本文選題：楊樹 + 潰瘍病菌��； 參考：《中國(guó)林業(yè)科學(xué)研究院》2017年博士論文

【摘要】：一年生草本植物受到病原菌侵染時(shí),體內(nèi)的水楊酸甲基轉(zhuǎn)移酶(salicylic acid methyltransferase,SAMT)基因能將植物侵染部位產(chǎn)生的水楊酸(salicylicacid,SA)轉(zhuǎn)化為水楊酸甲酯(methyl salicylate,MeSA),部分MeSA揮發(fā)到空氣中,在植物的未侵染部位通過水楊酸結(jié)合蛋白2(salicylic acid-binding protein 2,SABP2)再次轉(zhuǎn)化為SA,這一過程在SA信號(hào)轉(zhuǎn)導(dǎo)和植物系統(tǒng)獲得抗性(systemic acquired resistance,SAR)中起著重要作用。而多年生木本植物中SAMT和SABP2基因的功能還有待進(jìn)一步驗(yàn)證。本研究對(duì)SABP2和SAMT基因在楊樹與潰瘍病菌(Botryosphaeria dothidea)互作中的功能進(jìn)行了研究,首先對(duì)該兩個(gè)基因進(jìn)行了克隆和遺傳轉(zhuǎn)化,并對(duì)轉(zhuǎn)基因苗木抗性進(jìn)行了接種驗(yàn)證分析,結(jié)果如下:84K楊SABP2基因ORF全長(zhǎng)cDNA序列789 bp,編碼263個(gè)氨基酸。序列分析和比對(duì)顯示84K楊SABP2基因與其他植物的相關(guān)序列同源性高達(dá)76%~98%。比對(duì)結(jié)果一定程度上也體現(xiàn)了SABP2在結(jié)構(gòu)上的保守性。用生物信息學(xué)相關(guān)軟件對(duì)其蛋白質(zhì)功能進(jìn)行預(yù)測(cè),顯示其可能具有中間代謝功能,屬于α/β折疊水解酶家庭成員中的酯酶。84K楊SAMT基因的cDNA序列全長(zhǎng)1095bp,開放閱讀框1094bp,編碼364個(gè)氨基酸;對(duì)該基因編碼的蛋白的理化性質(zhì)、疏水性、結(jié)構(gòu)域、功能、二級(jí)結(jié)構(gòu)及亞細(xì)胞定位等進(jìn)行了生物信息學(xué)分析,結(jié)果顯示:該基因位于細(xì)胞質(zhì)中,屬于甲基轉(zhuǎn)移酶7家族,為親水性蛋白。利用Gateway克隆技術(shù),成功構(gòu)建了SABP2和SAMT的超表達(dá)和RNAi載體,通過農(nóng)桿菌介導(dǎo)的“葉盤轉(zhuǎn)化法”獲得了各自的轉(zhuǎn)基因苗木,SABP2和SAMT超表達(dá)株系分別命名為OE-SABP2和OE-SAMT,SABP2和SAMT沉默株系分別命名為R-SABP2和R-SAMT,挑選合適的株系進(jìn)行接種試驗(yàn)。接種結(jié)果表明:SAMT在楊樹中與SA和MeSA密切相關(guān),在接種部位和未接種部位均可以將SA轉(zhuǎn)化為MeSA。OE-SAMT植株在接種病原菌B.dothidea后,接種期內(nèi),組織中SAMT和MeSA的變化趨勢(shì)保持一致,接種后轉(zhuǎn)基因植株與野生型相比組織中的MeSA含量和氣化的MeSA均呈顯著或極顯著上升,同時(shí)SA含量顯著降低;而R-SAMT植株則在接種后顯著降低了植株組織中的MeSA含量,同時(shí)揮發(fā)到空氣中的MeSA降低。SABP2在楊樹中與SA和MeSA的關(guān)系同樣密切相關(guān),能在接種部位和未接種部位將MeSA轉(zhuǎn)化為SA。OE-SABP2植株接種期間與野生型相比,接種部位組織中SA含量上升,而且接種期內(nèi)各時(shí)間段揮發(fā)的MeSA和組織中的MeSA含量與野生型相比均顯著或極顯著下降,與前人研究認(rèn)為SABP2能催化MeSA合成SA相一致。OE-SABP2楊樹中MeSA的降低應(yīng)是受SABP2的催化部分形成SA所致。OE-SABP2未接種部位MeSA與野生型相比顯著降低,但SA的含量也顯著降低,是由于接種部位揮發(fā)的MeSA較少,相應(yīng)地在未接種部位轉(zhuǎn)化的SA也較少有關(guān)。R-SABP2在接種后期SA的含量和野生型相比也顯著降低。PR-1在楊樹中不能作為獲得SAR的標(biāo)志,楊樹中植株的抗病和感病和SA的含量關(guān)系更為密切。各株系在接種結(jié)束后,與對(duì)照相比,OE-SABP2植株接種結(jié)束后顯示比野生型抗病與植株中存在SA的含量最高有關(guān);OE-SAMT植株接種結(jié)束后沒有野生型抗病是由于該植株中SAMT的高表達(dá)帶動(dòng)了PR-1的高表達(dá),而PR-1高表達(dá)的植株一般更易感病,且PR-1是否抗病和病原菌的種類有關(guān)。R-SAMT植株和R-SABP2植株與野生型相比也更易感病,也與接種后PR-1大量上調(diào)表達(dá)有關(guān)。轉(zhuǎn)基因植株在遺傳轉(zhuǎn)化過程中存在將某個(gè)基因超量表達(dá)或干擾后,引起其他基因表達(dá)改變的情況,試驗(yàn)中在OE-SAMT植株中更顯著,SAMT在植株中的超量表達(dá),接種前就使得PR-1響應(yīng)SAMT的表達(dá)而大量上調(diào)表達(dá),接種后也發(fā)現(xiàn)SAMT的大量上調(diào)表達(dá)帶動(dòng)了SABP2的大量上調(diào)表達(dá)。對(duì)試驗(yàn)中測(cè)定的相關(guān)基因的影響使得接種結(jié)束OE-SAMT植株變得比野生型更易感病,可能也與自身其他基因的改變有關(guān)。轉(zhuǎn)基因植株中未發(fā)現(xiàn)SAMT和SABP2存在平衡制約關(guān)系,與前人推測(cè)接種后SAMT的高表達(dá)是由于接種點(diǎn)SA的升高對(duì)植株的毒害作用,需要通過SAMT將其中的一部分轉(zhuǎn)化為MeSA從而揮發(fā)到空氣中相矛盾。研究表明,接種后SAMT和SABP2基因的表達(dá)在楊樹中和MeSA和SA的生物合成相關(guān),接種B.dothidea后SAMT的超表達(dá)植株能顯著提高揮發(fā)到空氣中的MeSA和組織中MeSA的含量,同時(shí)降低SA在組織中的含量;而對(duì)其進(jìn)行RNAi后則降低MeSA的合成,OE-SABP2也顯著降低了MeSA的合成,提高了組織中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.
【學(xué)位授予單位】：中國(guó)林業(yè)科學(xué)研究院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S763.7

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本文編號(hào)：1991005