【作者】 孙浩然；

【导师】 尤江峰；

【作者基本信息】 吉林大学 ， 农业硕士（专业学位）， 2018， 硕士

【摘要】 铝(Aluminum,Al)毒是酸性土壤上限制作物生产的最大因素之一。细胞壁是植物感知铝毒害的第一屏障,也是累积铝的重要部位。植物细胞壁的基础结构以及多糖构成与铝毒及抗铝的发生紧密相关。尿苷二磷酸葡萄糖基转移酶(UDP-Glucosetransferase,UGTA)广泛存在于植物体中,主要负责将糖基从活化的供体分子移动到接受体上,直接参与多糖、二糖的生物合成。根据前期研究发现,铝胁迫下大豆根系UGTA基因转录表达上调,据此,本研究旨在研究UGTA是否能够调控细胞壁多糖的结构或组分从而影响铝毒的发生。本研究以大豆耐铝品种吉育70作为实验材料,利用前期大豆根尖转录谱中发现的响应于铝胁迫的UGTA基因信息,从大豆基因组中筛选得到三个基因(Glyma.13G090300,Glyma.11G000500和Glyma.08G244700),分别命名为Gm UGTA1,Gm UGTA2和Gm UGTA3,利用生物信息学分析、表达模式分析以及超表达于大豆发根后的细胞壁部分糖类分析解析三个UGTA基因的功能以及与铝毒的关系。具体研究结果如下。1.利用实时荧光定量PCR实验,发现Gm UGTA1(Glyma.13G090300),Gm UGTA2(Glyma.11G000500)和Gm UGTA3(Glyma.08G244700)基因都对铝有一定的响应。在铝处理的24小时内,Gm UGTA1的转录峰度变化不大,只在4小时时有约3倍的提高化。Gm UGTA2和Gm UGTA3的转录丰度从12小时提高,前者在24小时约为18倍,后者则达到27倍。2.将携带UGTA基因的植物表达载体p EGAD-3XHA-LUC,利用发根农杆菌K599转化至大豆发根中。通过LUC标签检验,得到超表达的发根。然后通过LC-MS代谢组学初步分析,发现木糖、葡萄糖等糖类改变最为显著。因此对大豆发根细胞壁进行提取,对细胞壁多糖和木糖、木质素、葡萄糖含量等糖类含量测定。结果表明,无铝处理条件下,Gm UGTA1-OE和Gm UGTA2-OE的细胞壁多糖、胼胝质、木糖含量改变较显著,木质素含量改变不显著,葡萄糖含量与野生型相比明显下降;Gm UGTA3-OE的细胞壁多糖、胼胝质含量改变较显著,但与WT发根相比,其木糖、葡萄糖含量有显著下降,木质素含量改变不显著。有铝处理条件下,Gm UGTA1-OE和Gm UGTA3-OE的细胞壁多糖、胼胝质含量进一步提高,木糖、木质素含量改变不大,葡萄糖含量与野生型相比明显下降;Gm UGTA2-OE的细胞壁多糖、胼胝质、木糖、木质素含量改变不大,葡萄糖含量与野生型相比明显下降。更多还原

【Abstract】 Al toxicity is one of the most important constraints limiting crop yield on acid soils.Cell wall is the first barrier for Al toxicity and also have capacity to accumulate more Al.The basic structure of plant cell wall and the composition of polysaccharides,such as cellulose,uronic acid,and lignin,are closely related to the occurrence of aluminum toxicity and aluminum resistance.Uridine diphosphate glucosyltransferase(UGTA)is widely present in plants and is mainly responsible for the transfer of glycosyl groups from activated donor molecules to receptors,and directly involve in the biosynthesis of polysaccharides and disaccharides.Our previous microarray analysis showed that the expression of Gm UGTAs was upregulated under Al stress.Thus,the present study aimed to clarify if composition of cell wall polysaccharides was disrupted under Al stress in soybean root apices.Three soybean genes Gm UGTA1(Glyma.13G090300),Gm UGTA2(Glyma.11G000500)and Gm UGTA3(Glyma.08G244700)were blasted from soybean genome with UGTA probe information of previous microarray.We analyze the function of three UGTA genes through bioinformatics analysis,expression pattern analysis,subcellular localization,and analysis of carbohydrates in the cell wall by overexpressing soybean hairy roots.The main findings are as follows.1.Using quantitative real-time PCR experiments,it was found that Gm UGTA1(Glyma.13G090300),Gm UGTA2(Glyma.11G000500)and Gm UGTA3(Glyma.08G244700)genes was verified to upregualted under Al stress in soybean root apices.The transcription abundance increased about 3 folds within 4 h Al treatement duration.The expression of Gm UGTA2 and Gm UGTA3 increased from 12 hours,the former was 18 folds and the latter reached27 folds at 24 h treatmentduration.2.The plant expression vector p EGAD-3XHA-LUC carrying each UGTA gene was transformed into soybean hairy root using Agrobacterium rhizogenes K599.LC-MS metabolomics preliminary analysis showed that xylose,glucose and other sugars changed greatly in the Gm UGTA1-OE hairy roots compared with the WT.The concentrations of cell wall polysaccharides,xylose,lignin,and glucose were compared within three transgenic Gm UGTAs-OE lines and WT.The results show thatunder the condition of non-aluminum treatment,the contents of cell wall polysaccharide,callose and xylose of Gm UGTA1-OE and Gm UGTA2-OE changed significantly,the content of lignin did not change significantly,and the content of glucose decreased significantly compared with the wild type;Contents of cell wall polysaccharides and calloses of the Gm UGTA3-OE were significantly changed,but compared with WT hair roots,the xylose and glucose contents were significantly decreased,and the lignin content was not significantly changed.Under the aluminum treatment conditions,the content of Gm UGTA1-OE and Gm UGTA3-OE cell wall polysaccharides and calloses were further increased,xylose and lignin contents were not changed significantly,but the glucose contents were significantly lower than that of the wild types;the cell wall content of the polysaccharide,callose,xylose and lignin of the Gm UGTA2-OE were changed little,but the glucose contents were decreased significantly compared with the wild type.更多还原