Cloning of cDNA encoding F5H from Brassica napus and regulation of lignin biosynthesis by expressing antisense gene in transgenic tobacco
SHI Zhujuan, LIU Guihua, SHEN Jinxiong, WANG Hanzhong, YANG Xiangdong
National Center for Oil Crops Genetic Improvement, Oil Crops Research Institute of CAAS,
Wuhan 430062, China Email: shizhujuan@; zyzy@
Abstract
The whole coding sequences of F5H were obtained by RT-PCR from Brassica napus cv. Zhongshuang 9. All the sequences
had the conserved functional domains and showed high identity with those of Brassica napus cv. Westar. From the sequences analysis, a new member of F5H gene distinguished from the published members was found. The result suggested there were at
least 3 members of F5H gene in Brassica napus. Antisense gene expression binary vector was constructed by inserting one F5H
gene after the xylem-specific-expression promoter C4H from Arabidopsis thaliana. With Agrobacterium-mediated transformation, many independent T0 antisense F5H transgenic tobacco plants have been obtained. The growth conditions of the transgenic plants
and the wildtype control were identical before flowering. In the reproductive growth stage, the transgenic plants grew slowly and
were slender with less leaves, but there were no obvious differences in florescence and seeding. Thin transverse sections were cut
from the basal-stem of some plants in uniform growth with a freezing microtome for histochemical coloration. The results of Wiesner reaction and Maüle reaction indicated lignin distribution of transgenic plants changing. Most mature transgenic plant roots had less fibres and obvious taproot.
Key words: Brassica napus; Lignin; Ferulate 5-hydroxylase (F5H); RT-PCR; Agrobacterium-mediated transformation
Introduction
Brassica napus is a major oilseed crop grown worldwide. A natural lodging event resulted in a yield reduction of 13-20% and more sensitive to pathogen infect (Goodman et al., 2001). Lignin is crucial for structural integrity of the cell wall and strength of the stem, and therefore plays a role in protecting plants against lodging and pathogens (Boerjan et al., 2003).
In dicotyledonous angiosperms, lignin is composed of two major monomeric phenolic constituents: guaiacyl (G) and syringyl (S) units. Increased G content leads to more highly condensed lignin composed of a greater proportion of biphenyl and other carbon-carbon linkages, whereas S subunits are commonly linked through more labile ether bonds at the 4-hydroxyl position (Li et al., 2000). Softwood gymnosperm lignin essentially lack S residue and therefore are highly condensed.
Ferulate 5-hydroxylase (F5H) is a rate-limiting enzyme determining syringyl-substitued lignin precursors (John et al., 2004). Earlier studies have demonstrated that lignin of the F5H mutant lacks the sinapic acid-derived components typical of wild-type lignin (Chapple et al., 1992), while over-expression of F5H increases lignin syringyl monomer content (Meyer et al., 1998; Franke et al., 2000). We intend to acquire more condensed lignin to impart dramatic resistance against lodging and pathogen by down-regulation of F5H activity. Here we report regulation of lignin biosynthesis by expressing antisense F5H gene under the control of the lignification-associated cinnamate-4-hydroxylase promoter in tobacco.
Materials and Methods
Plant material
Leaf and petiole tissues were collected from Brassica napus cv. Zhongshuang No.9 planted in Oil Crops Research Institute, Chinese Academy of Agricultural Sciences.
Tobacco was Nicotiana benthamiana, planted in chamber under 16-h light/25°C and 8-h dark/23°C cycle.
Cloning of cDNA encoding F5H from Brassica napus
Gene specific and degenerate primers were designed on the basis of comparing the published plant F5H sequences (Brassica napus: AF214007, AF214008, AF214009; Arabidopsis thaliana: At4g36220; Eucalyptus gunnii: AJ249093; Liquidambar styraciflua: AF139532), primarily referred to the sequences of Brassica napus and Arabidopsis thaliana. The primers are as following:
The forward primer: 5′-ATGGAGTCTTCTATATCACAAACACTAAG-3′;
The reverse primer: 5′-TTA(a,g)A(c,g)AG(a,c)ACA(a,g)AT(a,c)AGGCG(t,c)GTG-3′.
Vector construction and tobacco transformation
The plant expression vector was pBI121, which carried an nptII gene as a selection marker, the 35S promoter was substituted with Arabidopsis thaliana C4H promoter provided by Dr. Yang. The sequence encoding GUS of pBI121 was replaced by the F5H sequence in reverse orientation. The recombined plasmid was transformed into Agrobacterium tumefaciens LBA4404 by freezing-thawing methods. Nicotiana benthamiana tissue was transformed and regenerated into
whole plants as common methods.
Another primer corresponding to the NOS terminator region were used for detecting the regenerate tobaccos, that is: 5′-GATTGCTTTGATATTGTCACGGGT-3′.
Analysis of transgenic tobacco
In the reproductive growth stage, the lower stems of some plants in uniform growth were cut to 20µm transverse sections with freezing microtome for histochemical coloration. For Wiesner staining, sections were stained with 2% (w/v)