Directional Regulation of Lignin Monomer Synthesis in Tobacco by Using COMT Gene and CCoAOMT Gene of Eucalyptus urophylla

doi:10.13523/j.cb.20180304

China Biotechnology

2018, Vol. 38

Issue (3): 24-32 DOI: 10.13523/j.cb.20180304

Orginal Article

Directional Regulation of Lignin Monomer Synthesis in Tobacco by Using COMT Gene and CCoAOMT Gene of Eucalyptus urophylla

Bo-wen CHEN(

),Hai-long LIU,Yu-fei XIAO,Zi-hai QIN,Ye ZHANG,Xiao-ning ZHANG

Guangxi Forestry Research Institute, Key Laboratory of Central South Fast-growing Timber Cultivation of Forestry Ministry of China, Guangxi Key Laboratory of Superior Timber Trees Resource Cultivation, Nanning 530002, China

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Abstract

Directional regulation effect of Eucalyptus urophylla COMT gene and CCoAOMT gene on lignin monomer synthesis were studied by tobacco transformation system. Transgenic plants C-S(contain sense EuCOMT fragment), CR(contain full-length RNAi fragment of EuCCoAOMT), C-CR(contain EuCOM and EuCCoAOMT dual-gene) were obtained, and the expression of sense EuCOMT fragment was detected in transgenic tobacco, while the RNAi fragment of EuCCoAOMT caused strong inhibition on tobacco CCoAOMT gene. The growth morphology, lignin content, fiber content and anatomical structure of transgenic tobacco were not significantly different from those of wild type. The results of lignin monomer detection showed the G lignin content increased 17.72% and S/G ratio decreased 17.99% in transgenic plant C-S, meanwhile the S/G ratio increased 61.62% in CR. In C-CR, G lignin content decreased 57.38%, and S/G ratio increased 114.94%. The result showed that the inhibition of CCoAOMT had a significant inhibitory effect on lignin synthesis in G, and the dual-gene transformation of EuCOMT and EuCCoAOMT showing the best directional regulation effect.

Key words： Dual-gene transformation Lignin monomer synthesis Directional regulation Tobacco transformation

Received: 28 November 2017 Published: 04 April 2018

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	Bo-wen CHEN
	Hai-long LIU
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	Zi-hai QIN
	Ye ZHANG
	Xiao-ning ZHANG

Cite this article:

Bo-wen CHEN,Hai-long LIU,Yu-fei XIAO,Zi-hai QIN,Ye ZHANG,Xiao-ning ZHANG. Directional Regulation of Lignin Monomer Synthesis in Tobacco by Using COMT Gene and CCoAOMT Gene of Eucalyptus urophylla. China Biotechnology, 2018, 38(3): 24-32.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180304 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I3/24

Fig.1 Schematic of expression vectors

Table 1 Primers used in this study

Fig.2 PCR validation of transgenic tobacco seedling
(a) M:D2000 marker(TIANGEN);1-6:PCR identification results of transgenic plants contain pEuCOMT-S vector with COMT-F1/COMT-R1 primers, 3 and 6 were positive plants (b) M:D2000 marker(TIANGEN);1-6:PCR identification results of transgenic plants contain pEuCCoAOMT-RNAi vector with CCoAOMT-F1/ CCoAOMT-R1 primers, 2 and 3 were positive plants (c) M:D2000 marker(TIANGEN);1,3,5,7,9,11:PCR identification results of transgenic plants contain pEuCCoAOMT-EuCOMT vector with CCoAOMT-F1/CCoAOMT-R1 primers;2,4,6,8,10,12:PCR identification results of transgenic plants contain pEuCCoAOMT-EuCOMT vector with COMT-F1/COMT-R1 primers,3/4、7/8 and 9/10 corresponding plant were positive plants

Fig.3 Tobacco genetic transformation
(a) Callus differentiation (b) Bud strengthen cultivation (c)Rooting cultivation of transgenic plants (d) Transplanting of transgenic plants (e) Transgenic plants cultivated for 120 days (f) Transgenic plants cultivated for 270 days (g) Transgenic plants cultivated for 330 days blooming as normal

Fig.4 Effect of transforming on gene expression
Different letters represent a highly significant difference at 0.05 level

Table 2 Lignin and cellulose content in transgenic tobacco

Table 3 Measurement of stem section of transformed plants

Fig.5 M?ule stain of xylem

Table 4 Analysis of lignin monomer content in stem of transgenic plants


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