<br>使用多重 CRISPR/Cas9 突变苜蓿香豆酸 3-羟化酶可减少木质素沉积并提高饲料质量,Frontiers in Plant Science

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使用多重 CRISPR/Cas9 突变苜蓿香豆酸 3-羟化酶可减少木质素沉积并提高饲料质量
Frontiers in Plant Science ( IF 4.1 ) Pub Date : 2024-03-05 , DOI: 10.3389/fpls.2024.1363182
Tezera W Wolabu ₁ , Kashif Mahmood ₁ , Fang Chen ₂ , Ivone Torres-Jerez ₁ , Michael Udvardi ₃ , Million Tadege ₁ , Lili Cong ₄ , Zengyu Wang ₄ , Jiangqi Wen ₁

Affiliation

苜蓿（苜蓿L.) 草料质量受到成熟阶段细胞壁中木质素沉积的不利影响。通过 RNA 干扰或反义方法降低木质素含量已被证明可以提高苜蓿草料的质量和消化率。我们采用多重 CRISPR/Cas9 介导的基因编辑系统，通过靶向香豆酸 3-羟化酶 (MsC3H)基因，编码木质素生物合成中的关键酶。四个靶向第一个外显子的向导 RNA (gRNA)女士C3H被设计并聚类成 tRNA-gRNA 多顺反子系统，并通过农杆菌介导的转化。在 130 个转基因品系中，至少 73 个品系被证实在一个或多个等位基因中含有基因编辑事件。女士C3H 。选择 55 个品系进行木质素含量/成分分析。在这些品系中，三个独立的四等位基因纯合品系（ MSC3H-013、MSC3H-121 ，和MSC3H-158 ）与不同的突变事件女士C3H进行了详细的表征。纯合突变女士C3H在这三个品系中，显着降低了茎中的木质素含量并改变了木质素组成。此外，这些品系的酸性洗涤纤维和中性洗涤纤维水平显着较低，而总可消化营养物、相对饲料价值和体外真实干物质消化率。综上所述，这些结果表明 CRISPR/Cas9 介导的编辑女士C3H在不牺牲植物生长和生物量产量的情况下，成功降低了芽木质素含量，提高了消化率和营养价值。这些品系可用于苜蓿育种计划，以产生木质素减少和饲料质量提高的优良非转基因苜蓿品种。

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Mutating alfalfa COUMARATE 3-HYDROXYLASE using multiplex CRISPR/Cas9 leads to reduced lignin deposition and improved forage quality

Alfalfa (Medicago sativa L.) forage quality is adversely affected by lignin deposition in cell walls at advanced maturity stages. Reducing lignin content through RNA interference or antisense approaches has been shown to improve alfalfa forage quality and digestibility. We employed a multiplex CRISPR/Cas9-mediated gene-editing system to reduce lignin content and alter lignin composition in alfalfa by targeting the COUMARATE 3-HYDROXYLASE (MsC3H) gene, which encodes a key enzyme in lignin biosynthesis. Four guide RNAs (gRNAs) targeting the first exon of MsC3H were designed and clustered into a tRNA-gRNA polycistronic system and introduced into tetraploid alfalfa via Agrobacterium-mediated transformation. Out of 130 transgenic lines, at least 73 lines were confirmed to contain gene-editing events in one or more alleles of MsC3H. Fifty-five lines were selected for lignin content/composition analysis. Amongst these lines, three independent tetra-allelic homozygous lines (Msc3h-013, Msc3h-121, and Msc3h-158) with different mutation events in MsC3H were characterized in detail. Homozygous mutation of MsC3H in these three lines significantly reduced the lignin content and altered lignin composition in stems. Moreover, these lines had significantly lower levels of acid detergent fiber and neutral detergent fiber as well as higher levels of total digestible nutrients, relative feed values, and in vitro true dry matter digestibility. Taken together, these results showed that CRISPR/Cas9-mediated editing of MsC3H successfully reduced shoot lignin content, improved digestibility, and nutritional values without sacrificing plant growth and biomass yield. These lines could be used in alfalfa breeding programs to generate elite transgene-free alfalfa cultivars with reduced lignin and improved forage quality.

更新日期：2024-03-05

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