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调控柳枝稷SAMS和SAHH表达改变木质素合成的研究
Alternative Title生物工程
齐天雄
Thesis Advisor木质素,腺苷甲硫氨酸合成酶,腺苷高半胱氨酸水解酶,柳枝稷
2017-06
Degree Grantor中国科学院大学;中科院青岛生物能源与过程研究所
Place of Conferral北京;青岛
Degree Discipline植物细胞壁富含纤维素和半纤维素,是生物质能源和畜牧业生产的重要原材料。然而由于细胞壁中木质素的存在严重制约了上述细胞壁多糖的转化和利用,从而增加了生物质能源和畜牧业生产的成本。柳枝稷是重要的禾本科能源草和牧草,其木质素大分子主要由氧甲基化程度不同的羟苯基丙烷 (phydroxyphenyl propane,H)、愈创木基丙烷(guaiacyl,G)和紫丁香基丙烷(syringyl,S)三种单体组成,其中G型和S型木质素与细胞壁木质化程度紧密相关。在植物中G型和S型木质素单体甲基化主要通过3位和5位氧甲基化酶—咖啡酰辅酶 A-O-甲基转移酶(caffeoyl CoA-O-methyltransferase, CCoAOMT)和咖啡酸-O-甲基转移酶(caffeic acid-O-methyltransferase, COMT)进行催化,而反应所需要的甲基则由S-腺苷甲硫氨酸(S-adenosyl methionine, SAM)进行提供。先前的研究表明在柳枝稷中抑制COMT表达主要影响S型木质素合成,从而显著改变木质素成分和含量,增加细胞壁可降解性与生物乙醇的产量。然而在柳枝稷中抑制CCoAOMT的表达对G型和S型木质素的合成却无显著影响。因此为了达到同时调控G型和S型木质素的目的,本论文针对涉及SAM及其去甲基化产物S-腺苷高半胱氨酸(S-adenosyl homocysteine, SAH)合成的S-腺苷甲硫氨酸合成酶(S-adenosyl methionine synthetas, SAMS)和S-腺苷高半胱氨酸水解酶(S-adenosyl homocysteine hydrolase, SAHH)的基因表达进行分子调控。 本论文获得的数据表明,通过RNAi抑制柳枝稷PvSAHH表达能够显著增加SAH的水平,并导致SAM/SAH比值下降达到46.9%-72.8%;而在柳枝稷中过量表达PvSAHH1对SAH水平影响不一,推测柳枝稷中可能存在SAH的代谢补偿途径,从而抵消由于SAHH过量表达造成的SAH损失。进一步分析表明,在柳枝稷中过量表达PvSAHH1同样能够导致SAM/SAH比值一致下降。先前在微生物和动物中的研究表明SAH是SAM的竞争性抑制剂,因此SAM/SAH比值而非SAM的水平决定氧甲基化反应的速率。鉴于上述报道,本论文通过体外测定野生型柳枝稷粗蛋白提取物中COMT的酶活性,证明COMT的酶活随着SAM/SAH比值的下降而下降。因此上述结果暗示在柳枝稷中无论抑制还是上调SAHH的表达,均能够降低木质素单体合成途径氧甲基化酶的活性,从而达到同时干扰G型和S型木质素单体合成的效果。进一步GC-MS分析结果表明G型和S型木质素单体在SAHH-RNAi和SAHH-OE转基因柳枝稷植株中均发生了显著下降,从而证实了上述推测。另外,由于G型和S型木质素单体合成受阻,引起木质素总含量的下降17%-28%,而转基因柳枝植株细胞壁的糖化效率则增加了4.2%-11.2%。 除了在柳枝稷中调控SAHH表达外,本论文还对涉及SAM合成的SAMS的表达水平进行了分子调控,获得了SAMS表达显著下降的柳枝稷RNAi植株和SAMS表达显著提高的过表达转基因植株。木质素总量分析结果表明,抑制SAMS的表达也能够显著降低木质素的合成(下降7%-25%)。 先前木质素基因工程主要针对木质素合成途径本身的基因进行调控,从而限制了其可用靶基因库的容量。而本论文通过分子调控柳枝稷一碳代谢途径的SAHH和SAMS的表达,成功改变了木质素单体氧甲基化合成途径的甲基供体SAM及其去甲基产物SAH的水平,最终导致G型和S型木质素单体合成受阻,改变了木质素的结构和含量,从而在不影响柳枝稷正常生长发育的情况下显著提高了细胞壁的可降解性。因此今后进一步研究与木质素单体氧甲基化合成关联的植物一碳代谢途径中各基因的功能,不但能够拓宽木质素基因工程的研发思路,加深人们对木质素合成调控的认识,而且能够显著增加木质素基因工程靶基因库的数量,从而使这些基因(包括SAMS和SAHH)有望作为功能分子标记用于高细胞壁品质能源草和牧草作物的分子育种实践。
Keyword工科
Abstract中文
Other AbstractPlant cell walls contain abundant cellulose and hemicellulose that are important sources for producing bioenergy and forage. However, the presence of lignin in cell walls negatively impacts the bioconversion of these polysaccharides, and thereby increases the cost of biofuels and fodders. Switchgrass(Panicum virgatum L.)is a gramineous forage and energy grass. Lignin polymers of switchgrass mainly comprise three types of lignin monomers with different extent of methoxylation, which are designated as hydroxyl phenyl propane (H), guaiacyl propane (G) and syringyl propane (S). Large amounts of G and S monomers are deposited in switchgrass cell walls compared with a trace of H monomers. The methoxylation of G and S precursors is catalyzed by caffeoyl COA-O-METHYLTRANSFERASE (CCoAOMT) and CAFFEIC ACID-O-METHYLTRANSFERASE (COMT). Their methyl donors are provided by S-adenosyl methionine (SAM). Previous studies have shown that downregulation of COMT in switchgrass can result in significant changes in lignin composition and content, and therefore increase the efficiency of cell wall saccharification and the production of bioethanol. Additionally, recent studies in our lab indicate that downregulation of CCoAOMT in switchgrass has no effects on the biosynthesis of lignin monomers. Thus, the present work focused on two genes, namely S-ADENOSYL HOMOCYSTEINE HYDROLASE (SAHH) and S-ADENOSYL METHIONINE SYNTHETASE (SAMS), which are involved in the biosynthesis of SAM and its demethylation product S-adenosyl homocysteine (SAH), respectively. We genetically regulated the expression levels of