Differential Expression Analysis Of Proteins Under Verticillium Dahliae Stress In Cotton

Verticillium wilt is a worldwide destructive plant disease, which is recognized as a serious threat to cotton production. In this research, two-dimensional electrophoresis was used to study the differential expression proteins under Verticillium dahlia stress in cotton. Then MS was used to study the differential expression proteins. This is not only propitious to enrich the biochemical theory of the resistance of Verticillium wilt in cotton, but also can get the amino acid sequences of the resistant proteins. This can provide guidance in the breeding of resistant variety by genetic engineering.The variety of cotton named Yuwu620 was used in this research. Yuwu620 inoculated V.dahliae. A system of 2-DE that is suitable for cotton was established. The improved TCA-acetone was used to prepare protein sample. In this step, the time of using acetone to precipitate proteins was three. Through this improvement, protein sample had higher concentration and was pure. In IEF, the final voltage reached 120000V. This can make IEF get a good result. The time of equilibration of the IPG gel strips was made sure, which was helpful for avoiding vertical streaks. It contained two steps, the time of first step was 20min, and the second was 25min. Then the SDS-PAGE was carried out by the gel (T=12%) under lower and steady electric current.PDQuest was used to study the 2-DE maps of leaves proteins that were extracted from inoculated plant and CK. The results showed that there were many differences between the protein maps that inoculated and CK. Three spots appeared in the protein map of the leaves inoculated, but was not existed in CK. Sent these three spots to study by MALDI-TOF-MS, then we performed database search for these three differential expressed proteins. The result shows that these;proteins showed some similarity with known proteins in the database. They may have function in the resistance responses to Verticillium wilt.(1) The protein spot DB1 showed some similarity with DEAD/H box RNA helicase. The protein has an ATP binding site. It might have NBS domain and have important function in signal transduction pathway and the immune regulating of host. It may have function in the resistance responses to Verticillium wilt.(2) The protein spot DB2 protein showed some similarity with serine proteinase inhibitor.It belonged to potato proteinase inhibitor I . It will accumulate in plant when plant was infected by microbial and can inhibit the proteolytic enzymes of microbial. The protein has defensive characteristic and can effect the defensive responses of plant. (3) The protein spot DB3 showed some similarity with ODR-3. It belonged to the a subunit of G protein. This protein might combine with receptor and activate some signal transduction pathway, then induce some defensive responses of cotton.