期 刊 ：植物遗传资源学报 2014年 15卷 6期
关键词：1大豆；异黄酮；异黄酮合成相关酶基因；
Keywords:1soybean [Glycine max (L.) Merill], isoflavone, isoflavone synthesis relative enzyme genes,
摘 要 ：1大豆异黄酮是大豆生长过程中产生的一类重要的次生代谢产物，由于其在植物自身保护和改善人类健康方面的重要作用受到了越来越多的关注。本研究利用高效液相色谱法和实时定量PCR方法分别测定了两个异黄酮含量显著差异的大豆品种鲁黑豆2号（LHD2）和南汇早黑豆（NHZ）在籽粒发育过程中的异黄酮含量变化以及异黄酮合成相关酶基因的表达模式变化，试图分析异黄酮积累与各基因表达量变化的相关关系。结果表明在大豆籽粒发育过程中，异黄酮含量逐渐升高，而不同异黄酮合成相关酶基因的表达趋势不同，CHS7、CHS8、CHR、CHI1A和IFS2的表达趋势与异黄酮积累模式基本一致，而IFS1和CHI1B1的表达趋势与异黄酮积累模式相反。IFR的表达模式在两个大豆品种中存在相反的趋势，在LHD2中与异黄酮组分积累趋势相反，而在NHZ中与异黄酮组分积累趋势相同。结果还表明，同一基因家族中不同基因在籽粒发育过程中的表达量也存在差异。查尔酮合酶基因家族中CHS7和CHS8以及查尔酮异构酶基因家族的CHI1A的表达水平相对其他成员较高，异黄酮合酶基因家族中IFS2的表达量显著高于IFS1的表达量，预示这些基因家族在大豆籽粒异黄酮积累过程中存在功能分化。此外，各基因表达模式与异黄酮积累的相关分析结果表明，不同基因表达模式与异黄酮积累的相关性在两个品种中也不尽相同。LHD2中CHS7、CHS8和IFS2在籽粒发育过程中的表达量变化与不同异黄酮组分呈显著正相关，CHI1B1基因的表达量变化与不同异黄酮组分呈显著负相关。而在NHZ中，IFR在籽粒发育过程中的表达量变化与多个异黄酮组分呈显著正相关。这预示了不同大豆品种异黄酮含量差异的潜在遗传基础。各异黄酮合成相关酶基因表达量变化的相关分析表明，在两个品种中，苯丙氨酸水解酶PAL1与4CL，4CL与CHS2以及CHS1与IFS2基因的表达量均呈现显著正相关。表明这些基因可能通过协同作用共同调控异黄酮的合成与积累。这些结果为我们利用基因工程提高大豆异黄酮含量奠定了基础。
Abstract:1 Isoflavones are an important group of secondary metabolites in accompany with soybean development. Due to their important roles for plant protection and human health, more and more attention has been paid on soybean isoflavones. In this study, the HPLC and Real-time PCR analyses were used to determine the isoflavone content and relative expression levels of genes encoding isoflavone synthesis relative enzymes during the seed development in two soybean cultivars with significantly different isoflavone content (cv. LHD2 and NHZ), and their correlation was also analyzed. We found that the isoflavone content increased gradually during the development of soybean seeds. In the meanwhile, the expression trends of different isoflavone synthesis relative enzyme genes differed significantly. The expression trends of CHS7, CHS8, CHR, CHI1A and IFS2 were consistent with isoflavone accumulation, while the expression trends of IFS1 and CHI1B1 were opposite to isoflavone accumulation. The expression trend of isoflavone reductase (IFR) differed between two cultivars. In LHD2, it was opposite to isoflavone accumulation, while in NHZ, it was consistent with isoflavone accumulation. Our results also demonstrated that different gene members in the same gene family exhibited different expression levels during the seed development. For instance, CHS7 and CHS8 showed higher expression levels than other members in chalcone synthase gene family, as well as the CHI1A in chalcone isomerase gene family. The expression level of IFS2 was also significantly higher than IFS1 in isoflavone synthase gene family. This result suggested functional differentiations among different members of the same gene family. Additionally, the correlation analysis between expression level and isoflavone accumulation showed that the correlations differed between two cultivars for different isoflavone synthesis relative enzyme genes. In LHD2, the expression levels of CHS7, CHS8 and IFS2 showed significant positive correlations with multiple isoflavone components, CHI1B1 exhibited significantly negative correlation with multiple isoflavone components. On the other hand, in NHZ, the expression level of IFR was significantly positively correlated with the accumulations of multiple isoflavone components. That implied a potential genetic basis for the varying isoflavone contents between both of soybean varieties. The correlation analysis among the expression levels of isoflavone biosynthesis relative enzyme genes during soybean seed development showed that PAL1 and CHS2 had significantly positive correlation with 4CL, and that CHS1 was significantly positively correlated with IFS2. This result suggested that these genes co-regulated the biosynthesis and accumulation of isoflavones. Taken together, our results provided substantial genetic basis on isoflavone biosynthesis and accumulation during soybean seed development, which will be useful to improve soybean isoflavones through genetic engineering in the future.