CRISPR/Cas9基因组编辑苜蓿MtSAHH基因调控木质素合成的研究

Cell wall bioengineering is a valuable strategy to increase forage digestibility and therefore improve the utilization of lignocellulosic biomass. Previous studies have shown that the lignin biosynthetic pathway can be genetically modified for the transmission of desirable traits to agriculturally important crops. Plant one-carbon (C1) metabolism supplies methyl residues for methyltransferase-mediated reactions and has the potential to disrupt O-methylation of lignin metabolism. Thus, the regulation of the C1 metabolic intermediates could affect the extent of methylation of lignin precursors. However, the cross-talk between lignin and other metabolic pathways has yet to be investigated. SAH is a product derived fromO-methylation reaction that can compete the binding site of O-methyltransferase with SAM to inhibit its catalytic efficiency. Here we make a proposal to manipulate lignin by CRISPR/Cas9-mediated targeted mutagenesis of MtSAHH in hairy root system of Medicago truncatula R108. The two SAHHs, MtSAHH1 and 2, will be edited by the above technique and the progeny of genome-edited lines will be subjected to an analysis of the content of SAM and SAH. The effect of these C1 metabolic intermediates on expression levels of other pathway genes, lignin biosynthesis, and cell wall digestibility will be studied, and then a novel regulation mechanism controlling lignin biosynthesis through C1 metabolism will be drawn in this project. Our work will provide more targets for improving forage quality by molecular breeding in future.

木质素的含量和成分能够显著影响牧草细胞壁中纤维素、半纤维素等多糖类生物大分子在草食类牲畜瘤胃中的消化吸收效率，通过基因工程技术对木质素的合成进行分子调控，可以降低其对牧草细胞壁品质的干扰。先前的研究表明，木质素合成途径中关键的两个甲基化酶—CCoAOMT和COMT均需要一碳代谢关键酶S-腺苷甲硫氨酸（SAM）作为甲基供体，进而催化生成G型和S型木质素单体。而作为上述反应的竞争物S-腺苷同型半胱氨酸（SAH）则可显著抑制甲基化酶的反应效率。因此，本项目拟以豆科模式植物蒺藜苜蓿R108为研究对象，利用基于毛状根培养系统的苜蓿高效CRISPR/Cas9基因组编辑平台，对R108中两个同源基因MtSAHH1和MtSAHH2进行编辑，获得不含转基因痕迹的子代植株，研究改变一碳代谢中间体积累对木质素合成及细胞壁品质的影响，从而为高品质牧草的分子育种提供更多更新的靶基因。

牧草干物质消化率是衡量牧草品质的重要指标。细胞壁中木质素的含量和单体组成严重影响牧草多糖类生物大分子在草食类牲畜瘤胃中的消化和吸收。先前的研究表明，木质素合成途径中关键的两个甲基化酶—CCoAOMT和COMT均需要S-腺苷甲硫氨酸（SAM）作为甲基供体，进而催化生成G型和S型木质素单体。而作为上述反应的竞争物，可被S-腺苷同型半胱氨酸水解酶（MtSAHH）水解的S-腺苷同型半胱氨酸（SAH）则显著抑制甲基化酶的反应效率。本项目首先建立了基于蒺藜苜蓿毛状根系统的高效CRISPR/Cas9基因组编辑平台，利用该平台对豆科模式植物蒺藜苜蓿R108的MtSAHHs基因进行编辑，研究该基因对木质素合成及细胞壁品质的影响。通过实验分析发现，MtSAHH1基因编辑导致蒺藜苜蓿中SAH含量增加，G型和S型木质素单体以及总木质素含量降低，苜蓿的干物质消化率提高。该结果可以初步表明，一碳代谢途径中SAHH基因对蒺藜苜蓿木质素的生物合成具有重要调控作用。本项目的研究初步解析了MtSAHH1基因在蒺藜苜蓿木质素合成调控过程中的分子机制，为创制高细胞壁品质苜蓿种质新资源提供更多更新的靶基因。