The Construction Of Transgenetic System For Locusta Migratoria Manilensis (Meyen)

The Locusta migratoria manilensis (Meyen) is one of the most widespread agriculture pest in China with a broad geographic distribution covering both temperate and tropical climatic zones. The species also serves as an important model in the studies of morphology, physiology, developmental biology, and neuroscience and so on. With the development of molecular biology and biotechnology, molecular techniques have been widely used in research of locusts. So far, the investigation of the locust gene functions has become a hot spot with lots of genes cloned.Transgenetic technology is one of the most important approaches for determining the gene functions for insects. To date, transgenetic systems have been developed for some insects. However, the research on gene of L. migratoria manilensis (Meyen) with the technology at the molecular level has fall behind. No operative transgenetic vector, vector screening system and transgenetic method have been reported. Therefore, there is a urgent need to develop transgenic technological system for the gene function research of the locust.The precent study aims to develop an effective and rapid transgenetic technological system that lays a basis for the functional invesigation of genes for the locust. The currently available and widely used transposon piggBac was used to construct the vector, and the EGFP as a fluorescence marker gene. The microinjection was served to deliver the vecior with target genes and the helper vector. The various influence factors involved in the transgenetic process was optimized, and the transgenetic individuls were confirmed by microscopr, PCR and sequencing. The main results are summarized as follows.(1) The optimization of fixing and moisture keeping of eggs: The problems for fixing and moisture keeping of eggs were solved by comparing different measures. For the best choice, the eggs were fixed into channel made of humid sponge for microinjection that were housed in 90 x 15 mm size of pedri dishes, The sponge need to be maintenced with propert humidity, no water overflowing, but enough moistre. Sponges and pedri dishes were necessary to be sterilized prior to injection against microbiological infection.(2) The optimization of injection spot decision of eggs: The various points of egg's furface were used for microinjection to determine which spot was optimal for highest hatching rate and transgenetic effectiveness. The results showed that the position 1/3 to the egg's small end in ventral side is the best spot for microinjection.(3) The optimization of microinjection volume: The microinjecter (nanoject II) was used for microinjection in the research. No obvious linear relationship between injection volume of plasmid solution and survival rate was observed in the scope of nanoject II calibration. However, the elevation of injection volume increased the transformation rate. As a result, the injection volume each egg, 69 nl, was decided to be optimal, which is the biggest volume for single microinjection for the microinjector.(4) The optimization of culture condition after injection: Various culture measures were experimented to obtain high survival rate of the eggs after microinjection. Compared with steriled sandy soil as culture base, the capped petri dishes with sponges as culture base, directly kept in temperature-controlled incubators, could benefit the egg's survival.(5) The positive individuals were obtained and confirmed by fluorescence microscope and molecular technologies, which indicates that the constructed transgenetic system could work well for the locust species. The transgenetic individulas were screened with fluorescence microscope, and the compound eyes of the positive individuals produced green fluorescence stemed from the the EGFP fluorescent marker gene. The PCR and sequencing results with genomic DNA extracted from the positive individulas, using EGFP-anchored primers, confirmed that the individuls were successfully transformed with the EGFP genes.The further works for the transgenetic technological system includ: 1) the further test and optimization of the system with more individuls; 2) the method development for establishing homological strain; 3) the employment of Southern blot to check the copy of inserted genes and position in chrimosomes.