Molecular cloning, expression pattern, and putative cis-acting elements of a 4-coumarate: CoA ligase gene in bamboo (Neosinocalamus affinis)

Background: 4-coumarate:CoA ligase (4CL) plays an important role at the divergence point from general phenylpropanoid metabolism to several branch pathways. Although 4CL sin higher plants have been extensively studied, little has known about the 4CL gene of bamboo. Results: In current study, a Na4CL gene putative encoding 4-coumarate:CoA ligase (4CL) and its 5’-flanking region were isolated from bamboo (Neosinocalamus affinis) by RACE-PCR and genomic DNA walker, respectively. Na4CL encodes a predicted protein of 557 amino acids, with conserved motifs of adenylate-forming enzymes. Phylogenetic analysis showed that Na4CL shared 62~85 percent identity with other known plant 4CLs, and cluster closely with some known 4CLs in monocots. Sequence analysis revealed conserved cis-acting elements (Box A and AC-II element) present in the Na4CL promoter. Additionally, a Na4CL RNAi construct was transformed into tobacco. Transgenic tobaccos displayed significant down-expression of endogenesis 4CL and reduced lignin contents. Conclusion: These results contribute to the knowledge of the presence of Na4CL gene and its possible role in phenylpropanoid metabolism.

Isolation of total RNA from hard bamboo tissue rich in polyphenols and polysaccharides for gene expression studies

RNA isolation from hard and woody internodal bamboo (Bambusa balcooa) tissue is very difficult due to the presence of secondary metabolites, polysaccharides, and polyphenolics. These compounds often co-precipitate with isolated RNA and hinder downstream applications. We have developed an efficient, cost effective and reproducible RNA isolation method from hard tissue of bamboo internode. This protocol includes an additional organic solvent refinement steps to remove endogenous phenolic compounds and acidic phenol (pH 4.2) to critically stabilize RNA in extraction buffer. In addition to these, two 2M Lithium chloride washing steps were introduced to eliminate DNA and polysaccharides contamination. The RNA isolated from the present protocol was found to be superior, when compared to total RNA extracted by other available protocols. The A260/A280 absorption ratio of the isolated RNA was found ranging between 1.89-1.97. The integrity of 28S and 18S rRNA was highly satisfactory when analyzed in agarose denaturing gel. RNA was further used for RT PCR, northern hybridization, cDNA library and subtractive hybridization without any further refinement.