MASP And CDNA-AFLP Analysis Of Differentially Expressed Genes Under Chilling Stress Response In Brown Cotton

Cotton is a kind of thermophilic crop. Low temperture is a major environmental factor affecting cotton yield. The objective of this study was to determine temporal expression profiles of transcripts during cold acclimation of cotton by using MSAP、 cDNA-AFLP and semi-quantitative RT-PCR. The upland cotton line ZongCaiXuan No.1with chilling treatment was analyzed. The inducible genes under chilling stress were screened to provide a better understanding about the underlying molecular mechanisms of cotton in response to low temperature. Furthermore, the experiment analyzed the relationship between the chilling resistance and the changes of five physiological indices including SOD, CAT, POD activity, MDA, proline content. The results was applied to illustrate that whether the relationship was present among inducible gene under chilling stress and physiological index, which to facilitate cold resistant breeding of brown cotton.The results were as follows:(1) In the MSAP experiment,66-primer combinations were used. The patterns and levels of cytosine methylation are different among different treatments.29polymorphic fragments were identified. The result of BLAST showed that,11fragments (Seql、 Seq2、Seq4、Seq11、Seq15、Seq18、Seq19、Seq21、Seq23、Seq26、Seq27) were homologous with genes from cotton, including promoter, transcription factor, drought defense, celullose synthase and so on.(2) To study the roles of DNA methylation in regulation of gene expression, four genes, including GhARM, GhARK, GhCLSD and GhTPS, involved in abiotic stress response, were specifically amplified by semi-quantitative RT-PCR reaction. The results showed that cold treatment decreased the expression of GhARM, GhARK and GhCLSD. However, the expression of GhTPSwas up-regulated under cold stress.(3) In the cDNA-AFLP experiment, after screening with64primer combinations,2321bands were amplified and34polymorphic transcripts derived fragments (TDFs) were obtained. According to the difference of gene expression patterns,22polymorphic TDFs were isolated and cloned. Among the22TDFs,18TDFs were found to be related to low temperature treatment, including2TDFs up-regulated expression,9TDFs down-regulated expression and7TDFs transient expresstion. In addition, there were4 TDFs specifically expressed after two-day recovery under normal conditions. The results of BLAST indicated that21TDFs were found to be homologous with functional genes, including hypothetical protein, transposon, peroxisome proliferator activated receptor and stress defense and so on, but1TDF’s function remains unknown.(4) By MSAP and cDNA-AFLP analysis, we found that ten sequences (Seq3、Seq5、Seq7、 Seq9、Seq10、Seq13、Seq16、Seq17、Seq29、Seq49) were closely related to active oxygen scavenging, H2O2signal transduction and metabolism. Moreover, the Seq2and Seq4were associated with the cell osmotic potential regulation. The results showed that their specificity expression increased the activity of SOD, CAT, POD and the content of MDA. However, the content of proline decreased.