| Cassava has a prominent drought-resistant ability in comparison with the other plants. The drought-resistance physiological mechanism of cassava has been reported a lot, but the molecular mechanism has not yet been reported. cDNA-AFLP is a high-throughput and economic technique for discovery and distinguish differentially expressed gene fragment. This study improved traditional cDNA-AFLP technique by absorbing the SMART technique method. We insert a restriction enzyme cutting site of MmeⅠ in5’end of the double-strand cDNA, and then digested by MmeⅠ combination with other restriction endonucleases, so the5’end of differentially expressed transcript-derived fragments (DE-TDF) maybe begin at the transcription start site (TSS) of genes and we can get more information of5’ent by this method.This study established and applied the modified method to discovery the drought-resistance genes with the samples of KU50and SC124under drought treatment. The main results were as follows:1. Obtained differentially expressed gene TDFs of cassava:restriction endonucleases combination MmeⅠ and EcoRⅠ or MmeⅠ and Bst YI were used and obtained483TDFs.108of them were DE-TDFs. The proportion of DE-TDFs was21.9%. Sixty-eight DE-TDFs out of them were sequenced and51DE-TDFs have M and E adaptor,5DE-TDFs have M and M adaptor,8DE-TDFs have M and B adaptor,3DE-TDFs have E and E adaptor,1DE-TDF has B and E adaptor at both ends of the TDFs.2. Position effect of DE-TDFs with improved cDNA-AFLP technique:in order to investigate whether the DE-TDFs start from the transcription initiation site of genes, we analyzed the position of DE-TDFs in the genes. The results were as follows:4DE-TDFs strated at5’UTR or translation initiation site;8TDFs started at less than200bp away from TSS;19DE-TDFs are located at more than200bp away from TSS or near3’UTR. The results indicated that39%DE-TDFs obtaining by modified cDNA-AFLP technique that started at or near TSS, which matche the expectation of this mothed.3. Functional annotation of the drought-tolerance relate genes:using Phytozome v8.0and NCBI websites to annotate the DE-TDFs:33DE-TDFs have homologous genes with functional annotation,20DE-TDFs have homologous with no functional annotation and15DE-TDFs have no homologous genes. The33drought-tolerance relate TDFs participate in Transcription factors, Energy metabolism, Sugar metabolism, Lipid metabolism, Cell growth and apoptosis, Signal transduction, Transfer of transport, Genetic information processing, Transcriptional regulation, Peptides synthesis and metabolism and Photosynthesis respectively. Ten DE-TDFs found homologous sequence in Jatropha, Ricinus communis, Euphorbia tirucalli, Leafy Spurge in Euphorbiaceae.4. Discovery eight important drought-resistance related gene:analysis the function and confirmed by qRT-PCR with TDFs which may related in drought-tolerance and shows difference compare with the control. The8genes involved in photosynthesis, transcription, genetic information processing, signal transduction, carbohydrate metabolism process respectively and2DE-TDFs with unknown function. The qRT-PCR result shows that:with the drought stress increased, the8DE-TDFs all showed significant differentiation. Additionally, the differential expression of FBPase, Bin and Pho reached a highly significant level in SC124and Unknownl and Unknown2reached a highly significant level in KU50. So that we inferred that the rest60DE-TDFs were all differentially expressed genes under drought stress indeed.5. Establish an improved cDNA-AFLP technique. The improved cDNA-AFLP technique is a feasibility technique with high efficient screening differentially expressed genes. |
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