不同抗倒性甜荞茎秆木质素合成关键酶基因的表达分析

doi:10.3864/j.issn.0578-1752.2015.09.20

摘要/Abstract

摘要： 【目的】测定不同抗倒伏能力甜荞品种在不同时期的茎秆木质素含量及其相关合成酶基因表达量，探讨甜荞茎秆木质素合成积累的关键时期及其关键酶基因，为甜荞抗倒伏育种提供理论依据。【方法】用简并引物扩增得到基因CAD、CCR、F5H、COMT和CCOAOMT的部分CDS序列，在NCBI数据库中进行比对分析；用紫外分光光度法测定4个不同抗倒性甜荞品种在分枝期、盛花期和乳熟期茎秆基部第2节间木质素含量；用荧光定量PCR（qRT-PCR）方法分析木质素合成途径中9个相关基因PAL、4CL、C4H、C3H、CAD、CCR、F5H、COMT和CCOAOMT的表达，用2^-^△△CT法计算其相对表达量。用Microsoft Excel 2003整理数据和作图，用SPSS 19软件进行方差分析和相关性分析。【结果】扩增得到CAD、CCR、F5H、COMT和CCOAOMT的CDS序列可用于设计后续荧光定量引物。甜荞茎秆木质素含量从分枝期到乳熟期逐渐增加，不同抗倒性品种间，高抗倒伏品种木质素含量在各个时期均显著高于易倒伏品种。基因PAL、4CL、C4H和CCR表达量在品种间和时期间均差异显著或极显著，CCOAOMT在品种间不显著，时期间显著，F5H在品种间差异显著，时期间不显著，C3H、CAD和COMT在品种和时期间差异均不显著。从分枝期到乳熟期，各品种的PAL、4CL、C4H、CAD、CCOAOMT、F5H和CCR表达量均先增后减，在盛花期达最大值，高抗倒伏品种表达量显著高于易倒伏品种；从分枝期到盛花期COMT表达量逐渐下降，易倒伏品种表达量显著高于高抗倒伏品种，乳熟期高抗倒伏品种显著高于易倒伏品种；从分枝期到乳熟期C3H表达量无明显变化规律。木质素含量与PAL、4CL、C4H、CCOAOMT、CAD和CCR基因表达量呈显著正相关，与C3H呈负相关，与COMT在分枝期和盛花期呈负相关，乳熟期呈正相关，与F5H呈正相关。【结论】木质素含量与甜荞茎秆倒伏密切相关，抗倒伏能力强的甜荞品种其茎秆木质素含量高；盛花期是甜荞茎秆木质素合成积累的关键时期；PAL、4CL、C4H、CAD、CCOAOMT和CCR是甜荞茎秆木质素合成的关键酶基因。

关键词: 甜荞, 木质素, 关键酶基因, 荧光定量PCR, 抗倒性

Abstract: 【Objective】The objective of this study is to explore the critical period and key enzyme genes in lignin synthesis of stem in common buckwheat and to provide a theoretical basis for common lodging resistance breeding cultivation by determining the lignin content and related genes expression characteristics at different stages of different lodging resistance cultivars. 【Method】Degenerate primers were designed to amplify part of the CDS sequences of CAD, CCR, F5H, COMT and CCOAOMT genes and blast was conducted in the NCBI database. The lignin content at the 2nd internode from the bottom were analyzed at branching stage, full bloom stage and milk ripe stage among four different lodging resistances of common buckwheat by UV spectrophotometry. Quantitative real-time polymerase chain reaction was used to analyze the expression characteristics of nine related genes (PAL, 4CL, C4H, C3H, CAD, CCR, F5H, COMT, and CCOAOMT), and the relative expression level was defined as 2^-^△△CT. Microsoft Excel 2003 and SPSS 19 were used for sorting data, drawing and analysis of variance, correlation analysis, respectively.【Result】Amplified CAD, CCR, F5H, COMT and CCOAOMT genes sequences were used to design the subsequent qRT-PCR primers. Lignin content of stem gradually increased from branching stage to milk ripe stage among different lodging resistances of common buckwheat, and lodging resistance cultivar was significantly higher than that of lodging susceptible cultivar. The expression levels of four genes (PAL, 4CL, C4H, CCR) varied significantly or extremely significantly among different cultivars and stages, CCOAOMT, F5H varied significantly among stages and cultivars, respectively. The remaining three genes (C3H, CAD, COMT) showed no significant difference among the cultivars and stages. The expressions of genes PAL, 4CL, C4H, CAD, CCOAOMT, F5H and CCR increased and then decreased, and the maximum appeared at full bloom stage, and lodging resistance cultivar was significantly higher than that of lodging susceptible cultivar. For COMT, the expression level decreased gradually from branching stage to full bloom stage, compared with the front seven genes, it showed an opposite expression pattern between lodging resistance and lodging susceptible cultivars at the first two stages. There was no obvious changing rule in gene C3H from branching stage to milk ripe stage. The lignin content was significantly and positively correlated with expressions of genes PAL, 4CL, C4H, CCOAOMT, CAD, and CCR, negatively correlated with C3H, and negatively correlated at branching stage and full bloom stage with COMT, while positively correlated at milk ripe stage with COMT.【Conclusion】The common buckwheat cultivar with lodging resistance showed high lignin content in the stem. The PAL, 4CL, C4H, CAD, CCOAOMT and CCR genes play a crucial role in the pathway of lignin synthesis. Full bloom stage is the critical period in lignin synthesis of stem in common buckwheat.

Key words: common buckwheat, lignin, key enzyme gene, qRT-PCR, lodging resistance

胡丹，刘星贝，汪灿，杨浩，李鹤鑫，阮仁武，袁晓辉，易泽林. 不同抗倒性甜荞茎秆木质素合成关键酶基因的表达分析[J]. 中国农业科学, 2015, 48(9): 1864-1872.

HU Dan, LIU Xing-bei, WANG Can, YANG Hao, LI He-xin, RUAN Ren-wu, YUAN Xiao-hui, YI Ze-lin. Expression Analysis of Key Enzyme Genes in Lignin Synthesis of Culm Among Different Lodging Resistances of Common Buckwheat (Fagopyrum esculentum Moench)[J]. Scientia Agricultura Sinica, 2015, 48(9): 1864-1872.

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