SlNAC2提高转基因拟南芥抗逆性及机制分析

发布时间：2018-08-14 12:21

【摘要】：NAC(NAM、ATAF1/2、CUC)转录因子是植物特有的一类转录因子,它不仅参与植物的多个生长发育过程,还参与植物对生物以及非生物胁迫的响应。本实验室前期从盐生植物辽宁碱蓬(Suaeda liaotungensis K.)中克隆了一个NAC转录因子SlNAC2,并获得了转SlNAC2拟南芥T2代种子。本研究以转SlNAC2拟南芥T2代种子为实验材料,筛选出转基因拟南芥T3代纯合二倍体,并对转基因纯合二倍体植株进行非生物胁迫抗性及机制分析。主要结果如下:1.以实验室前期获得的转pBI121-SlNAC2拟南芥和转pBI121-GUS拟南芥T2代种子为材料,利用含卡那霉素(100 mg/L)的MS培养基进行筛选培养,获得转pBI121-SlNAC2拟南芥纯合二倍体株系:L1、L2、L3;转pBI121-GUS拟南芥纯合二倍体株系:VT1、VT2。半定量RT-PCR分析表明,SlNAC2基因已在转SlNAC2基因拟南芥中表达。2.非生物胁迫(干旱、高盐、低温)条件下,对转基因拟南芥及野生型拟南芥进行形态和生理特征分析,形态指标主要包括:存活率、种子萌发率、根部发育、组织含水量和气孔;生理指标主要包括:叶绿素荧光、可溶性蛋白含量、脯氨酸含量、电导率及抗氧化酶活性等。非生物胁迫下,两种拟南芥萌发均受到明显抑制,但转pBI121-SlNAC2株系萌发率明显优于野生型与转pBI121-GUS株系。在幼苗生长阶段,非生物胁迫下的存活率、根部发育、组织含水量和气孔的结果显示,转pBI121-SlNAC2株系比野生型和转pBI121-GUS株系具有更好的生存能力。非生物胁迫下叶绿素荧光与可溶性蛋白含量的变化表明,转基pBI121-SlNAC2拟南芥通过提高光合作用来应对非生物胁迫。转pBI121-SlNAC2基因拟南芥在非生物胁迫下的脯氨酸含量及抗氧化酶活性显著高于野生型与转pBI121-GUS拟南芥,且细胞膜的损伤程度要低于野生型与转pBI121-GUS拟南芥。结果表明,外源SlNAC2基因在拟南芥中表达,提高了拟南芥的抗逆境能力,在很大程度上减少了非生物胁迫对转基因拟南芥的损害。有效的证明了SlNAC2基因具有提高拟南芥对非生物胁迫耐受性的能力。3.利用安捷伦基因芯片分析转SlNAC2基因拟南芥与野生型拟南芥差异表达基因,并通过实时荧光定量PCR方法对部分差异表达基因进行验证。差异表达分析结果显示,在转SlNAC2基因拟南芥中共筛选到1258个差异表达基因,其中上调表达的基因706个,下调表达基因552个。差异倍数大于50倍的基因22个,GO分类结果显示,这些基因主要参与植物细胞的生长发育、激素信号传递、酶活性的调控以及胁迫响应过程中涉及的离子转运、抗氧化剂的合成、转录因子的相互作用等复杂的生理生化过程。差异表达基因包括MYB、WRKY类转录因子基因,并且存在与精氨酸和脯氨酸有关的信号通路基因。SlNAC2是NAC转录因子家族成员之一,通过调控下游胁迫相关的基因提高转基因拟南芥在非生物胁迫下的耐受性。本研究揭示了SlNAC2在提高植物抗逆性中的作用,初步分析了其调控的下游基因,为植物抗逆机理研究提供了新资料。
[Abstract]:NAC (NAM, ATAF 1/2, CUC) transcription factors are plant-specific transcription factors. NAC transcription factors not only participate in many plant growth and development processes, but also participate in plant responses to biological and abiotic stresses. In this study, T3 homozygous diploids of transgenic Arabidopsis thaliana were screened from the seeds of T2 generation of transgenic Arabidopsis thaliana SlNAC2, and the abiotic stress resistance and mechanism of transgenic homozygous diploid plants were analyzed. The main results were as follows: 1. The transgenic Arabidopsis thaliana pBI121-SlNAC2 and transgenic pBI121-GUS were obtained in the early stage of the laboratory. Transgenic Arabidopsis thaliana homozygous diploid lines: L1, L2, L3 and pBI121-GUS homozygous diploid lines: VT1, VT2. Semi-quantitative RT-PCR analysis showed that SlNAC2 gene was expressed in transgenic Arabidopsis thaliana. The morphological and physiological characteristics of transgenic Arabidopsis and wild Arabidopsis thaliana under drought (drought, high salinity, low temperature) stress were analyzed. The morphological indexes mainly included survival rate, seed germination rate, root development, tissue water content and stomata; physiological indexes mainly included chlorophyll fluorescence, soluble protein content, proline content, electrical conductivity and resistance. The germination rate of transgenic pBI121-SlNAC2 was significantly higher than that of wild-type and trans-pBI121-GUS strains. At seedling growth stage, the survival rate, root development, tissue water content and stomata of trans-pBI121-SlNAC2 strains were higher than that of wild-type. The changes of chlorophyll fluorescence and soluble protein content under abiotic stress indicated that the transgenic Arabidopsis thaliana pBI121-SlNAC2 could cope with abiotic stress by increasing photosynthesis. The results showed that the expression of exogenous SlNAC2 gene in Arabidopsis improved the stress resistance of Arabidopsis thaliana and reduced the damage of transgenic Arabidopsis to a great extent. Genes have the ability to improve the tolerance of Arabidopsis to abiotic stress. 3. Differentially expressed genes of transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana were analyzed by Agilent microarray, and some differentially expressed genes were verified by real-time fluorescence quantitative PCR. A total of 1,258 differentially expressed genes were screened, of which 706 were up-regulated and 552 were down-regulated. 22 genes were more than 50-fold differentially expressed. GO classification showed that these genes were mainly involved in plant cell growth and development, hormone signaling, enzyme activity regulation and ion transport involved in stress response. Differentially expressed genes include MYB and WRKY transcription factor genes, and there are signal transduction genes associated with arginine and proline. SlNAC2 is a member of the NAC transcription factor family that enhances transgenic mimicry by regulating downstream stress-related genes. This study revealed the role of SlNAC2 in improving stress tolerance of Arabidopsis thaliana under abiotic stress, and preliminarily analyzed the downstream genes regulated by SlNAC2, which provided new information for the study of stress resistance mechanism of plants.
【学位授予单位】：辽宁师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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