Genes And Study Of The Resistance To Pests In Transgenic High Generation Of Inbred Lines In Cabbage

Cabbage(Brassica oleracea L. var. capitata) is an important vegetable in China. The damage on growth of cabbage is mainly caused by Lepidopteran pests such as cabbage worm(Pieris rapae),diamondback moth(Plutella xyllostella), beet armyworm(Spodoptera exigua) and cabbage moth(Mamestra brassicae) in production. The lack of insect-resistant germplasm resources is an obstacle in traditional breeding. Insect-resistant cabbage can be obtained through genetic engineering, which may provide new germplasm resources for cabbage breeding.Bt cry1Ia8 and cry1 C gene was cloned from Bacillus thuringiensis by the Institute of Plant Protection, Chinese Academy of Agriculture Sciences. Cry1Ia8 toxin has obvious insecticidal efficacy to diamondback moth and the pests didn’t have cross-resistance with Cry1 Ac which is widely used in production. The new cry1 C sequence was artificially synthesized with optimal codon and Cry1 C toxin has insecticidal activity to noctuids pests. In this study, plant bivalent expression vector cry1Ia8-cry1 C was constructed and transferred into cabbage line by Agrobacterium tumefaciens-mediated transformation method. We hope new transgenic plants can effectively restrain diamondback moth,cabbage worm and noctuids pests. Agronomic traits, insect-resistance and influence on A. mellifera were investigated. The main results were as follows:1. Construction of plant bivalent expression vector cry1Ia8-cry1 C. The obtained PBI121 vector was further verified by PCR analysis, and sequencing.2. Optimization of the regeneration system and genetic transformation system in cabbage F13J85.Both hypocotyls and cotyledons with petiole of F13J85 were perfect explants for regeneration. Explants had highest regeneration frequency using buds of 5 days from sowing, putting explants completely horizontal on medium, diluting bacterium solution with OD600 being 0.6-0.8 for infection and controlling infection time for 12-15 min. Transformation of cabbage with pyramided Bt cry1Ia8 and cry1 C genes. A total of 16 kanamycin-resistant plants transformants were obtained. 12 of the kanamycin-resistant plants showed PCR positive and 8 southern blot positive plants, 7 RT-PCR and western blot positive plants were obtained.3. In the fall of 2014, we investigated agronomic traits and the amount and type of insect on T4 generation of cry1Ia8 and cry1Ia8-cry1Ba3 cabbage in the field. The date showed that there were not significant difference in most agronomic traits between transgenic plants and the controls. T4 generation of cry1Ia8 and cry1Ia8-cry1Ba3 cabbage still effectively control both diamondback moth and cabbage worm, but were susceptible to beet armyworm and cabbage moth.4. The survival and body weight of the bees were not influenced by Cry1Ia8 cabbage. Although some changes in activities of metabolic and digestive enzyme happened. However, no significant differences were found in growth of the bees. The results indicated that Cry1Ia8 cabbage pollen did not pose deleterious effects to worker honeybees.