甜菜M14品系BvM14-APX、BvM14-DHAR3、BvM14-MDAR基因响应NaCl胁迫的表达分析

Expression analysis of Bv M14-APX,Bv M14-DHAR3 and Bv M14-MDAR responsive to Na Cl stress in sugar beet M14 line

甜菜M14品系是在二倍体栽培甜菜染色体组上附加野生白花甜菜第9号染色体的单体附加系,具有耐盐性、无融合生殖等优良性状,是发掘优质基因资源的良好研究材料。实验室前期已利用i TRAQ串联LC-MS/MS技术获得0、200、400 m M Na Cl胁迫下叶中差异表达蛋白质76个;与利用Hiseq 2000高通量测序技术构建的Na Cl(0、200、400 m M)胁迫下甜菜M14品系转录组数据库相匹配,得到了3条参与抗氧化酶系统的Bv M14-APX、Bv M14-DHAR3及Bv M14-MDAR的Unigenes序列。对3条Unigenes序列进行生物信息学分析;采用实时荧光定量PCR技术对0、200、400 m M Na Cl胁迫下叶和根中的3条基因进行组织特异性分析,探究3条基因在叶和根中的表达趋势,并比较了叶中3条基因在转录水平和蛋白水平的表达量变化是否一致,借以评估3条基因对Na Cl胁迫的响应。实时荧光定量PCR结果表明,Bv M14-APX基因在200 m M Na Cl胁迫下叶和根中分别上调21.56、3.56倍;400 m M Na Cl胁迫下叶和根中分别上调51.27、11.47倍。Bv M14-DHAR3基因在200 m M Na Cl胁迫下叶和根中分别上调14.52、1.83倍;400m M Na Cl胁迫下叶和根中分别上调22.78、5.1倍。Bv M14-MDAR基因在200 m M Na Cl胁迫下叶和根中分别上调29.04、1.33倍;400 m M Na Cl胁迫下叶和根中分别上调59.3、3.81倍。3条基因在响应Na Cl胁迫过程中,叶中的表达量变化均显著于根,同时叶中转录水平与蛋白表达水平的变化具有一致性,说明这3条基因在抗氧化酶系统中起到了重要的作用,这也为进一步探究Bv M14-APX、Bv M14-DHAR3及Bv M14-MDAR基因间互作关系奠定了基础。

Sugar beet monosomic addition line M 14 is a unique germplasm , which contains the Beta vulgaris L. genome with the addition of chromosome 9 of Beta corolliflora Zoss..The M14 line exhibits some good phenotypes such as tolerance to salt stress and apomixes .It is a high quality genetic material to explore good genes resources . In previous study , iTRAQ LC-MS/MS had been employed to identify 76 differential expression proteins in leaves under 0, 200, 400 mM NaCl stress.Combining a new generation of Hiseq 2000 high-throughput sequencing technology, transcriptome database of M14 under 0, 200 and 400 mM NaCl had been built and matched each other.Three BvM14-APX, BvM14-DHAR3 and BvM14-MDAR Unigenes involved in antioxidant enzyme system had been obtained .This study , three unigenes sequences had been analyzed by bioinformatics method .The tissue specificity analysis of three genes in M 14 leaves and roots under 0, 200 and 400 mM NaCl stress had been detected by Real-time PCR.In order to explore the expression trend of three genes in leaves and roots , we compared the transcription level and protein expression level whether were consistent or not , which evaluated the level of three genes response to salt stress .The conclusion of Real-time PCR have showed that compared with control , the expression of BvM14-APX gene was upregulated 21.56 and 3.56 times respectively in leaves and roots under 200 mM NaCl stress.What is more, it also had the same trend under 400 mM NaCl treatment with the times of 51.27, 11.47 .And the expression of BvM14-DHAR3 gene was also up-regulated 14.52 times and 1.83 times in leaves and roots under 200 mM NaCl treatment .As the same trend under 400 mM NaCl stress treatment , it was up-regulated 22.78 times and 5.1 times.Moreover, the expression of BvM14-MDAR gene showed 29.04 times and 1.33 times increase under 200 mM NaCl treatment .Under 400 mM NaCl treatment , the multiples are 59.3 and 3.81 .To three genes in response to NaCl stress , the expression trends of the three genes in leaves were more significant compared in roots and the trends of transcription level and protein expression level of the three genes in leaves were consistent , which illuminated that three genes played an important role in the antioxidant enzyme system .The research opened a new path for the interaction relationship among BvM14-APX, BvM14-DHAR3 and BvM14-MDAR genes.