Characterization of sugarcane mutants developed through gamma irradiations for their lignin content and caffeic acid-O-methyl transferase (COMT) gene mutations

Bagasse, the residue left after extracting juice from sugarcane stalks, is rich in lignocellulosic biomass. The lignin present in this plant biomass is the key factor that hinders the efficient extraction of ethanol from the bagasse. In the current study, γ-irradiated sugarcane mutants were evaluated for variation in lignin content and its corresponding caffeic acid-O-methyl transferase (COMT) gene. The acetyl bromide method was used to estimate lignin content in sugarcane mutants. PCR-based cloning of the COMT gene was performed in low lignin mutants as well as control plants in E. coli (strain DH5α) to understand the mechanism of variation at the molecular level. The Sanger sequencing for cloned gene was performed to check variation in gene sequence. In comparison to the control (21.5%), the mutant plants’ lignin content ranged from 13 to 28%. The Sanger sequencing revealed approximately the same length of the gene from mutants as well as a control plant. In comparison to the reference gene, the mutated gene showed SNPs and indels in different regions, which may have an impact on lignin content. Therefore, γ-irradiated mutagenesis is an acceptable approach to develop novel mutants of sugarcane with low lignin content to enhance bioethanol production from waste material using bioprocess technology.

蔗渣（Bagasse）是甘蔗茎秆经榨汁后剩余的副产物，其富含木质纤维素类生物质。该植物生物质中含有的木质素，是阻碍从蔗渣中高效提取乙醇的关键制约因素。本研究针对经γ射线辐照获得的甘蔗突变体，评估了其木质素含量以及对应咖啡酸-O-甲基转移酶（caffeic acid-O-methyl transferase，COMT）基因的变异情况。本研究采用乙酰溴法测定甘蔗突变体的木质素含量。为从分子层面解析变异产生的机制，本研究以低木质素含量突变体及对照植株为材料，在大肠杆菌（E. coli，菌株DH5α）中完成了COMT基因的PCR介导克隆，并通过桑格测序（Sanger sequencing）检测克隆基因的序列变异。对照组的木质素含量为21.5%，而各突变体植株的木质素含量介于13%至28%之间。桑格测序结果显示，突变体与对照植株的COMT基因长度基本一致。与参考基因相比，突变后的基因在不同区域存在单核苷酸多态性（Single Nucleotide Polymorphisms，SNPs）与插入缺失变异（insertions-deletions，indels），这些变异或对木质素含量产生影响。因此，γ射线辐照诱变是一种可行的手段，可用于培育低木质素含量的新型甘蔗突变体，从而借助生物过程技术提升以该废弃物为原料的生物乙醇生产效率。