【摘要】 WRKY转录因子在植物抵御盐胁迫中发挥重要功能。实验室前期发现小麦(Triticum aestivum)TaWRKY46基因(GenBank No. EF368365)对盐胁迫产生明显应答,并构建了烟草(Nicotiana tabacum)过表达转基因系(overexpression lines, OE)。本研究对TaWRKY46蛋白的亚细胞定位特征分析表明其定位于核内。为研究TaWRKY46的功能,应用蛭石培养和Murashige&Skoog (MS)营养液水培法,对野生型(wild type, WT)和OE烟草植株表型进行了分析。盐胁迫下,OE1和OE5较WT呈现明显的生长优势,叶面积显著大于WT,植株鲜重及可溶性糖和可溶性蛋白含量显著高于WT。对植株叶片细胞保护酶超氧化物歧化酶(superoxide dismutase, SOD)、过氧化物酶(peroxidase, POD)和过氧化氢酶(catalase, CAT)活性测定表明,NaCl处理下,过表达植株中上述酶的活性均显著高于WT;利用逆转录PCR (reverse transcription PCR, RT-PCR)技术分析上述保护酶基因在根系中的表达量发现,盐胁迫下OE1和OE5中部分保护酶基因的表达量也较WT高。进一步采用四唑氮蓝(nitroblue tetrazolium,NBT)和二氨基联苯胺(diaminobenzidine, DAB)对超氧阴离子自由基(superoxide radical, O2-·)和过氧化氢(hydrogen peroxide, H2O2)进行组织定位发现,盐处理后,过表达株系OE5的O2-·和H2O2含量均较WT低。脱落酸(abscisic acid, ABA)受体基因(pyrabactin resistance-like ABA, PYL) NtPYL8以及蔗糖非发酵相关蛋白激酶家族2 (sucrose non-fermenting-1-related protein kinase 2, SnRK2)基因在根中的表达分析表明,盐胁迫下NtPYL8和部分SnRK2基因的表达量在OE中高于WT。叶片的气孔特征观察发现,OE5叶片的气孔开度在盐处理前比WT大,但盐处理后叶片气孔闭合速度快于WT。应用酵母双杂交技术还发现TaWRKY46与胁迫相关蛋白(stress associated protein, SAP) TaSAP1-1可能存在互作关系。结果表明,TaWRKY46通过增强渗透调节和保护酶系统、与ABA信号途径交叉以及蛋白质互作等途径在介导植株耐盐性中发挥重要作用。本研究丰富了对小麦WRKY家族成员耐盐功能的认识,为作物抗逆的遗传改良提供了理论依据。更多还原

【Abstract】 The members of the WRKY transcription factor(TF) family play important roles in mediating plant tolerance to salt stress. Previously, TaWRKY46, a member of WRKY gene family, was revealed to obviously respond to salt stress. Thus transgenic tobacco(Nicotiana tabacum) lines overexpressing this wheat gene were generated. The experiment indicated that TaWRKY46 targeted onto nucleus at subcellular level. Using the culture methods of vermiculite-based and Murashige & Skoog(MS) hydroponic solution, the phenotype of wild type(WT) and overexpression lines(OE) under salt stress treatment was investigated. OE1 and OE5, two OE lines overexpression TaWRKY46, displayed increased growth vigor and leaf area of plants, together with enhanced plant fresh weight and contents of soluble sugar and proteins upon salt stress with respect to WT. Assays on the activities of superoxide dismutase(SOD), peroxidase(POD), and catalase(CAT), the enzymes worked as cellular protector, showed the higher activities in OE lines than those in WT plants. Reverse transcription PCR(RT-PCR) analysis indicated the expression levels of some protection enzymes mentioned above from roots were higher in OE1 and OE5 than those in WT under salt stress treatment. Further histochemical staining study via nitroblue tetrazolium(DAB) and diaminobenzidine(NBT) dyeing methods revealed that the accumulative amounts of O2-·and H2 O2 were lower in the OE plants than that of WT plants.The expression analysis revealed that the pyrabactin resistance-like abscisic acid receptor gene(PYL8) and a suite of sucrose non-fermenting-1-related protein kinase 2(SnRK2) family genes were upregulated in expression in the OE plants. Observation on stomata movement showed that the OE plants possessed enlarged stomata aperture and increased closure rate upon salt stress compared with the WT plants. Using yeast-two hybridization assay, TaWRKY46 was identified to be interacted with TaSAP1-1, a stress-related protein in T. aestivum. Therefore, TaWRKY46 played an important role in salt tolerance through enhancing osmotic regulation and protective enzyme system, crossing with ABA signaling pathway, as well as initiating protein interaction.The results enrich the knowledge as to wheat plants coping with salt stress and provide theoretical guidance for breeding stress tolerant cultivars of crops.更多还原