SABP2和SAMT基因在杨树与溃疡病菌（Botryosphaeria dothidea）互作中的功能分析

发布时间：2018-06-07 11:25

本文选题：杨树 + 溃疡病菌　；参考：《中国林业科学研究院》2017年博士论文

【摘要】：一年生草本植物受到病原菌侵染时,体内的水杨酸甲基转移酶(salicylic acid methyltransferase,SAMT)基因能将植物侵染部位产生的水杨酸(salicylicacid,SA)转化为水杨酸甲酯(methyl salicylate,MeSA),部分MeSA挥发到空气中,在植物的未侵染部位通过水杨酸结合蛋白2(salicylic acid-binding protein 2,SABP2)再次转化为SA,这一过程在SA信号转导和植物系统获得抗性(systemic acquired resistance,SAR)中起着重要作用。而多年生木本植物中SAMT和SABP2基因的功能还有待进一步验证。本研究对SABP2和SAMT基因在杨树与溃疡病菌(Botryosphaeria dothidea)互作中的功能进行了研究,首先对该两个基因进行了克隆和遗传转化,并对转基因苗木抗性进行了接种验证分析,结果如下:84K杨SABP2基因ORF全长cDNA序列789 bp,编码263个氨基酸。序列分析和比对显示84K杨SABP2基因与其他植物的相关序列同源性高达76%~98%。比对结果一定程度上也体现了SABP2在结构上的保守性。用生物信息学相关软件对其蛋白质功能进行预测,显示其可能具有中间代谢功能,属于α/β折叠水解酶家庭成员中的酯酶。84K杨SAMT基因的cDNA序列全长1095bp,开放阅读框1094bp,编码364个氨基酸;对该基因编码的蛋白的理化性质、疏水性、结构域、功能、二级结构及亚细胞定位等进行了生物信息学分析,结果显示:该基因位于细胞质中,属于甲基转移酶7家族,为亲水性蛋白。利用Gateway克隆技术,成功构建了SABP2和SAMT的超表达和RNAi载体,通过农杆菌介导的“叶盘转化法”获得了各自的转基因苗木,SABP2和SAMT超表达株系分别命名为OE-SABP2和OE-SAMT,SABP2和SAMT沉默株系分别命名为R-SABP2和R-SAMT,挑选合适的株系进行接种试验。接种结果表明:SAMT在杨树中与SA和MeSA密切相关,在接种部位和未接种部位均可以将SA转化为MeSA。OE-SAMT植株在接种病原菌B.dothidea后,接种期内,组织中SAMT和MeSA的变化趋势保持一致,接种后转基因植株与野生型相比组织中的MeSA含量和气化的MeSA均呈显著或极显著上升,同时SA含量显著降低;而R-SAMT植株则在接种后显著降低了植株组织中的MeSA含量,同时挥发到空气中的MeSA降低。SABP2在杨树中与SA和MeSA的关系同样密切相关,能在接种部位和未接种部位将MeSA转化为SA。OE-SABP2植株接种期间与野生型相比,接种部位组织中SA含量上升,而且接种期内各时间段挥发的MeSA和组织中的MeSA含量与野生型相比均显著或极显著下降,与前人研究认为SABP2能催化MeSA合成SA相一致。OE-SABP2杨树中MeSA的降低应是受SABP2的催化部分形成SA所致。OE-SABP2未接种部位MeSA与野生型相比显著降低,但SA的含量也显著降低,是由于接种部位挥发的MeSA较少,相应地在未接种部位转化的SA也较少有关。R-SABP2在接种后期SA的含量和野生型相比也显著降低。PR-1在杨树中不能作为获得SAR的标志,杨树中植株的抗病和感病和SA的含量关系更为密切。各株系在接种结束后,与对照相比,OE-SABP2植株接种结束后显示比野生型抗病与植株中存在SA的含量最高有关;OE-SAMT植株接种结束后没有野生型抗病是由于该植株中SAMT的高表达带动了PR-1的高表达,而PR-1高表达的植株一般更易感病,且PR-1是否抗病和病原菌的种类有关。R-SAMT植株和R-SABP2植株与野生型相比也更易感病,也与接种后PR-1大量上调表达有关。转基因植株在遗传转化过程中存在将某个基因超量表达或干扰后,引起其他基因表达改变的情况,试验中在OE-SAMT植株中更显著,SAMT在植株中的超量表达,接种前就使得PR-1响应SAMT的表达而大量上调表达,接种后也发现SAMT的大量上调表达带动了SABP2的大量上调表达。对试验中测定的相关基因的影响使得接种结束OE-SAMT植株变得比野生型更易感病,可能也与自身其他基因的改变有关。转基因植株中未发现SAMT和SABP2存在平衡制约关系,与前人推测接种后SAMT的高表达是由于接种点SA的升高对植株的毒害作用,需要通过SAMT将其中的一部分转化为MeSA从而挥发到空气中相矛盾。研究表明,接种后SAMT和SABP2基因的表达在杨树中和MeSA和SA的生物合成相关,接种B.dothidea后SAMT的超表达植株能显著提高挥发到空气中的MeSA和组织中MeSA的含量,同时降低SA在组织中的含量;而对其进行RNAi后则降低MeSA的合成,OE-SABP2也显著降低了MeSA的合成,提高了组织中SA的含量,植株抗病性提高。
[Abstract]:When the annual herbaceous plants are infected by pathogenic bacteria, the salicylic acid methyltransferase (SAMT) gene in the body can convert the salicylic acid (salicylicacid, SA) from the site of plant infection to Methylis salicylas (methyl salicylate, MeSA), some MeSA volatiles into the air, and water is passed by water at the uninfected site of the plant. The salicylic acid binding protein 2 (salicylic acid-binding protein 2, SABP2) is converted to SA again. This process plays an important role in SA signal transduction and plant system acquired resistance (systemic acquired resistance, SAR), while the work energy of SAMT and SABP2 genes in perennial woody plants remains to be further verified. The function of the interaction between poplar and Botryosphaeria dothidea was studied. First, the two genes were cloned and genetically transformed, and the resistance of the transgenic seedlings was verified by inoculation. The results were as follows: the ORF full length cDNA sequence of the 84K poplar SABP2 gene was 789 BP, and 263 amino acids were encoded. Sequence analysis and comparison showed 8 The homology of the SABP2 gene of 4K Poplar with other plants is higher than that of other plants to a certain extent, to a certain extent the conservatism of SABP2 in the structure. The function of protein is predicted by the software of bioinformatics, which shows that it may have intermediate metabolic function, which belongs to the esterase.84K poplar of family members of alpha / beta foldable hydrolase. The cDNA sequence of the SAMT gene is full length 1095bp, open reading frame 1094bp, encoding 364 amino acids, and bioinformatics analysis on the physicochemical properties, hydrophobicity, domain, function, two grade structure and subcellular localization of the protein encoded by the gene. The result shows that the gene is located in the cytoplasm, belongs to the 7 family of methyltransferase and is a hydrophilic egg. Using Gateway cloning technology, the overexpression and RNAi vector of SABP2 and SAMT were successfully constructed. The transgenic seedlings were obtained by the "leaf disc transformation" mediated by