Construction And Preliminary Analysis Of Suppression Subtractive Hybridization Library For Cotton Induced By Spodoptera Litura

The research of plant allelopathy that caused by target pest is a hot subject in the agricultural research field now. The physiological process of plant defence that caused by insects is very complicated. The study of the action mode of plants allelopathy increase can help people find the pest natural control factors, make sure the defence mechanism, and construct the system of an environment friendly approach to control pest.To study differential expression of genes in the healthy cotton plant and in the cotton induced by Spodoptera liture, a subtracted library of cotton was constructed by using the suppression subtractive hybridization (SSH). We compared the kinetic expression of genes involved in plant responses under natural growing and herbivore stress, and studied the molecular mechanism and the new insect-resistant genes of cotton.After the differential screening, the results obtained are as following:After similarity analysis based on BLASTx in GenBank, 59 differentially expressed genes were obtained, and 40 genes of them were similar with the sequences of the genes with known function, and the left 19 genes were similar with those with unknown function. Thirty-five ESTs were obtained from the forward library, and 24 ESTs were obtained from the reverse library. According to the physiological effects of those genes, the sequences can be divided into 9 types. They were related to signaling pathway, gene regulation, anti-stress ability, defense responses, energy synthesis and metabolism, proliferation of cells. These genes defended against Spodoptera liture directly or indirectly.Physiological stress of Spodoptera liture could increase the content of O2.- in cotton. During the treatment period, the activity of enzyme systems, such as SOD, POD and MFO, increased at the beginning, then decreased. The scavenging system of active oxygen of zhong12 started earlier than that of 33B, which might be related with resistance of Bt gene. ADH might be involved in energy synthesis, which acted in fermentation metabolism, and the activity of ADH also increased after herbivore stress. By quantitative analysis of polyubiquitin and magnesium-chelatase subunit H family protein by real-time PCR, we found they had a similar changing trend, that is, they increased at first, then decreased extremently slowly. The gene expression levels of polyubiquitin reached the highest after 8h of the pest stress, which is 40 times compared to that of the control cotton, and still maintained in a higher level after 10h and 12h of the pest stress Magnesium-chelatase H subunit was the receptor of ABA, and involved in the stress defense as a signaling component. The gene expression levels of magnesium-chelatase H subunit reached a small climax after 4h of pest stress, then decreased, but after 10h, rapidly reached the big climax , which was 30 times compared to that of the control, then decreased to some expression level.This study provides the basis for the gene regulation involved in the target pest inducing the responses in cotton, and provides support for searching the induced plant defense genes against insect pests and more modes of action of insect resistance transgenic plants.