Bhanupriya Ch, Kar Satarupa
Advanced Laboratory for Plant Genetic Engineering, Advanced Technology Development Centre, Indian Institute of Technology, Kharagpur, India.
Planta. 2025 Jan 10;261(2):30. doi: 10.1007/s00425-024-04603-y.
This study seeks to improve the biomass extractability of Sorghum bicolor by targeting a critical enzyme, 4CL, through metabolic engineering of the lignin biosynthetic pathway at the post-transcriptional level. Sorghum bicolor L., a significant forage crop, offers a potential source of carbohydrate components for biofuel production. The high lignin content in sorghum stems often impedes the extractability of desired carbohydrate components for industrial use. Thus, the present study aimed to develop an improved variety of S. bicolor with reduced lignin through RNA interference of the endogenous 4-coumarate:CoA ligase (4CL) gene involved in the lignin biosynthetic pathway. The S. bicolor gene was isolated, characterized, and used to construct the RNAi-inducing hpRNA gene-silencing construct. Two independent transgenic sorghum lines were produced by introducing an hpRNA-induced gene-silencing cassette of the Sb4CL through Agrobacterium-mediated transformation in the shoot tips of S. bicolor. This was confirmed by PCR amplification of the hygromycin-resistance gene and Southern hybridization. The Sb4CL gene transcript and its enzymatic activity were found to reduce to varying degrees, as shown by northern hybridization and enzyme activity in the independent transgenic samples. Endogenous Sb4CL downregulation in sorghum stem tissue correlates with reduced lignin content to a maximum range of 25%. The transfer of the transgene in the second generation was also analyzed. Decreased lignin content in the transgenic lines was compensated by increased total cell wall carbohydrates such as cellulose (36.56%) and soluble sugars (59.72%) compared to untransformed plants. The study suggests that suppressing the Sb4CL gene effectively develops better sorghum varieties with lower lignin content. This can be useful for industrial purposes, as the enhanced carbohydrate content and favorable alteration of lignin content can lead to economic benefits.
本研究旨在通过在转录后水平对木质素生物合成途径进行代谢工程改造，靶向一种关键酶4CL，以提高双色高粱的生物质可提取性。双色高粱L.是一种重要的饲料作物，为生物燃料生产提供了潜在的碳水化合物成分来源。高粱茎中高含量的木质素常常阻碍了工业用所需碳水化合物成分的可提取性。因此，本研究旨在通过对参与木质素生物合成途径的内源性4-香豆酸：辅酶A连接酶（4CL）基因进行RNA干扰，培育出一种木质素含量降低的改良双色高粱品种。分离并鉴定了双色高粱基因，并用于构建RNA干扰诱导的hpRNA基因沉默构建体。通过农杆菌介导的转化，将Sb4CL的hpRNA诱导基因沉默盒导入双色高粱的茎尖，产生了两个独立的转基因高粱品系。通过潮霉素抗性基因的PCR扩增和Southern杂交对此进行了证实。如在独立转基因样品中的Northern杂交和酶活性所示，发现Sb4CL基因转录本及其酶活性有不同程度的降低。高粱茎组织中内源性Sb4CL的下调与木质素含量降低至最大25%的范围相关。还分析了转基因在第二代中的传递情况。与未转化的植株相比，转基因品系中木质素含量的降低被总细胞壁碳水化合物如纤维素（36.56%）和可溶性糖（59.72%）的增加所补偿。该研究表明，抑制Sb4CL基因可有效培育出木质素含量较低的更好的高粱品种。这对于工业用途可能是有用的，因为碳水化合物含量的增加和木质素含量的有利改变可带来经济效益。