Agrobacterium tumefaciens. The SABP2 and SAMT overexpressed lines were named OE-SABP2 and OE-SAMT respectively. SABP2 and SAMT silenced lines were named R-SABP2 and R-SAMT, and selected suitable plants. Inoculation test. The results of inoculation showed that SAMT was closely related to SA and MeSA in Yang Shuzhong. SA could be transformed into MeSA.OE-SAMT plant at inoculation and uninoculated sites after inoculation of pathogenic bacteria B.dothidea. The change trend of SAMT and MeSA in the tissue was kept in the inoculation period, and the transgenic plants were compared with the wild type after inoculation. The contents of MeSA and MeSA in the gasification were significantly or significantly increased, while the content of SA decreased significantly, while the R-SAMT plant decreased significantly the MeSA content in the plant tissue after inoculation, and the MeSA decreased by the volatilization of.SABP2 in the relationship between Yang Shuzhong and SA and MeSA, and the M could be M in the inoculation and uninoculated sites. Compared with the wild type, the content of SA increased in the inoculation period of eSA to the SA.OE-SABP2 plant, and the content of the volatile MeSA in the inoculation period and the MeSA content in the tissues were significantly or significantly lower than those of the wild type. And the previous study suggested that SABP2 could catalyze the decrease of MeSA in the.OE-SABP2 poplar tree of the MeSA synthesis SA phase. It should be caused by the formation of SA from the catalytic part of SABP2, which was significantly lower than that of the wild type, but the content of SA decreased significantly, because the MeSA content volatilized from the inoculation site was less, and the SA in the uninoculated site was less related to the SA content of.R-SABP2 in the later period of inoculation than in the wild type and also significantly reduced.PR-1. Yang Shuzhong could not be used as a sign of obtaining SAR, and the relationship between the resistance and the sense of disease and the content of SA was more closely related. After the inoculation, the OE-SABP2 plants showed the highest content of SA in the wild type resistance and the plant after the end of inoculation, and there was no wild type resistance after the end of inoculation of OE-SAMT plants. The high expression of SAMT in this plant leads to high expression of PR-1, and the plants with high expression of PR-1 are generally more susceptible to disease, and whether PR-1 is resistant to disease and the species of pathogenic bacteria is related to.R-SAMT plants and R-SABP2 plants more susceptible to disease than in the wild type, and is related to a large number of up-regulated PR-1 expressions after inoculation. Transgenic plants are genetically transformed. The presence of an overexpression or interference of a gene, causing a change in other gene expression, is more significant in the OE-SAMT plant, the overexpression of SAMT in the plant. Before inoculation, the expression of PR-1 in response to SAMT is up to a large number of expression. After inoculation, a large number of up-regulated expressions of SAMT have also led to a large number of up-regulated expressions of SABP2. The effect of the related genes determined in the test made the OE-SAMT plant more susceptible to the disease than the wild type and may be related to the change of other genes. The balance restriction between SAMT and SABP2 was not found in the transgenic plants. The high expression of SAMT after inoculation was due to the increase of the inoculation point SA to the plant. The study shows that the expression of SAMT and SABP2 genes after inoculation is related to the biosynthesis of MeSA and SA in poplar and MeSA and SA after inoculation. After inoculation, the overexpressed plants of SAMT can significantly increase the MeSA in the air and the content of MeSA in the tissues after the inoculation of B.dothidea, as well as the content of MeSA in the tissues. The content of SA in tissue was reduced and the synthesis of MeSA was reduced after RNAi, and OE-SABP2 significantly decreased the synthesis of MeSA, increased the content of SA in the tissue, and increased the resistance of the plant.
【学位授予单位】：中国林业科学研究院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S763.7

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