拟南芥种子特异性表达双向基因对AtBGLU19/AtMBL1的共表达调控研究

A bidirectional gene pair is defined as two adjacent genes which are located on opposite DNA strands and transcribed divergently. This organization has been known for many years, which is common in different organisms, including bacteria, yeasts, plants and animals. Microarray-based studies indicated that the majority of bidirectional gene pairs are co-expressed. To our knowledge, the mechanisms involved in the co-expression regulation of bidirectional gene pairs have not been well understood until now. Based on bioinformatics studies and our findings, we propose that the transcriptional factor interacting with the orientation- independent cis-acting element controls the co-expression of a bidirectional gene pair..To test our hypothesis, an Arabidopsis bidirectional gene pair (At3g21370 and At3g21380) was chosen for the present study. At3g21370 and At3g21380 loci encode beta glucosidase 19 (BGLU19) and a mannose-binding lectin superfamily protein (MBL), respectively. This bidirectional gene pair is then named as AtBGLU19/AtMBL1 by us. In semi-quantitative RT-PCR analysis, transcripts of these two genes showed almost identical expression profiling in the presence of low temperature and ABA stresses. Using a GFP:Promoter:GUS transgene approach has revealed that the intergenic region of divergent genes AtBGLU19 and AtMBL1 is a seed-specific bidirectional promoter, which exhibits similar tissue-specific expression patterns in both orientations. These above results indicated that the bidirectional genes AtBGLU19/AtMBL1 are co-expressed genes and regulated by a functional bidirectional promoter..To further verify our hypothesis, we will perform a comprehensive analysis of the tissue-specific expression and induction patterns of the AtBGLU19/AtMBL1 bidirectional promoter, and then use the promoter deletion and mutation and electrophoretic mobility shift assays to identify the cis-regulatory elements controlling the tissue-specific expression and induction patterns of the AtBGLU19/AtMBL1 bidirectional promoter. Next, we will use the yeast one-hybrid assay to screen the transcriptional factors which bind to these cis-elements, and perform EMSA and CHIP-qPCR assays to examine whether these factors directly bind to the cis-elements in vitro and in vivo. Finally, by using stably expressed transgenic lines, we will investigate how these transcriptional factors regulate the coordinated expression of the AtBGLU19/AtMBL1 bidirectional genes through interaction with the cis-acting elements. In summary, these studies provide new insight into the mechanism of co-expression of the bidirectional genes in higher plants, establish novel approachs and methods for research on the bidirectional gene pairs, and provide bidirectional promoters used for genetic engineering-based crop improvement.

双向基因对是指相邻且转录方向相反的两个基因。生物信息学研究表明双向基因对存在共表达现象，但其具体机制尚未阐明。基于已完成项目实验结果，我们认为转录因子与不依赖于方向的共享顺式元件互作调控双向基因对共表达。为证实该假说，本项目以拟南芥双向基因对β-葡萄糖苷酶19基因/甘露糖结合凝集素基因为研究对象，首先证实双向基因对的两个基因在相同组织或胁迫下共表达，两基因的间隔序列为种子特异性双向启动子。后续我们拟详细分析双向启动子活性，利用启动子双向缺失和启动子突变等实验鉴定重要调控元件，通过酵母单杂交、EMSA和CHIP-qPCR筛选验证调控元件结合蛋白，利用转基因稳定表达系统分析调控元件与结合蛋白互作对双向基因对转录活性的影响。研究结果将在调控元件与转录因子互作这一更高层次上阐释双向基因对共表达机制，为双向基因对实验研究提供新方法建立新途径，为作物遗传改良提供合适的双向启动子。

生物信息学研究发现，在不同生物基因组中存在大量的双向基因对，并且大多数双向基因对存在共表达现象，但其具体机制尚未阐明。本项目以拟南芥双向基因对AtBGLU19/AtMBL1为研究对象，首先明确了AtBGLU19/AtMBL1双向基因对的共表达模式，证实两基因的间隔序列为种子特异性双向启动子。然后利用启动子缺失和启动子突变等实验鉴定了调控元件RY的作用，通过EMSA和CHIP-qPCR筛选验证与RY元件结合的AtABI3蛋白，最终证实AtABI3蛋白能够正调控AtBGLU19/AtMBL1双向基因对的转录活性。以上研究结果为双向基因对转录调控机理阐释和新基因功能鉴定提供了重要的实验依据和理论指导。