桑树抗寒冷基因的鉴定与功能分析

Chilling and freezing seriously affect sericultural production, and it is very important to improve chilling-resistance of mulberry for sericulture production. The newly identified molecular chaperone AKR2A mutant , akr2a, impairs plant resistance to chilling under certain conditions. To determine the proteins interacted with mAKR2A, a yeast two-hybrid assay was performed. The chilling-resistance gene library were selected by bioinformatics analysis, and the protein-protein interactions were further determined by Co-immunoprecipitation and protein colocalization. Compared with the library of arabidopsis chilling-resistance gene library, the newly identified genes were tranformed into the akr2a mutant, respectively, and plant resistance to chilling was detected. To determine the chilling-resistance mechanism of the gene, physiologic and biochemical assays were performed, and the expression of chilling-related genes were also detected. The mulberry tree genome is completely sequenced, bioinformatics, promoter activity, time course analysis, and subcellular location assays will further interpret the chilling-resistance gene. This project is in favor of identification of chilling-resistance gene of mulberry, realization the different chilling-resistance mechanism between woody and herbaceous plants, enrichment the chilling-resistant network of plant. This project provides new ideas for identification and utilization of chilling-resistance gene, improvement the utilization of gene library, and enhancement the chilling-resistance of crop.

寒冷冻害严重影响我国蚕桑生产，提高桑树抗寒能力对蚕桑生产具有很大的意义。Ankyrin Repeat-containing Protein 2A (AKR2A)是最新鉴定到的一个伴侣蛋白，目前研究发现AKR2A处于抗寒基因网络上游，调控抗寒基因表达。本项目以抗寒能力较强的蒙古桑为研究材料，利用酵母双杂交技术，以桑树AKR2A（mAKR2A）为诱饵从桑树cDNA文库筛选互作蛋白。通过生物信息学分析，筛选新的桑树抗寒冷基因。通过将筛选基因转化拟南芥，验植株抗寒能力是否增强。通过检测相关生理生化指标以及相关基因的表达状况，明确基因的抗寒冷机理。本项目将有助于揭示分子伴侣在植物抗逆性方面的重要调控作用，对阐述木本植物抗寒机制、明确木本植物和草本植物抗寒冷机制的差异和丰富植物抗寒冷基因网络有重要意义。本项目为挖掘、鉴定和利用新的抗寒基因，进一步提高基因库资源的利用水平，增强农作物抗寒能力提供新的思路

桑树AKR2A (mAKR2A)与拟南芥AKR2A蛋白的相似性高达65%，两者具有相似的功能。我们成功构建了桑树酵母双杂交cDNA文库，并通过酵母双杂交技术，筛选到296个与mAKR2A互作的蛋白。将mAKR2A转化入拟南芥AKR2A突变体，可以恢复突变体抗寒能力，表明桑树AKR2A在桑树抗寒性中发挥重要作用。我们从AKR2A互作蛋白中挑选出3个基因，通过免疫共沉淀和双分子荧光互补实验（BIFC）进一步验证所筛选基因与mAKR2A的互作关系。构建重组质粒转化拟南芥，通过检测相关生理生化指标、调查抗寒冷相关基因的表达状况，分析这些基因在抗寒过程中的作用，阐述基因的抗寒冷机理，并进一步揭示这些基因的分子功能。通过对抗寒能力明显差异的三个桑树品种分析显示，抗寒能力强的桑树品种SOD活性较高并且具有较高的不饱和脂肪酸组分。酵母双杂交与BIFC结果表明桑树AKR2A与mSOD、KCS1以及mFADI互作。另外抗寒能力强的桑树品种AKR2A、SOD、KCS1和FAD的表达水平。即使在寒冷条件下，抗寒能力强的桑树品种AKR2A表达水平高于抗寒能力弱的品种。而较高的AKR2A表达水平使SOD、KCS1和FAD表达水平较高。这表明高水平表达AKR2A有利于维持或促进抗寒相关基因表达水平，进而促进植物抗寒能力。