EuCAD基因小片段RNAi对烟草木质素合成的影响

Effect of RNA Interference Triggered by Small Fragment of EuCAD Gene on Lignin Synthesis in Tobacco

【目的】深入研究尾叶桉(Eucalyptus urophylla)肉桂酰乙醇脱氢酶(CAD)基因序列中的关键片段并利用其进行木质素合成调控。【方法】采用生物信息学工具对EuCAD基因进行序列分析,根据编码蛋白保守结构域的分布情况进行筛选,获得一段44 bp关键序列用于构建RNA干扰载体并转化烟草,获得转基因植株并对基因表达水平、木质素含量等表型进行检测。【结果】获得28个转基因株系,转基因植株正常生长发育,与野生型无异,随机挑选3个株系进行检测,发现CAD基因表达水平均较野生型显著降低,以R1株系降幅最大,降至野生型的12.51%。对R1株系进一步检测发现,其茎部直径与野生型无异,但木质部厚度较野生型显著降低,其中第4节处降低11.75%,第6节处降低10.06%,茎部整体木质素含量较野生型降低15.26%。表明该RNAi片段显著抑制烟草中CAD基因表达并影响烟草木质素合成。【结论】通过序列分析筛选出的EuCAD基因小片段,可通过RNA干扰显著影响烟草木质素合成,为尾叶桉木质素合成调控研究提供了技术参考。

【Objective】The study aimed to explore the key sequence of cinnamoyl alcohol dehydrogenase(CAD)gene of Eucalyptus urophylla and use them to regulate lignin synthesis.【Method】Sequence analysis of EuCAD gene was carried out by bioinformatics method.According to the distribution of conservative domain of coding protein,a 44 bp key sequence was obtained to construct RNA interference vector and transformed to tobacco.Transgenic plants were obtained and phenotypes such as gene expression and lignin content were detected.【Result】Twenty-eight transgenic lines were obtained,which grew normally as same as the wild type.Three transgenic lines were randomly selected for detection.The results showed that the expression level of CAD gene was significantly lower than that of wild type.The largest decline was observed in line R1 which decreased to 12.51%of the wild type.It was found that the stem diameter of line R1 was the same as that of the wild type,but the xylem thickness was significantly lower than that of the wild type,which decreased by 11.75%at the 4th node and 10.06%at the 6th node.The lignin content of the stem in line R1 decreased by 15.26%compared with that of the wild type.The results showed that the RNAi fragment significantly inhibited the expression of CAD gene in tobacco and affected the lignin synthesis of tobacco.【Conclusion】The small fragment of EuCAD gene screened by sequence analysis can significantly affect lignin synthesis in tobacco by triggering RNA interference,which provides a technical reference for the study on the regulation of lignin synthesis in E.urophylla.