Alfalfa (Medicago sativa L.) is a legume forage that is widely cultivated owing to its high biomass yield and favorable nutrient values. However, alfalfa contains relatively high lignin, which limits its utilization. Downregulation of two transcriptional factors, Transparent Testa8 (TT8) and Homeobox12 (HB12), has been proposed to reduce lignin content in alfalfa. Therefore, silencing of TT8 (TT8i) and HB12 (HB12i) in alfalfa was achieved by RNAi technology. The objective of this project was to determine effect of gene modification through silencing of TT8 and HB12 genes in alfalfa plants on lignin and phenolic content, bioenergic value, nutrient supply from rumen degradable and undegradable fractions, and in vitro ammonia production in response to the silencing of TT8 and HB12 genes in alfalfa. All gene silenced alfalfa plants (5 TT8i and 11 HB12i) were grown under greenhouse conditions with wild type as a control. Samples were analyzed for bioactive compounds, degradation fractions, truly digestible nutrients, energetic values and in vitro ammonia productions in ruminant systems. Furthermore, relationships between physiochemical, metabolic and fermentation characteristics and molecular spectral parameters were determined using vibrational molecular spectroscopy. Results showed that the HB12i had higher lignin, while TT8i had higher phenolics. Both silenced genotypes had higher rumen slowly degraded carbohydrate fractions and truly digestible neutral detergent fiber, but lower rumen degradable protein fractions. Moreover, the HB12i had lower truly digestible crude protein, energetic values and ammonia production compared with other silenced genotypes. In addition, in relation to the nutritive values of alfalfa, structural carbohydrate parameters were negatively correlated, whereas alpha/beta ratio in protein structure was positively correlated. Furthermore, good predictions were obtained for degradation of protein and carbohydrate fractions and energy values from molecular spectral parameters. In conclusion, silencing of the TT8 and HB12 genes decreased protein availability and increased fiber availability. Silencing of the HB12 gene also increased lignin and decreased energy and rumen ammonia production. Moreover, nutritional alterations were closely correlated with molecular spectral parameters. Therefore, gene modification through silencing the TT8 and HB12 genes in alfalfa influenced physiochemical, metabolic and fermentation characteristics.

苜蓿（Medicago sativa L.）是一种豆科牧草，由于其高生物量产量和良好的营养价值而被广泛种植。然而，苜蓿含有较高的木质素，限制了其利用。下调透明 Testa8 ( TT8 ) 和 Homeobox12 ( HB12 ) 两种转录因子可降低苜蓿中的木质素含量。因此，通过RNAi技术实现了苜蓿中TT8 ( TT8i )和HB12 (HB12i )的沉默。该项目的目的是确定通过沉默TT8和HB12进行基因修饰的效果苜蓿植物中的基因对木质素和酚含量、生物能值、瘤胃可降解和不可降解部分的养分供应以及苜蓿中TT8和HB12基因沉默的体外氨产量的影响。所有基因沉默的苜蓿植物（5 株TT8 i 和 11 株HB12 i）均在温室条件下生长，并以野生型作为对照。分析样品的生物活性化合物、降解分数、真正可消化的营养物质、能量值和反刍动物系统中的体外氨产量。此外，利用振动分子光谱确定了理化、代谢和发酵特性与分子光谱参数之间的关系。结果表明，HB12 i 具有较高的木质素，而TT8 i 具有较高的酚类物质。两种沉默基因型都具有较高的瘤胃缓慢降解碳水化合物组分和真正可消化的中性洗涤纤维，但瘤胃可降解蛋白质组分较低。此外，与其他沉默基因型相比， HB12 i 的真正可消化粗蛋白、能量值和氨产量较低。此外，与苜蓿的营养价值相关，碳水化合物结构参数呈负相关，而蛋白质结构中的α/β比值呈正相关。此外，根据分子光谱参数，对蛋白质和碳水化合物组分的降解以及能量值进行了良好的预测。总之，TT8的沉默HB12基因降低了蛋白质的利用率，增加了纤维的利用率。HB12基因的沉默还增加了木质素并减少了能量和瘤胃氨的产生。此外，营养变化与分子光谱参数密切相关。因此，通过沉默苜蓿中的TT8和HB12基因进行基因修饰会影响其理化、代谢和发酵特性。