甘蓝型油菜F5H基因的克隆及反义调控烟草木质素生物合成_英文_(2)

phloroglucinol (dissolved in 95% alcohol) in 12% HCl. For Maüle staining, sections were immersed in 1% (w/v) potassium permanganate solution for 5 min at RT, then washed twice with 3% hydrochloric acid until the color turned from black or dark brown to light brown.

Results and Discussion

Sequence analysis of the cDNAs encoding F5H

The whole coding sequences were obtained by RT-PCR from Brassica napus cv. Zhongshuang 9. All the deduced amino acid sequences of the F5H clones contained the motifs known to be conserved (Meyer et al., 1996). Immediately following the inferred initiator methionine was a 17-amino acid sequence containing nine hydroxy amino acids. The subsequent 15-amino acid sequence was rich in hydrophobic amino acids. An RRRR putative stop transfer sequence followed the hydrophobic stretch immediately. Following the stop transfer sequence was the sequence PPGPRGWP, which obeyed the consensus for the proline-rich sequence found in many P450s. The most obvious of these conserved regions was the heme-binding domain between residues Pro-450 and Gly-460, including Cys-458, the presumed heme-binding cysteine.

The clones could be classified to 3 groups. The sequences in one group showed over 99% identity each other at protein level (Nuances of amino acids were some similar amino acids replacements), so randomly choose one clone each group to analyze at protein level. The NO.1 clone only showed 94% identity with the NO.2 as those of Brassica napus cv. Westar (Remesh et al., 2000). The NO.1 clone contained a 1563 bp open reading frame coding 520 amino acids, while the NO.2 and the NO.3 both contained a 1560 bp open reading frame coding 519 amino acids. The similarity scores of the NO.3 clone with the other two were all 96%. The result suggested there were at least 3 members of F5H gene in Brassica napus. So a new member of F5H gene might be found in this study.

Antisense plant expression binary vector was constructed by inserting the NO.1 clone after xylem-specific-expression promoter C4H from Arabidopsis thaliana. The C4H promoter sequence was identity with the sequence reported by Mizutani M et al (1997), contained three cis-acting elements (box P, YTYYMMCMAMCMMC; box A, CCGTCC; and box L, YCYYACCWACC), which were conserved among the genes involved in the core reactions of the phenylpropanoid pathway of several plant species. These elements might be involved in coordinating C4H gene expression with regulation of the PAL and 4CL in response to wounding and light.

Analysis of antisense-F5H-transgenic tobacco

With Agrobacterium-mediated transformation, many independent T0 antisense F5H transgenic tobacco plants had been obtained. The growth of the transgenic plants and the wildtype control were almost 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.

In the reproductive growth stage, thin transverse sections were cut for histochemical coloration (as showed in Fig 1). Wiesner reaction is the method to detect the total extent of lignin in the rough through shade of color, and Maüle reaction is the way to determine the content of S lignin (presented in red). In Wiesner reaction, the vascular bundle of some transgenic plants presented different shade of mauve strip, the vascular bundle near the marrow of some other transgenic plants showed visible deep mauve. However, the vascular bundle of the wildtype control were with even mauve. In Maüle reaction, the vascular bundle of some transgenic plants exhibited red strips and inter-phase yellow strips, the vascular bundle near the marrow of some other transgenic plants showed visible deep brown yellow and the periphery of the vascular bundle showed visible deep red, while the vascular bundle of the wildtype control were yellow changing to red from the periphery to the middle part gradually.

Most mature transgenic plant roots were with less fibres and obvious taproot than the control (Fig 2).

The F5H mutants exhibit a characteristic red fluorescence under UV, whereas wild-type plants have a blue-green appearance (Chapple et al., 1992). In the present study, all the T0 antisense F5H transgenic tobacco plants were not different from the wildtype in any growth stage to UV. This indicated the F5H activity existed in the transgenic tobacco plant. Referrences

1. Boerjan W, Ralph J, Baucher M. 2003. Lignin Biosynthesis. Annals Reviews of Plant Biol, 54: 519–549.

2. Chapple C C, Vogt T, et al. An Arabidopsis mutant defective in the general phenyl -propanoid pathway. Plant Cell, 1992, 4: 1413-1424.

3. Franke R, Mcmichael C M, Meyer K, et al. Modified lignin in tobacco and poplar plants over-expressing the Arabidopsis gene encoding ferulate

5-hydroxylase. Plant J, 2000, 22: 223-234.

4. Goodman A.M, Crook M.J, Ennos A.R. Anchorage Mechanics of the Tap Root System of Winter-sown Oilseed Rape. Annals of Bot, 2001, 87: 397-404.

5. Li L, Popko JL, Umezawa T, et al. 5-Hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of

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