Expression of β-glucosidase from Neocallimastix patriciarum in rice chloroplast or apoplast

• Main Authors: Pei-yu Huang, 黃佩玉
• Other Authors: Maurice S. B. Ku
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/75742261721581375891

碩士 === 國立嘉義大學 === 生物農業科技學系碩士班 === 100 === Traditional energy derived from fossil oil will be exhausted in less than fifty years. Use of lignocellulosic crops or agricultural wastes to produce ethanol will not only help solve energy crisis but also protect the environment in a sustainable manner. The limitation in cellulosic ethanol production is the high cost to produce cellulose hydrolytic enzymes. The improvement in producing cellulose hydrolytic enzymes in large quantity will make cellulosic ethanol production cost effective. Use of transgenic crops, such as rice, to produce cellulose hydrolytic enzymes in large amounts will be an excellent choice with many advantages. The overall goal of this study was to develop rice as a bioreactor for large-scale production of cellulose hydrolytic enzyme β-glucosidase with the protein stored in the chloroplast or apoplast and at the same time to improve rice straw as an efficient biomass feedstock. Several lines of transgenic rice plants expressing a β-glucosidase gene isolated from the fungus Neocallimastix patriciarum were obtained in TNG67, a japonica rice cultivar (Oryza sativa L.), by Agrobacterium tumefaciens mediated transformation. Gene integration and expression were analyzed by PCR, RT-PCR, TAIL-PCR, Southern blot and western immunoblot. For transformation with pHLC-196 (PHC) to target the β-glucosidase in the chloroplast, we obtained many albino plantlets and 3 green transgenic plants from one independent line. For transformation with pHLA-196 (PHA) to target the β-glucosidase in the apoplast, we obtained 24 green transgenic plants from 8 independent lines. Molecular analysis indicated that the β-glucosidase gene was integrated into the genome of the transgenic rice lines at 1-2 copies. In both cases, transgenic plants with higher β-glucosidase activities exhibited poor growth and abnormal phenotypes (albino, zero tillering). It’s most likely that if the higher β-glucosidase activities cause the severe reduction in growth. The overall β-glucosidase expression level in the transgenic rice plants was low, and further improvement for its expression may include codon optimization and adoption of a senescence inducible promoter. These improvements may enhance its expression after the plants reaching maturity without affecting plant growth.