荷花R2R3-MYB转录因子NnMYB4对拟南芥木质素合成的影响

[目的]本文旨在研究荷花R2R3-MYB转录因子NnMYB4的功能，探讨其在木质素合成过程中的作用。[方法]利用RT-PCR技术从荷花品种‘黑龙江红莲’中克隆得到1个MYB转录因子基因cDNA全长开放阅读框（ORF），命名为NnMYB4。采用实时荧光定量PCR（qRT-PCR）法分析NnMYB4在荷花不同组织中的表达情况。构建pMDC43-NnMYB4过表达载体转化拟南芥，采用乙酰溴法测定转基因植株的木质素含量，通过qRT-PCR技术对木质素合成关键酶基因的表达进行分析。[结果]NnMYB4基因ORF全长726 bp，编码241个氨基酸，属于R2R3-MYB转录因子G4亚组，与拟南芥AtMYB4亲缘关系较近。组织定量结果表明，NnMYB4基因在荷花根、茎、叶柄、叶中均有表达，且在幼叶中表达量相对最高，在剑叶中最低。与野生型拟南芥相比，转NnMYB4拟南芥植株花序茎变矮，花期延迟，茎木质部染色面积较小、染色程度变浅，木质素含量显著降低。过表达NnMYB4分析结果显示，转基因拟南芥AtC3H、At4CL1、AtF5H、AtCOMT1等木质素合成关键酶基因的表达量显著下调。[结论]荷花NnMYB4可能是木质素合成途径中的一个负调控因子，对植物木质素合成具有负调控作用。
[Objectives] The paper was aimed to study the function of Nelumbo nucifera R2R3-MYB transcription factor NnMYB4,and explore its role in lignin synthesis process. [Methods] We cloned a complete cDNA open reading frame(ORF) of MYB by RT-PCR method,designated as NnMYB4. The expression of NnMYB4 in different tissues of Nelumbo nucifera was analysed by real-time quantitative PCR(qRT-PCR). pMDC43-NnMYB4 overexpression vectors were constructed and transformed into Arabidopsis thaliana by agrobacterium-mediated transformation. Lignin content was determined by acetyl bromide. The expression of the key genes involved in lignin synthesis were analyzed by qRT-PCR. [Results] The full length of the open reading frame was 726 bp,encoding 241 amino acids. It belongs to the R2R3-MYB transcription factor G4 subgroup and it was closely related to Arabidopsis AtMYB4. NnMYB4 was expressed in the root,stem,leaf,but most strongly in the young leaves and most weakly in the flag leaves. The transgenic plants showed inflorescence stems shorter,flowering delay,xylem stain area and the degree of staining shallow smaller,and lignin content was significantly reduced when compared to wild-type A.thaliana. Over expression of NnMYB4 analysis showed that the key enzyme gene(including At4CL1,AtC3H,AtF5H and AtCOMT1) of the lignin biosynthesis pathway was significantly reduced in transgenic lines. [Conclusions] NnMYB4 is a negative regulator of genes involved in the lignin pathway,and it can negatively regulate the synthesis of lignin