Suppression Subtractive Hybridization Analysis of Genes Regulated by Application of Exogenous Abscisic Acid in Pepper Plant (Capsicum annuum L.) Leaves under Chilling Stress

Low temperature is one of the major factors limiting pepper (Capsicum annuum L.) production during winter and early spring in non-tropical regions. Application of exogenous abscisic acid (ABA) effectively alleviates the symptoms of chilling injury, such as wilting and formation of necrotic lesions on pepper leaves; however, the underlying molecular mechanism is not understood. The aim of this study was to identify genes that are differentially up- or downregulated in ABA-pretreated hot pepper seedlings incubated at 6°C for 48 h, using a suppression subtractive hybridization (SSH) method. A total of 235 high-quality ESTs were isolated, clustered and assembled into a collection of 73 unigenes including 18 contigs and 55 singletons. A total of 37 unigenes (50.68%) showed similarities to genes with known functions in the non-redundant database; the other 36 unigenes (49.32%) showed low similarities or unknown functions. Gene ontology analysis revealed that the 37 unigenes could be classified into nine functional categories. The expression profiles of 18 selected genes were analyzed using quantitative RT-PCR; the expression levels of 10 of these genes were at least two-fold higher in the ABA-pretreated seedlings under chilling stress than water-pretreated (control) plants under chilling stress. In contrast, the other eight genes were downregulated in ABA-pretreated seedlings under chilling stress, with expression levels that were one-third or less of the levels observed in control seedlings under chilling stress. These results suggest that ABA can positively and negatively regulate genes in pepper plants under chilling stress.

低温是制约非热带地区冬季及早春辣椒（*Capsicum annuum L.*）生产的主要限制因子之一。外施脱落酸（abscisic acid, ABA）可有效缓解辣椒叶片的冷害症状，如萎蔫与坏死斑形成，但其背后的分子调控机制尚未明确。本研究采用抑制性消减杂交（suppression subtractive hybridization, SSH）技术，对经6℃低温处理48小时的脱落酸预处理辣椒幼苗中差异表达的基因进行筛选。最终共得到235条高质量表达序列标签（expressed sequence tags, ESTs），经聚类与组装后获得73个单基因簇（unigenes），其中包含18个重叠群（contigs）与55个单拷贝序列（singletons）。其中37个单基因簇（占比50.68%）与非冗余蛋白数据库中具有已知功能的基因存在同源相似性，剩余36个单基因簇（占比49.32%）同源性较低或功能未知。基因本体（gene ontology, GO）分析结果显示，这37个单基因簇可划分为9个功能类别。本研究选取18个基因开展定量反转录PCR（quantitative RT-PCR）表达谱分析，结果显示，其中10个基因在脱落酸预处理的低温胁迫幼苗中的表达量较清水预处理（对照组）低温胁迫幼苗至少提升2倍；与之相反，另外8个基因在脱落酸预处理的低温胁迫幼苗中表达下调，其表达水平仅为对照组低温胁迫幼苗的1/3及以下。上述研究结果表明，脱落酸可在低温胁迫下对辣椒植株的基因表达同时发挥正向与负向调控作用。