[Objective]The study was to analyze the expression of the deletion fragments from the promoter of a glycosyltransferase gene induced both by MeJA and SA cloned from tobacco W38（sm-Ngt） in transgenic tobacco plants.[Method]Using T1 seedlings of sm-Ngt transgenic tobacco lines containing Gus gene controlled by five 5＇ flank deletion promoter fragments different in length as experimental materials,GUS histochemical staining and fluorometric analysis of T1 seedlings treated with MeJA and SA for 16 h were conducted to analyze the effect of MeJA and SA treatment on the expression of 5＇ flank deletion promoter fragments.[Result]Of five 5＇ flank deletion promoter fragments transgenic plant lines,30 d old T1 seedlings containing 220-0 bp promoter fragment performed worst in GUS staining（showing least staining spots）,those containing-524-0 bp and-468-0 bp promoter fragment both performed best.In the plants not treated with MeJA and SA,activities of GUS driven by-524-0 bp and-468-0 bp deletion promoter fragments were enormously higher than that driven by-1 150-0,-800-0 or-220 0 bp,and which were proved to be not resulted from insert copy number by Southern blot.For GUS expression,promoter fragment-800-0 bp expression was doubly induced by both MeJA and SA,while fragment-1 150-0 was induced by MeJA.[Conclusion]There are activity enhancement elements within-524--220 bp of the sm-Ngt in promoter and activity down regulation elements within-1 150--524 bp region,as well as MeJA and SA doubly inducing activity regulation elements in this promoter.
DNA methylation is known to play a crucial role in regulating plant development and or- gan or tissue differentiation. In this study, we as- sessed the extent and pattern of cytosine methylation during rapeseed (Brassica napus L.) seed germina- tion, and compared the methylation level of various tissues in seedling, using the techniques of methyla- tion-sensitive amplified polymorphism (MSAP) and HPLC separation and quantification of nucleosides. In all, 484 bands, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified by 12 pairs of selective primers in DNA ob- tained from dry seeds. A total of 76 sites were found to be differentially digested by the isoschizomers, indicating that approximately 15.7% of 5′-CCGG-3′ sites in the genome were cytosine methylated. Four classes of patterns were observed in a comparative assay of cytosine methylation in the dry and germi- nating seeds; a small number of hypermethylation events occurred at 5′-CCGG-3′ sites in germinating seeds compared with dry seeds, while many more hypomethylation events were detected after seed germination. Differences in DNA methylation level in various tissues were also detected; radicel was less methylated than hypocotyl and cotyledon. These observations were further confirmed by HPLC analy- sis. In addition, sequencing of eleven differentially methylated fragments and the subsequent blast search revealed that cytosine methylated 5′-CCGG- 3′ sequences were equally distributed between cod- ing and non-coding regions. These results clearly demonstrate the power of MSAP technique for large-scale DNA methylation detection in rapeseed genome, and the complexity of DNA methylationchange during seed germination. DNA Hypomethyla- tion going with seed germination appears to be a necessary step toward transcriptional activation in gene expression, and may well contribute to the de- velopmental gene regulation.
LU Guangyuan WU Xiaoming CHEN Biyun GAO Guizhen XU Kun LI Xiangzhi
