Construction Of Aphid Resistance Gene PPA Expression Vector And Validation Of Expression In Wheat Protoplast

The pest is a worldwide problem threating to agricultural production. It's significant to create new insect-resistant plant strains with transgenic technology for agriculture pest control, environmental protection and sustainable development of agriculture. Currently, the plant lectin insect-resistant genes are mostly focused on the Galanthus nivalis Agglutinin (GNA) gene. Pinella is one endemic specie of Chinese herbal medicine, the crude extract from Pinellia pedatisecta has a strong lethal effect on aphids, and the main component is Pinellia pedatisecta agglutnin, PPA. Tobaccos with the agglutinin gene from Pinellia ternata harbor more resistant aphid activity than those with GNA gene.The maize Ubiquitin promoter Ubi that was widely implicated in monocots, retains many advantages such as high promoting efficiency, low level of methylation, and stable genetic traits, and so on., moreover, the Ubi promoter has a higher activity in transgenic wheat, the expression efficiency up to 4-5 times than the CaMV35S promoter. Green fluorescent protein (gfp) was derived from marine organism Aequorea victoria in a light-emitting proteins, it has many advantages as reporter molecule: stability, high sensitivity, fluorescence reaction requiring no any substrate and cofactor to simplify the expression detection efficiency, and it could express in cells autonomously, has no species-specific, non-toxic to the receptor cells, and has no effect on the target gene. GFP gene was selected as a reporter gene in this study.The objective of this study is to transform PPA gene into the plant expression vector pCAMBIA1380 (promoter: Ubi, reporter gene: GFP), to create a highly effective anti-aphid plant expression vector. It formed the material foundation for genetic transformation, and provided new genetic resources for plants especially for Pest control.In this study, PPA gene was cloned with genetic engineering techniques, and transferred into the eukaryotic expression vector. The results: firstly, the plant expression vector pUGP was successfully constructed, it contained Ubi promoter, PPA gene, GFP genes, providing an experimental platform and basic materials for subsequent protein expression and functional experiments. Next, wheat protoplasts were the successfully prepared, and transient transformed.