黄瓜自毒作用关键基因CsARR-9的负调控机理分析

Autotoxicity is a serious problem for continuous cropping of cucumber. Cinnamic acid (CA) derivatives are a class of autotoxins that can mimic autotoxic effects in cucumber. We screened an Arabidopsis long hairpin RNA (lhRNA) library under CA-mimicked autotoxicity stress to obtain autotoxicity-resistant mutant lines. A new gene, AtCARR-9 (Arabidopsis thaliana Cinnamic Acid Resistance Related gene), whose function is unknown, was found to be silenced in the resistant mutant line HCA350-597, and the homologous AtCARR-9 gene in cucumber was cloned and named CsARR-9 (Cucumis sativus Autotoxicity Resistance Related gene), whose function is also unknown. Here, we silenced the CsARR-9 gene using an established tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) system in cucumber and showed decreased CsARR-9 expression in both leaves and roots, as well as an enhanced tolerance to the autotoxin stress mimicked by CA. The alleviation of the damage caused by the CA treatment was probably achieved through reducing CA concentration and inhibiting CA transport from root to stem and leaf. The results indicated that AtCARR-9 is a crucial gene of CA stress resistence. The project intends to RNA interference and overexpress CsARR-9 to analyze the negative regulation mechanism of CsARR-9 on CA-induced stress, and the tissue-differential expression patterns and subcellular localization of CsARR-9 will also be analyzed. In addition, the interaction genes and proteins of CsARR-9 were will be screened by transcriptome sequencing and yeast two-hybrid (Y2H), and the upstream and downstream signaling mechanisms of CsARR-9 gene were will be analyzed, so as to clarify the regulating mechanism of CsARR-9 in cucumber autotoxicity. These researches will provide a valuable genetic resource for research regarding the continuous cropping obstacle, especially in monoculture facility cultivation.

自毒作用是黄瓜连作障碍发生的一个重要问题，肉桂酸（CA）是黄瓜主要自毒物质之一。我们利用CA诱导自毒作用筛选拟南芥RNAi文库，得到CA胁迫抗性提高的突变体株系，在其中功能未知的新基因AtCARR-9被沉默，进一步克隆黄瓜AtCARR-9的同源基因CsARR-9，利用建立的TRV介导的VIGS技术沉默CsARR-9显著增加了黄瓜对CA胁迫的抗性，且试验表明CsARR-9参与了CA运输途径，表明CsARR-9是CA胁迫抗性反应的关键基因。项目拟获得沉默（RNAi）和过表达CsARR-9黄瓜植株，进一步验证该基因对CA胁迫的负调控作用。对CsARR-9组织差异表达模式和亚细胞定位分析，利用转录组测序和酵母双杂交技术筛选CsARR-9的互作基因和蛋白，并分析其上下游信号调控通路，明确CsARR-9调控黄瓜自毒作用的机理，以期丰富黄瓜自毒作用相关理论，为黄瓜抗连作障碍育种提供基因资源和理论依据。

自毒作用是造成作物单一栽培中生长抑制的主要原因。黄瓜自毒作用限制了黄瓜植株的生长，导致黄瓜产量和品质明显下降。肉桂酸（Cinnamic Acid, CA）是一种黄瓜分泌的主要自毒物质，被用来模拟黄瓜的自毒作用。利用CA模拟自毒作用筛选拟南芥长发夹RNA (lhRNA)文库，获得抗CA诱导自毒作用抗性的突变体株系。我们发现在抗性突变体株系HCA350-597中沉默了一个拟南芥功能未知的新基因命名为AtARR-9，并在黄瓜中克隆了拟南芥AtARR-9的同源基因命名为CsARR-9 (Cucumis sativus Autotoxicity Resistance Related gene)，CsARR-9是一个功能未知的黄瓜新基因。我们利用建立的烟草脆裂病毒(Tobacco Rattle Virus, TRV)介导的病毒诱导基因沉默(Virus-Induced Gene Silencing, VIGS)系统沉默CsARR-9基因，并利用RNA干扰在黄瓜中沉默CsARR-9基因。结果表明，CsARR-9在根系和叶片中的表达量均下调，沉默CsARR-9基因缓解了CA诱导自毒作用对黄瓜植株生长的抑制作用。对CA模拟的自毒性的抗性可能是通过降低根系对CA吸收积累的浓度和抑制CA从根到茎和叶的运输来实现的；同时，沉默CsARR-9维持黄瓜根系和叶片细胞和组织的结构完整性，促进细胞活力，增强叶片细胞叶绿体中类囊体堆积，增加叶绿体中光合产物（淀粉颗粒）的储存，从而促进黄瓜植株生长。此外，沉默CsARR-9显著抑制开花结果期黄瓜侧枝生长，提早坐瓜并促进早熟。这些发现为研究连作障碍，特别是单一栽培的连作障碍提供了重要的遗传资源和分子机制，对延长种植年限和提高设施农业的高效利用，以及节约劳动成本具有重要的现实意义。