SAMS and SAHH in switchgrass to aim at simultaneous inhibition of the turn-over efficiency of both CCoAOMT and COMT in vivo. Our results showed that downregulation of PvSAHH in switchgrass resulted in a dramatic increase in SAH content, and thereby led to a 46.9%-72.8% decrease in the ratio of SAM/SAH. In contrast, overexpression of PvSAHH1 in switchgrass had various influences on SAH accumulation. We suspected that other biosynthetic pathways might compensate for the loss of SAH due to SAHH overexpression in switchgrass. Further analysis showed that overexpression of PvSAHH1 was able to reduce the ratio of SAM/SAH as well. Previous studies in animals and microorganisms have shown that SAH is a competitive inhibitor of O-methyltranferases. Therefore, the turn-over efficiency of O-methyltransferases depends on the ratio of SAM/SAH rather than the content of SAM in cells. To test this hypothesis in switchgrass, we measured the enzyme activity of crude enzyme extracts of COMT prepared from wild type switchgrass plants. We found that COMT activities in vitro were declined accompanying the decrease of SAM/SAH ration. Thus, our results suggest that both downregulation and upregulation of PvSAHH in switchgrass can inhibit the enzyme activity of methyltransferases in lignin biosynthetic pathway, and ultimately reduce the accumulation of G and S monomers simultaneously. Lignin composition analysis of PvSAHH-RNAi and -OE transgenic switchgrass lines revealed a substantial decrease in both G and S monomers which further confirmed our hypothesis. Moreover, total lignin contents of the above transgenic switchgrass lines were reduced by 17%-28%, and their saccharification efficiency were improved by 4.2%-11.2%. In addition, genetic regulation of SAMS in switchgrass was fulfilled in the present work. We generated PvSAMS1RNAi transgenic switchgrass lines in which expression levels of SAMS were significantly altered. AcBr lignin content analysis showed that downregulation of SAMS in switchgrass was able to reduce total lignin contents by 7%-25%. In summary, previous studies in lignin genetic engineering have been focused on the lignin biosynthetic pathway, which limited the number of target genes. In this study, genetic regulation of SAHH and SAMS were successfully performed in switchgrass. The results indicate that alternation of SAM and SAH accumulation can reduce the biosynthesis of both G and S monomers in switchgrass cell walls, and change the structure and content of lignin. Although the lignin biosynthesis was dramatically suppressed, the transgenic switchgrass plants were similar in growth with wild type plants. Due to lignin reduction, the cell wall digestibilities were significantly elevated in transgenic switchgrass plants. Therefore future researches will focus on the effects of other one-carbon genes on lignin biosynthesis in switchgrass, which can improve our understanding for the regulation mechanism of lignin biosynthesis, but also can increase the number of target genes in lignin engineering. Moreover, the target genes including SAMS and SAHH have potential to be employed as molecular markers for developing the cultivars of bioenergy and forage grass with high cell wall quality.
Department作物分子育种团队
Subject Area
Date Available2022
Subtype硕士 ; 学位论文
Language中文
Document Type学位论文
Identifierhttp://ir.qibebt.ac.cn/handle/337004/9979
Collection作物分子育种团队
Affiliation1.中国科学院大学
2.中科院青岛生物能源与过程研究所
Recommended Citation
GB/T 7714
齐天雄. 调控柳枝稷SAMS和SAHH表达改变木质素合成的研究[D]. 北京;青岛. 中国科学院大学;中科院青岛生物能源与过程研究所,2017.
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