DataSheet_1_GhBRX.1, GhBRX.2, and GhBRX4.3 improve resistance to salt and cold stress in upland cotton.docx

IntroductionAbiotic stress during growth readily reduces cotton crop yield. The different survival tactics of plants include the activation of numerous stress response genes, such as BREVIS RADIX (BRX).
MethodsIn this study, the BRX gene family of upland cotton was identified and analyzed by bioinformatics method, three salt-tolerant and cold-resistant GhBRX genes were screened. The expression of GhBRX.1, GhBRX.2 and GhBRXL4.3 in upland cotton was silenced by virus-induced gene silencing (VIGS) technique. The physiological and biochemical indexes of plants and the expression of related stress-response genes were detected before and after gene silencing. The effects of GhBRX.1, GhBRX.2 and GhBRXL4.3 on salt and cold resistance of upland cotton were further verified.
Results and discussionWe discovered 12, 6, and 6 BRX genes in Gossypium hirsutum, Gossypium raimondii and Gossypium arboreum, respectively. Chromosomal localization indicated that the retention and loss of GhBRX genes on homologous chromosomes did not have a clear preference for the subgenomes. Collinearity analysis suggested that segmental duplications were the main force for BRX gene amplification. The upland cotton genes GhBRX.1, GhBRX.2 and GhBRXL4.3 are highly expressed in roots, and GhBRXL4.3 is also strongly expressed in the pistil. Transcriptome data and qRT‒PCR validation showed that abiotic stress strongly induced GhBRX.1, GhBRX.2 and GhBRXL4.3. Under salt stress and low-temperature stress conditions, the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and the content of soluble sugar and chlorophyll decreased in GhBRX.1-, GhBRX.2- and GhBRXL4.3-silenced cotton plants compared with those in the control (TRV: 00). Moreover, GhBRX.1-, GhBRX.2- and GhBRXL4.3-silenced cotton plants exhibited greater malondialdehyde (MDA) levels than did the control plants. Moreover, the expression of stress marker genes (GhSOS1, GhSOS2, GhNHX1, GhCIPK6, GhBIN2, GhSnRK2.6, GhHDT4D, GhCBF1 and GhPP2C) decreased significantly in the three target genes of silenced plants following exposure to stress. These results imply that the GhBRX.1, GhBRX.2 and GhBRXL4.3 genes may be regulators of salt stress and low-temperature stress responses in upland cotton.

引言 生长过程中的非生物胁迫极易降低棉花作物的产量。植物的不同生存策略包括激活众多胁迫响应基因，例如BREVIS RADIX（BRX）。方法 本研究通过生物信息学方法对陆地棉（Gossypium hirsutum）的BRX基因家族进行鉴定与分析，筛选得到3个耐盐耐寒的GhBRX基因。采用病毒诱导基因沉默（virus-induced gene silencing, VIGS）技术对陆地棉中的GhBRX.1、GhBRX.2和GhBRXL4.3进行沉默。检测基因沉默前后植株的生理生化指标以及相关胁迫响应基因的表达水平，进一步验证GhBRX.1、GhBRX.2和GhBRXL4.3对陆地棉耐盐和耐寒性的影响。结果与讨论 本研究分别在陆地棉（Gossypium hirsutum）、雷蒙德氏棉（Gossypium raimondii）和亚洲棉（Gossypium arboreum）中鉴定到12、6和6个BRX基因。染色体定位分析表明，GhBRX基因在同源染色体上的保留与丢失并未表现出对亚基因组的明显偏好。共线性分析显示，片段重复是BRX基因家族扩张的主要驱动力。陆地棉基因GhBRX.1、GhBRX.2和GhBRXL4.3在根中高表达，且GhBRXL4.3在雌蕊中也呈现高表达水平。转录组数据与实时定量聚合酶链反应（quantitative real-time PCR, qRT-PCR）验证结果显示，非生物胁迫可强烈诱导GhBRX.1、GhBRX.2和GhBRXL4.3的表达。在盐胁迫和低温胁迫条件下，与对照（TRV: 00）相比，GhBRX.1、GhBRX.2和GhBRXL4.3沉默的棉花植株中超氧化物歧化酶（superoxide dismutase, SOD）、过氧化物酶（peroxidase, POD）和过氧化氢酶（catalase, CAT）活性以及可溶性糖和叶绿素含量均出现下降；同时，这些沉默植株的丙二醛（malondialdehyde, MDA）水平高于对照植株。此外，在胁迫处理后，沉默植株的三个靶基因对应的胁迫标记基因（GhSOS1、GhSOS2、GhNHX1、GhCIPK6、GhBIN2、GhSnRK2.6、GhHDT4D、GhCBF1和GhPP2C）的表达量显著下调。上述结果表明，GhBRX.1、GhBRX.2和GhBRXL4.3基因可能作为陆地棉盐胁迫和低温胁迫响应的调控因子。