外源酚酸诱导枳菌根共生相关基因筛选

Screening of genes induced by exogenous phenolic on mycorrhized Poncirus trifoliate

摘要:

[目的]基于丛枝菌根（AM）真菌改变柑橘酚类现象，筛选外源酚酸诱导菌根共生差异基因，分析酚相关基因功能。[方法]以枳（Poncirus trifoliate L.）接种AM真菌，并施加外源酚酸，采用随机引物扩增，获得差异表达片段；继而克隆酚相关基因并进行生物信息学分析。[结果]试验采用20条随机引物和3条锚定引物扩增cDNA，共获得了154条差异显示的表达片段；对其中16条Northern杂交显示阳性的片段测序与比对，发现9条有相关功能注释，参与环境因子及内源信号的识别，调节共生体的形成；DD-11基因只有在外源酚酸添加和接种AM真菌的根系中表达，该片段cDNA全长序列长度为1382 bp，编码454个氨基酸，分子式为C₂₂₁₀H₃₄₂₇N₆₀₃O₆₅₇S₃₁，其理论等电点和相对分子量分别为7.81和49950.03；磷酸化分析发现，该蛋白有22个丝氨酸残基、14个苏氨酸残基及7个酪氨酸残基可能成为蛋白激酶磷酸化位点。通过PredictProtein服务器对DD-11蛋白质的二级结构进行分析，该蛋白含有α螺旋22%、无规则卷曲58%、延伸链20%，不含有β折叠结构。[结论]本试验获得菌根共生过程酚相关基因，其功能与植物内源信号识别密切相关，为探讨酚类在菌根共生过程中的分子机制提供了新的视角。

Abstract:

[Objective] It has been reported that phnolics contents could be changed by the inoculation with arbuscular mycorrhizal (AM) fungi. In the present study, the differentially expressed genes were screened, and the genes function was analyzed with the exogenous phenolic addition. [Methods] Poncitrus trifoliata (L.) inoculated with AM fungi was treated by exogenous phenolic. We obtained the differentially expressed bands by differential-display RT-PCR (DDRT-PCR), and then cloned the related genes and analyzed the bioninformation. [Results] A total of 154 differentially accumulated transcript-derived fragments were identified. After the sequencing and blast of 16 fragments, 9 genes were found related to putative functions which participated into the recognition of environment factors and internal signals, and the adjust of mycorrhizal formation. The bioinformatic analysis showed that the cDNA sequences of DD-11 was 1382 bp in length, containing a complete 1080 bp open reading frame and 454 amino acids were encoded, which was only expressed under mycorrhizal inoculation and exogenous phenolic. The molecular weight, theoretical pI of the DD-11 gene deduced protein were 49950.03 Da, 7.81, C₂₂₁₀H₃₄₂₇N₆₀₃O₆₅₇S₃₁. The deduced DD-11 protein included 22 Ser, 14 Thr, 7 Tyr, which became the phosphorylation sites of protein kinase. The secondary structure of the deduced DD-11 protein mainly included Alpha helix, Random coil, Extended strand, Beta turn, which were 21.81%, 57.71%, 20.48% and have no Beta turn. [Conclusion] The genes related to phenolic expressed in mycorrhizal formation were obtained in our study, which were related closely to the plants signal recognition. Our study provide a new perspective of molecular mechanism of phnolics occurring in mycorrhizal formation.