Study On Transgenic Turfgrass Against Chafer With Cry8Eal/cry8Gal Genes

Creeping bentgrass is an important coolseason turfgrass species with a wide distribution in keeping water and soil, regulating climates, beautifying environment throughout the world, which is commonly used for parks, home lawns and athletic field. But creeping bentgrass is subjected to the threat of insect easily, especially scarabs. It usually leads to dying away of turfgrass and makes appreciate value and use value discounted greatly.The cry8-type genes from Bacillus thuringiensis are toxic to a great deal of Coleopteran pests such as leaf beetles, scarabs and so on. cry8Ea1, cry8Ga1 genes which were cloned from Bacillus thuringiensis strains 185 had special insecticidal activity against scarabs.In allusion to cry8-type genes'characteristic of resistent to underground insect, the study started from construction of effective gramineous expression vector. Consequently cry8Ea1, cry8Ga1 genes were tranformed into creeping bentgrass through Agrobacterium umefaciens, and creeping bentgrass resistent to insect was gained successfully.Firstly the study cloned promoter of monocotyledonous catalase gene from rice by PCR method, sequence of CatB promoter was the same as published in Genbank. Then four expression vectors were constructed, p3300-Ubi-8G (Ubiquitin : cry8Ga1 ), p3300-CatB-8G (CatB : cry8Ga1), p3300-Ubi-8E (Ubiquitin : cry8Ea1), p3300-CatB-8E (CatB : cry8Ea1). They were transformed into creeping bentgrass by Agrobacterium umefaciens, resistant lines were obtained atfter phosphinothricin selection.On basis of getting resistent lines, molecular analysis was done. By PCR analysis, 19 plantlets with p3300-Ubi-8G, 22 plantlets with p3300-CatB-8G, 22 plantlets with p3300-Ubi-8E, and 28 plantlets with p3300-CatB-8E were obtained respectively. The sequence analysis results of PCR product matched with cry8-type genes, which testified cry8Ea1 and cry8Ga1 were inserted into genome of creeping bentgrass primarily. Southern blot was under way. RT-PCR results of PCR positive lines showed that cry8Ea1, cry8Ga1 genes were transcripted normally. Semi quantitative RT-PCR analysis show that there were notable difference between different lines. Expression of gene driven by CatB promoter in root is more than in leaf. Expression level of the latter was extremely weak. Trasformation plantlets could translate into active protein through ELIASA analysis. Cry8Ea1 protein expressed by plantlets with CatB promoter took 5.37% of soluble protein in root. Compared with untransformed plants those transformed with cry8Ea1 gene could grow normally after inoculated Holotrichia parallela Motschulsky larvae 30 days and showed good resistence against scarabs.The study based a solid foundation for a large scale selection of new turfgrass species with resistence against pest and had great importance for controlling turfgrass chafers and cultivating new turfgrass species.