毛白杨融合基因4CL1-CCR对烟草木质素沉积的影响

doi:10.11707/j.1001-7488.20201007

Abstract

Abstract:

Objective: The genes encoding 4CL1(4-coumarate:coenzyme A ligase) and CCR (coumaryl:coenzyme A reductase) were ligated to form the fusion gene 4CL1-CCR. The complex enzyme 4CL1-CCR encoded by the fusion gene was analyzed in vitro, it was found that the complex enzyme has catalytic activity of 4CL1 and CCR. It was proven that in Escherichia coli the 4CL1-CCR has efficiently catalyzed the formation of the corresponding aldehyde from phenolic acids such as coumaric acid. On this basis, we studied the function of the fusion gene in tobacco(Nicotiana tabacum), mainly revealing the influence of the fusion gene on the total amount of tobacco lignin, monolignols composition and morphological of tobacco xylem. Method: The content of monolignols in tobacco stems were analyzed using GC-MS. The lignification of tobacco stems were observed by phloroglucinol-HCl staining and autofluorescence of lignin. The expression analysis of key genes involved in lignin biosynthesis in transgenic tobacco was carried out by semi-quantitative PCR. Result: The total amounts of G+S in the stem of transgenic tobacco plants were increased 10.89%-36.44%; the monolignols tests showed a significant increase of G-unit in all transgenic tobacco plants, but S-unit change was not significant. Through the anatomical analysis of the tobacco stems, it was found that the number of xylem cells and the cell wall thickness were increased in the transgenic tobacco plants. However, the lignin deposition pattern did not change. The region with high lignin density was still the cell corner and the compound middle lamella, and the region with low lignin density was the secondary cell wall. Semi-quantitative PCR analysis showed that the expression of C3H, CAD and COMT in the transgenic tobacco plants was increased compared with WT, while the expression of F5H, the key gene for the conversion of G-unit monolignol to S-unit, did not change significantly. Conclusion: Fusion gene expression increased the total amount of G+S lignin and the number of xylem cells, but did not change the distribution patterns of lignin deposition density.

Key words: 4CL1, CCR, fusion gene, lignin

CLC Number:

Jiaqi Hu,Qi Qi,Xiangning Jiang,Ying Gai. Effect of Fusion Gene 4CL1-CCR of Populus tomentosa on Lignin Deposition in Tobacco[J]. Scientia Silvae Sinicae, 2020, 56(10): 63-69.

Figures/Tables 6

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References 0

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引物名称 Primer name	引物序列 Primer sequence (5′—3′)
Actin-antisense	GGTGTTATGGTTGGTATGGGTC
Actin-sense	CCTCTCTGAGCCAATGGTAAT
F5H-F	CAACAGACCAGCCAACATAGCCATAAGC
F5H-R	GTTGCATTCCAGGGCAAGACCTCCGAC
COMT-F	AGATGCTCCAACTTACCCCG
COMT-R	GCCTTGCGAATCCAGTAAAACC
C3H-F	GACGAGTGGTGCCAAACAAC
C3H-R	ATTGAGCACTCTGTCCCTGC
4CL1-CCR-F	GTTTGGCCTTGGCCTGAAAG
4CL1-CCR-R	AAAACCAGGTGCATGTGGGA
CAD-F	AGTGGGACACATGGGAGTGA
CAD-R	AAACAAAAGGATACAAAAGACGACA

株系 Line	G型木质素单体 G-unit monolignol/ (μg·mg^-1)	S型木质素单体 S-unit monolignol/(μg·mg^-1)	G:S
WT	10.51±0.01	9.94±0.03	1.06±0.04
WT	10.65±0.59	9.81±0.28	1.09±0.41
WT	10.51±0.03	9.95±0.29	1.06±0.18
4CL1-CCR 2	12.32±0.07^*	10.49±0.66^*	1.17±0.05
4CL1-CCR 5-2	14.34±0.52^*	11.10±0.36^*	1.29±0.27^*
4CL1-CCR 10-1	12.96±0.45^*	9.85±0.65	1.32±0.47^*
4CL1-CCR 16-1	13.03±0.03^*	9.03±0.09	1.17±0.04
4CL1-CCR 20-2	11.81±0.13^*	9.35±0.86	1.26±0.01^*

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Effect of Fusion Gene 4CL1-CCR of Populus tomentosa on Lignin Deposition in Tobacco

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