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Construction And Analysis Of Cotton SSH Library Induced By Sucking Stress Of The Apolygus Lucorum Meyer-Dür

Posted on:2012-10-29Degree:MasterType:Thesis
Country:ChinaCandidate:J LiFull Text:PDF
GTID:2143330335479331Subject:Biosafety
Abstract/Summary:PDF Full Text Request
After the Bt cotton cultivated in large area, the cotton plant bugs damaged more and more severely to the cotton industry and also difficult to control. In this paper, we chose transfer (Bt+CpTI) insect resistance cotton (CCRI41) and non-transgenic cotton (CCRI23) as materials, constructed the Apolygus lucorum sucking stress induced cotton SSH libraries. We got some special genes, and analyzed them by semi-quantitative PCR and fluorescence quantitative PCR, so that we defined the expression quantitative of those genes under sucking and non-sucking stress conditions. The differences of ESTs of insect resistance and non-transgenic cotton SSH libraries were analyzed by bioinformatics tools. This study was very important for us to deeply study the resistance mechanisms of cotton to insects, and also we got some resistance gene of cotton, this can help us to cultivate resistant cotton, and explore new method for Apolygus lucorum management.The main results are as follows:1. We constructed suppression subtractive hybridization library (SSH) of cotton (non-transgenic cotton CCRI23 and transgenic cotton CCRI41) under sucking stress of A. lucorum, and obtained 92 unigene in SSH libraries of CCRI23, in which 75 in forward library, and 17 in reverse library, there were 96 effective unigene CCRI41 SSH library, in which 78 in forward library, and 18 in reverse library.2. Blast the Unique EST sequences in CCRI23 and CCRI41, in CCRI23 SSH library, there were 60 ESTs which can not find similarity sequences, and 32 ESTs can find the similar sequences. While in SSH library of CCRI41, there were 23 ESTs that can not find similar sequences, and 73 ESTs had the similar sequences. By analysis the different genes from SSH libraries of CCRI23 and CCRI41, we found the stress responses including stress resistance, energy synthesis and metabolism, signal transduction, gene regulation, and other biological pathways to the sucking stress induced by A. lucorum, which had directly or indirectly defense A. lucorum sucking.3. Analysis of forward and reverse ESTs in the CCRI23 and CCRI41 libraries by semi-quantitative RT-PCR, we obtained 8 significant difference genes in which there 5 was up-regulated and 3 was down-regulated. By qPCR four plant related tolerance genes PPO, LRR-RLK, HSP70 and LHCⅡexpression changes under the A.lucorum sucking stress and mechanical damage were analysis. In CCRI41 under the sucking stress, the expression quantitative of PPO, HSP70 increased and changed sharply, increased about 1082 times and 6237 times, respectively. And the expression quantitative of LRR-RLK reduced and changed small, the expression quantitative of LHCⅡwas down-regulated and changed significantly, decreased about 188 times. In CCRI23, the expression quantitative of PPO had not changed in trend, the expression quantitative of HSP70 was down-regulated and change not significant, LRR-RLK was up-regulated and the expression increased by about 134 times, the expression quantitative of LHCⅡwas down-regulated, and the expression trends like (CCRI41), but the variation was smaller. In mechanical damage condition, the expression quantitative of 4 genes in front had no significant changes in CCRI23 and CCRI41, which show that the expression changes of four genes were related with sucking stress, but no correlation with mechanical damage.?4. GO and COG analysis the known ESTs in forward and reverse libraries of CCRI23 and CCRI41. GO analysis showed that it was more types in CCRI41 than CCRI23 SSH libraries, which including cellular component, biological processes and molecular function. In CCRI23, COG protein cluster analysis showed that the ESTs in the positive and negative libraries were clustered into 3 categories and 1 protein group, respectively. In CCRI41, COG protein cluster analysis showed that the ESTs in the positive and negative libraries were clustered into 10 categories and 3 protein groups, respectively, the result showed that the types of the known functions ESTs were more in CCRI41 SSH libraries.
Keywords/Search Tags:cotton, green plant bugs, suppression subtractive hybridization (SSH), sucking stress, fluorescence quantitative
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