Cloning and Activity Analysis of the HvSWEET4 Gene Promoter
YOU En1,2, XU Jin-Qing1,3, WANG Lei1,3, WANG Han-Dong1,3, CHEN Tong-Rui1,2, Tang You-Lin1,3, DENG Chao1,2, BIAN Hai-Yan1,2, LU Lu1,2, WANG Yi-Hang1,2, SHEN Yu-Hu1,3,*
1 Key Laboratory of Adaptation and Evolution of Plateau Biota, Qinghai Provincial Key Laboratory of Crop Molecular Breeding, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China; 2 University of Chinese Academy of Sciences, Beijing 100049, China; 3 Laboratory for Research and Utilization of Qinghai-Tibetan Plateau Germplasm Resources, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China
摘要HvSWEET4是青稞(Hordeum vulgare var. nudum)中SWEET (sugars will eventually be exported transporters)糖转运蛋白家族的重要成员,参与籽粒发育过程中己糖的转运调控。为阐明青稞HvSWEET4基因转录调控的分子机制,本研究选取籽粒大小差异显著的2个青稞品种'北青3号' (大粒)和'3917' (小粒),扩增其编码框上游约2 000 bp的启动子序列并进行系统分析。结果表明,2个品种HvSWEET4的启动子序列高度保守,仅存在10处SNPs和5处插入/缺失变异(insertions/deletions, InDels),差异位点仅占总碱基数的0.75%。启动子元件分析揭示,HvSWEET4启动子含有参与多种逆境响应、糖代谢、淀粉代谢、花粉、胚乳及糊粉层发育调节的调控元件。启动子活性分析确定其核心功能区域位于-310~-212 bp。进一步分析表明,启动子活性的差异可能与-1 999~-310 bp区域的抑制子效应,以及-212~-1 bp区域SNP位点的变化有关,这些差异可能影响转录因子结合的种类和位点。本研究为深入解析HvSWEET4在籽粒大小调控中的分子机制及其在作物育种中的潜在应用提供了重要的理论依据。
Abstract：HvSWEET4, a member of the sugars will eventually be exported transporters (SWEET) sugar transporter family in hulless barley (Hordeum vulgare var. nudum), is involved in hexose transport regulation during grain development. To elucidate the transcriptional regulatory mechanism of HvSWEET4, approximately 2 000 bp of promoter sequences in upstream of the coding region were systematically analyzed from 2 hulless barley cultivars with significantly different grain sizes: 'Beiqing 3' (large grain) and '3917' (small grain). Sequence comparison revealed that the HvSWEET4 promoters from these 2 cultivars were highly conserved, containing only 10 SNPs and 5 InDels, which accounted for merely 0.75% of the total nucleotides. Cis-element analysis indicated that the HvSWEET4 promoter contained regulatory elements associated with stress responses, sugar and starch metabolism and developmental regulation of pollen, endosperm and aleurone layers. Promoter activity assays identified the core functional region at -310~-212 bp. Further analyses suggested that differences in promoter activity could be attributed to suppressor effects within the region from -1 999~-310 bp, as well as SNP variations in the region from -212~-1 bp, potentially altering the types and sites of transcription factor binding. These findings provide essential theoretical foundations for further dissecting the molecular mechanisms underlying HvSWEET4 role in grain size regulation and highlight its potential applications in crop breeding.
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