Lignin reduction in jute by down-regulating the lignin biosynthetic gene(s) through RNAi technique

Abstract

Lignin, a complex, three-dimensional aromatic polymer, serves as a matrix around the polysaccharide components of a plant cell wall by providing additional rigidity, comprehensive strength and hydrophobicity. As a bio-polymeric phenolic macromolecule, lignin is unusual because of its heterogeneity and lack of a defined primary structure. Plants are genetically engineered to produce less lignin so that they would be more suitable for conversion into bio-fuels or high quality paper. Jute, a naturally rich lingo-cellulosic fiber yielding crop, delineates a new promising sphere with respect to lignin manipulation. From an overabundance of experimentations, it is evident that the role of RNAi is immense in gene regulation. Likewise siRNA, artificial microRNA (amiRNA) appends a new prospect in the era of RNAi. Thus this study aimed at designing siRNA and amiRNA based hairpin constructs to introduce into jute by Agrobacterium tumefaciens mediated in planta transformation in order to develop jute variety with reduced lignin content. Apropos to this perspective, monolignoid biosynthetic genes, caffeic acid O-methyltransferase (COMT), ferulic acid 5-hydroxylase (F5H), coumarate 3-hydroxylase (C3H) and cinnamante 4-hydroxylase (C4H) were considered to be engineered. Initially, effective C3H-amiRNA and F5H-amiRNA based constructs were designed and cloned into pGEM-T entry vector. Then constructs were mobilized into destination vector pBI121. After confirmation of the functionality of the amiRNA constructs by agro-infiltration assay in tobacco, the constructs were introduced into C. olitorius by a tissue culture independent Agrobacterium tumefaciens mediated transformation process. At the same time, effectual COMT-siRNA and C4HsiRNA silencing constructs were designed and cloned into vector, pENTR11. Then the recombinant pENTR11 plasmids were mobilized into pK7GW1WG2 destination vector using LR reaction followed by Agrobacterium tumefaciens mediated in planta transformation. The transgenic generations for all four gene(s) were then analyzed through Southern, RT-PCR and northern assays. As a proof of concept, the decrease in lignin content was estimated (Klason Method) for the transgenic lines and compared with the wild type. The transgenic lines for both strategy viz. amiRNA (C3H-amiRNA & F5H-amiRNA) and siRNA (COMT-siRNA&C4H-siRNA) showed reduced level of gene expression and ~ 16-27 % reduction in acid insoluble lignin content compared to the normal control lines. The results indicate successful amiRNA/siRNA transgenesis in jute, which is likely to have far-reaching commercial implications. This study illustrates the role of RNAi (amiRNA/siRNA) with respect to jute lignomics in order to capitulate new insights into the fine-tuned alteration of lignin biosynthetic pathway of jute. Commercial usability of jute is expected to be boosted from a reduction in the lignin content of jute leading to its economic acceleration.