Cao Xinyou, Chen Xueyan, Liu Yangna, Xu Zhaoshi, Li Liancheng, Zhou Yongbin, Liu Jianjun, Zhao Zhendong, Chen Ming, Ma Youzhi
National Key Facility for Crop Genetic Resources and Genetic Improvement, Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, P.R. China.
Crop Research Institute, Shandong Academy of Agricultural Sciences/National Engineering Laboratory for Wheat and Maize/Key Laboratory of Wheat Biology and Genetic Improvement in North Yellow and Huai River Valley, Ministry of Agriculture, 250100, Jinan, China.
BMC Genomics. 2016 Mar 5;17:189. doi: 10.1186/s12864-016-2548-y.
Nuclear proteins play critical roles in regulating mRNA transcription and processing, DNA replication, and epigenetic genome modification. Therefore, the ability to monitor changes in nuclear proteins is helpful not only to identify important regulatory proteins but also to study the mechanisms of actions of nuclear proteins. However, no effective methods have been developed yet. Rye is strongly resistant to various biotic and abiotic stresses; however, few genes have been functionally characterized to date due to the complexity of its genome and a lack of genomic sequence information.
We developed an integrative Nuclear Transportation Trap (iNTT) system that includes an improved nuclear transportation trap and utilizes the "after suppression subtraction" method. Oligonucleotides encoding a nuclear localization signal (NLS) or a transcription factor, GmAREB, were inserted into pLexAD or pLexAD-NES, respectively, and then transformed into yeast cells (EGY48). We showed that the pLexAD vector expressing a cDNA library in the iNTT system was more efficient for screening than the vector pLexAD-NES, which has previously been used in an NTT system. We used the iNTT system to screen a cDNA library of cold-treated rye. A total of 241 unique genes were identified, including 169 differentially expressed proteins; of these, 106 were of known and 63 were of unknown function. Moreover, 82 genes (49 %) among the 169 differentially expressed genes were predicted to contain an NLS domain. Thirty-three (31 %) of the 106 functionally known proteins have DNA-binding activity. To test the specificity of the nuclear proteins identified using the iNTT screen, four of the proteins differentially expressed in response to temperature stress, ScT1 (a heat shock protein), ScT36 (a MYB-like transcription factor), ScT133 (an ERF-like transcription factor) and ScT196 (a protein of unknown function), were studied in more depth. These proteins were shown to exclusively localize to the nucleus, and their expression levels were increased in response to low-temperature stress. To identify the function of these screened nuclear proteins, ScT1- and ScT36-transgenic Arabidopsis plants were constructed, and ScT1 or ScT36 overexpression was found to enhance tolerance to high-temperature or freezing stresses, respectively.
The newly developed iNTT system provides an effective method for identifying nuclear-targeted proteins and monitoring induced expression levels. ScT1 and ScT36 might be good candidate genes for improving the stress tolerance of plants by genetic transformation.
核蛋白在调节mRNA转录与加工、DNA复制以及表观遗传基因组修饰过程中发挥着关键作用。因此，监测核蛋白变化的能力不仅有助于识别重要的调节蛋白，还能用于研究核蛋白的作用机制。然而，目前尚未开发出有效的方法。黑麦对多种生物和非生物胁迫具有很强的抗性；然而，由于其基因组的复杂性以及缺乏基因组序列信息，迄今为止很少有基因得到功能鉴定。
我们开发了一种整合的核转运陷阱（iNTT）系统，该系统包括一个改进的核转运陷阱，并采用了“抑制后减法”方法。将编码核定位信号（NLS）或转录因子GmAREB的寡核苷酸分别插入pLexAD或pLexAD-NES中，然后转化到酵母细胞（EGY48）中。我们发现，在iNTT系统中表达cDNA文库的pLexAD载体比先前在NTT系统中使用的pLexAD-NES载体在筛选方面更有效。我们使用iNTT系统筛选了经冷处理的黑麦的cDNA文库。总共鉴定出241个独特基因，包括169个差异表达蛋白；其中，106个功能已知，63个功能未知。此外，169个差异表达基因中有82个（49%）预计含有NLS结构域。106个功能已知的蛋白中有33个（31%）具有DNA结合活性。为了测试使用iNTT筛选鉴定出的核蛋白的特异性，对4个在温度胁迫下差异表达的蛋白进行了更深入的研究，它们分别是ScT1（一种热休克蛋白）﹑ScT36（一种MYB样转录因子）﹑ScT133（一种ERF样转录因子）和ScT196（一种功能未知的蛋白）。这些蛋白被证明仅定位于细胞核，并且它们的表达水平在低温胁迫下升高。为了鉴定这些筛选出的核蛋白的功能，构建了ScT1和ScT-36转基因拟南芥植株，发现ScT1或ScT36的过表达分别增强了对高温或冷冻胁迫的耐受性。
新开发的iNTT系统为鉴定核靶向蛋白和监测诱导表达水平提供了一种有效方法。ScT1和ScT36可能是通过基因转化提高植物胁迫耐受性的良好候选基因。
