以NIA1基因为靶点的药用植物次生代谢分子调控研究

Secondary metabolites are the main bioactive substances of medical plants. Nitric oxide (NO) is an important signaling molecule in plants. Previous studies showed that environmental factors such as UVB radiation may stimulate biosynthesis of secondary metabolites in plants by triggering NO signaling, which suggests that NO might be a potential target for controlling secondary metabolism in medical plants. Recently, we found that NIA1 gene is responsible for UVB-triggered NO burst in Betula pendula leaves. Based on the previous findings, we proposed that overexpression of NIA1 gene in medical plants may stimulate secondary metabolite biosynthesis by triggering endogenous NO signaling. In this project, we plan to generate transgenic Ginkgo Biloba L. overexpressing NIA1 protein under the estradiol-inducible promoter and assay the effects of NIA1 on contents of flavonoids and terpenoids and expression levels of the genes in terpenoid and flavonoid biosynthetic pathways such as GbCHS、GbF3H、GbFLS、GbHMGR and GbMVD. Furthermore, the effects of NIA1 on nitrate reductase (NR) activity and endogenous NO levels in Ginkgo Biloba will be determined to check if overexpression of NIA1 could induce NR activity and trigger NO burst. The roles of NR and NO signaling in NIA1-induced terpenoid and flavonoid biosynthesis will also be investigated by examining the effects of NR inhibitors and NO specific scavenger on the contents of terpenoids and flavonoids and the expression levels of the genes in terpenoid and flavonoid biosynthetic pathways in transgenic ransgenic Ginkgo Biloba. The functions of other signaling molecules such as jasmonic acid (JA), reactive oxygen species (ROS) and protein kinase and their cross-talking in mediating NIA1-inudced terpenoid and flavonoid biosynthesis will also be studied.

次生代谢产物是药用植物的主要有效成分，也是决定中药材品质的物质基础。NO是植物中一种重要信号分子。前期研究结果表明，UVB等环境因子通过激活植物细胞中NO信号途径促进次生产物合成，提示NO是药用植物次生代谢的潜在调控位点。最近，我们发现NIA1基因表达上调是介导UVB触发植物内源NO迸发的主要机制。在此基础上，提出通过调控NIA1基因表达，激活药用植物细胞中NO信号途径促进次生产物合成的研究设想。拟以银杏为材料，构建转NIA1基因细胞，考查NIA1基因对银杏黄酮、萜类等次生产物含量及GbF3H、GbFLS、GbMVD等次生代谢基因表达水平的影响，验证本项目的研究设想。同时，通过考察NIA1基因对胞内NO、JA等信号分子的激活作用以及不同信号分子在介导NIA1诱发次生产物合成中的功能及cross-talk机制，解析NIA1基因的作用机理。研究结果将为药用植物次生代谢分子调控提供一种新的策略。

黄酮和内酯是银杏中两类重要的活性组分。由于二者生物合成途径不同，因此很难用一般的分子调控技术同时提高银杏黄酮和内酯的含量。本项目提出了利用拟南芥NIA1基因（AtNIA1）同时促进银杏黄酮和内酯生物合成的研究设想。项目的主要研究结果如下：(1)构建EST诱导型转AtNIA1基因的银杏细胞系6个,其中可以高效诱导表达AtNIA1基因的细胞系3个；（2）AtNIA1基因诱导表达可以同时显著提高银杏细胞中黄酮和内酯含量，5天龄的转基因细胞经过EST诱导处理10天后中银杏内酯A、银杏内酯B、白果内酯和总内酯的含量分别增加38.1%, 104.5%, 51.2%和54.3%，而槲皮素、异鼠李素和山奈酚及总黄酮含量比野生型细胞分别提高了114.9%, 50.2%, 34.8%和79.2%；（3）AtNIA1基因诱导表达可以同时上调银杏细胞中GbDXR、GbHMGR、GbF3H、GbFLS、GbMVD等黄酮及内酯生物合成途径关键酶基因表达,说明AtNIA1基因可以同时激活银杏细胞黄酮和内酯生物合成途径；（4）AtNIA1基因诱导表达提高了银杏细胞中几丁质酶和β-1,3-葡聚糖酶的活性，提示AtNIA1基因可以激活银杏细胞的防御反应；（5）AtNIA1基因诱导表达提高了银杏细胞中NR活性和NO水平，证实AtNIA1基因依赖NR介导的NO信号途径诱发银杏细胞黄酮和内酯合成；（6）发现AtNIA1基因通过细胞壁NADPH氧化酶途径提高银杏细胞H2O2水平，证实H2O2是介导AtNIA1基因诱发银杏细胞黄酮和内酯合成的重要信号分子；（7）发现AtNIA1基因依赖NO激活银杏细胞内H2O2信号，初步揭示了H2O2和NO在介导AtNIA1基因诱发银杏细胞黄酮和内酯合成中的cross-talk机制；（8）发现AtNIA1过表达可以降低重金属对银杏细胞膜的损伤，提高细胞活力，促进细胞生长；（9）证实AtNIA1基因通过激活细胞内酶促和非酶促抗氧化活性，降低逆境氧化胁迫，提高抗逆性的作用机制。综上所述，本项目研究结果表明NIA1是一种可以同时促进银杏细胞黄酮和内酯合成的一种新型分子调控工具，实验结果同时表明，NIA1是一种提高植物抗重金属污染等逆境胁迫的潜在靶点。项目研究结果为利用分子生物学技术同时提高药用植物中多种活性组分，并改善药用植物抗逆性提供了一种新的策略。