Construction And Analysis Of A Normalized CDNA Library From Immue Tissues Of Locust Migratory And The Central Nervous System Transcriptome Analysis Of Locust Migratory

The migratory locust (Locusta migratoria) is a widespread agriculture pest in China. It can cause significant damage to pastures and crops; it's also a major threat for food safety, social stability and economic development. So we should strengthen on the research and control of agriculture pest. Because of the environmental pollution and some other reasons, chemical pesticide must be instead of new generation biological insecticide. However, slow speed of killing is perceived as a potential drawback to use biological insecticide against locust due to the immune reactions of the insect and the lack of specificity efficient targets.Metarhizium anisopliae, a kind of entomopathogenic fungus widely applied in domestic and abroad, has an important role in biological control of insect, The migratory locust is an orthopteran pest and a reprensentative member of hemimetabolous insects, in the course of long evolution, it adopt own immune system with great significance for biological studies. The transcriptomic data of locust infected by fungi would provide invaluable information for molecular entomology and pave a way for the comparative research of other medically, agromically, and ecologically relevant insect. A better understanding of fungal-induced immune responses would identify the insect defense genes, and hence help establish new strategies to accelerate host death in a biological control scenario to fight against important pests.Nervous system is very important for the regulation of the growth and development, ecdysis, pheromone biosynthesis and release and the expression of the genetic information in insect, also, there are amount of chemical insecticide targets in insect nervous system. So, it's a good approach to find specificity efficient targets for biological pesticide by way of the analysis of the transcriptome of L. migritoria central nervous system.A normalized cDNA library based on SMART Method combined with DSN treatment was constructed using mRNA extracted from the fat body and hemocyte of L. 6-24 h after naturally infected with M. anisopliae. Analysis of 259 unigenes out of 303 sequenced inserts from the cDNA library revealed that the cDNA library was not contaminated with any M. anisopliae transcripts and contained currently characterized immune-related genes. These results suggest that this method is reliable and feasible for constructing host cDNA library from mycosed insect. Another normalized cDNA library was also constructed based on SMART Method combined with DSN treatment using mRNA extracted from the brain and ventral nerve cord of L. migratoria. Large scale sequencing was executed and results in 5089 high quality unigenes which was blasted respectively against the public databases Nt, Nr, SWISSPROT and KEGG. Finally, GO annotation and domain prediction was completed by using INTERPRO program.The main results were as follows:1. A large number of mRNA was obtained through two steps. At first, high quality total RNA was extracted with Trizol reagent. Then mRNA was isolated from total RNA by using Oligo(dT)-Cellulose columns. The isolated mRNA was very intact and fit for the construction of cDNA library.2. Immune reaction of the L. migratoria was induced successfully by natural inoculation of the M. anisopliae conidium.3. To date, for the first time, a normalized cDNA library was constructed from fat body and hemocyte of the host insect naturally infected with entomopathogenic fungus. Clones from this cDNA library were randomly picked and sequenced. Unigenes were analyzed against Genebank using Blast-X and Blast-N revealed that the cDNA library was not contaminated with any M. anisopliae transcripts and contained currently characterized immune-related genes.4. In addition, another normalized cDNA library was also constructed using mRNA extracted from the brain and ventral nerve cord of L. migratoria. Large scale sequencing was executed and results in 5089 high quality unigenes which was blasted respectively against the public databases Nt, Nr, SWISSPROT and KEGG. Finally, GO annotation and domain prediction was completed by using INTERPRO program. A total of 994 unigenes have significant similarity with sequences from present public database, the predicted function of these unigenes contained a lot of important genes in nervous system of insect, such as ion channels, neuropeptides, hormone receptor, signal transduction factors and so on.