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

摘要/Abstract

摘要：

目的: 将毛白杨编码4CL1（4-香豆酸辅酶A连接酶）和CCR（香豆酰辅酶A还原酶）的基因连接在一起形成融合基因4CL1-CCR，在体外对其编码的融合酶4CL1-CCR进行活性分析，发现此融合酶具有4CL1和CCR的催化活性，并在大肠杆菌体内证明4CL1-CCR可以高效催化香豆酸等酚酸生成相应的醛。在前期研究基础上，本研究将融合基因转化烟草，对转基因烟草植株的木质素各单体的组成和木质部形态进行分析，以揭示融合基因在植物体内的功能。方法: 用硫酸解法结合GC-MS分析烟草茎中各个单体的含量；间苯三酚染色法和木质素由紫外激发荧光法用于观测细胞的木质化状态；并通过半定量PCR法对转基因烟草中参与木质素合成关键基因的表达进行分析。结果: 与野生型烟草相比，转基因烟草茎中木质素G+S总量增加10.89%~36.44%；其中转基因烟草中G型木质素单体含量显著增多，S型单体含量无明显变化。显微结构观察可见，转基因烟草木质部宽度增加，细胞壁显著增厚，木质素沉积状态没有变化，表现为高密度木质素区域仍然为细胞角隅和复合胞间层，而低密度区域为次生细胞壁。半定量PCR分析显示在转基因烟草中C3H、CAD和COMT的表达量较野生型烟草均有不同程度的提高，而由G型木质素单体向S型木质素单体转化的关键基因F5H的表达没有显著变化。结论: 人工融合基因4CL1-CCR可以在植物中表达并发挥其功能，在未改变木质素密度分布的前提下，增加烟草茎细胞中木质部细胞数量和木质素的含量，并使细胞壁增厚。

关键词: 4CL1, CCR, 融合基因, 木质素

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

中图分类号:

胡嘉祺,齐芪,蒋湘宁,盖颖. 毛白杨融合基因4CL1-CCR对烟草木质素沉积的影响[J]. 林业科学, 2020, 56(10): 63-69.

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.

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参考文献 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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