Construction Of Plant Expression Vector Of CryⅢA Gene And Its Genetic Transformation Of Potato

Colorado potato beetle (CPB), belonging to Coleopter.The harm caused by CPB in general losses can reach30%-50%, and in severe cases can reach more than90%, or even crop failure.It can also spread diseases such as brown patch, ring rot, CPB is a worldwide devastating quarantine pest, one of the China’s foreign major quarantine objects and important invasive species.So far,the potato beetle distribute the northern of Xinjiang potato producing area of260000km2.China is potato producing countries.Effective control of the Colorado potato beetle hazards, delay and prevent its spread, is of great significance for ensuring security and sustainable development of China’s potato production.Traditional chemical pesticides can play the role of crop protection in the short term, but long-term use can cause many problems, such as food safety, environment pollution, pest resistance. These problems will reduce the efficacy and increase cost.Biological insecticide is not widely used because of the high production costs, unstable field trials and so on.Cultivating resistant varieties using genetic engineering has opened up a new path for pest control.Transgenic Bt plants in production has demonstrated a good prospect, but the problem of insect resistance to Bacillus thuringensis insecticidal crystal proteins is becoming increasingly serious.This experiment takes two strategies, first is to increase the amount of gene expression in plants to kill resistant insects by selecting the strong constitutive promoter CaMV35S to control Bt gene.Secondly, use of inducible and tissue-specific promoter makes Bt insecticidal crystal protein gene highly expressed in specific tissue to reduce the selection pressure.The experiment used potato leaf and stem-specific promoter ST-LS1, the target gene CryⅢA gene is only expressed in the leaf and stem where is susceptible potato beetle damage, but not expressed in tubers.Then the edible part of the transgenic potato is more secure.The results are as follows:l.The plant expression vectors were constructed for transsformation of the CryⅢA gene, which was under control of the constitutive promoter CaMV35S or potato leaf and stem-specific promoter ST-LS1in the plant expression vector pBI121, via the use of In-Fusion technology.2.The recombinant plasmids were introduced into Agrobacterium tumefaciens strain LBA4404by freeze-thaw method. The putative transgenic plants of potato cultivars Longshu3and Gannongshu2were obtained by Agrobacterium-mediated transformation system. Results from selection of the transformed plants on the culture media containing kanamycin and PCR assay showed that the CryⅢA gene was integrated into potato genome. 3.Real-time fluorescence quantitative PCR (qRT-PCR) analysis indicated that transgenic potato plants typical fluorescence has amplification curve and the Ct values are less than40.However, non-transgenic plants and the blank control’s fluorescence amplification curve are presented straight, and located under the threshold line. The CryⅢA gene, driven by the promoter CaMV35S, expressed in roots, stems and leaves, and higher expression in leaves of the transgenic plants. The CryⅢA gene, driven by the promoter ST-LS1, expressed only in stems and leaves, and higher expression in leaves of the transgenic potato plants. However, no traces have been found in roots of the transgenic potato plants.