内生镰刀菌BDF09促进白花丹参毛状根中酚酸类成分积累的机制研究

Phenolic compounds is a potential biological agent with anti-diabetic nephropathy, anti-AIDS and anti-hepatoma effects, but it has low yield through plant tissue culture. One of the method improving yield is the addition of abiotic elicitors and yeast elicitor. But these elicitors can inhibit the growth of host plants, thus affecting the yield of secondary metabolites. We previously isolated an endophytic Fusarium BDF09 strain from the roots of Salvia miltiorrhiza bge.f.alba. Research results indicateded that, when BDF09 were co-cultured with hairy roots of Salvia miltiorrhiza bge.f.alba, the biomass and total phenolic compounds content of hairy roots were increased, and the content of endogenous signal molecules NO and jasmonic acid were also increased. Therefore, we hypothesize that NO and jasmonic acid play an important role in phenolic compounds biosynthesis of Salvia miltiorrhiza bge.f.alba induced by endophytic fungi. In support of this scientific hypothesis, we intend to establish a co-culture system of Salvia miltiorrhiza bge.f.alba hairy roots and endophytic Fusarium BDF09, from the three angles of gene expression, metabolites and signaling molecules, researching the effect of BDF09 on phenolic compounds biosynthesis and its signal transduction pathway. This research will, from a new perspective, reveal fungal induced mechanism of phenolic compounds synthesis of Salvia miltiorrhiza bge.f.alba, enrich the theory system of relationship between endophytic fungi and hosts, lay the foundations on Salvia miltiorrhiza bge.f.alba resource utilization and phenolic compounds large-scale production.

酚酸类化合物是一种有潜力的新型生物制剂，具有抗糖尿型肾病、抗艾滋病和抗肝癌等功效，但通过植物组织培养法生产其产量较低。目前提高产量的主要方法是添加非生物诱导子和酵母菌诱导子，但这些诱导子会抑制植物的生长，从而影响次生代谢产物产量的提高。我们从白花丹参根中分离到一株内生镰刀菌BDF09，发现其与白花丹参毛状根共培养后，可明显提高毛状根的生物量和总酚酸的含量，同时内源信号NO和茉莉酸含量也相应升高。因此，推测NO和茉莉酸在BDF09诱导白花丹参酚酸类成分的合成中起着重要的作用。为了证实这一假说，我们拟建立BDF09诱导子与白花丹参毛状根共培养体系，从基因表达、代谢产物和信号分子三个角度，研究内生真菌对白花丹参酚酸类成分合成的影响及其信号转导通路。本课题将从新的视角揭示白花丹参酚酸类物质的代谢调控机制，充实内生菌和宿主关系的理论体系，为白花丹参资源开发利用及酚酸类物质的规模化生产奠定理论基础。

酚酸类化合物是一种有潜力的新型生物制剂，具有抗糖尿型肾病、抗艾滋病和抗肝癌等功效，但通过植物组织培养法生产其产量较低。我们从白花丹参根中分离到一株内生镰刀菌BDF09，采用响应面法优化了其胞外多糖和胞内多糖诱导子提取条件，将多糖诱导子与白花丹参毛状根共培养后，可以诱发毛状根产生NO和茉莉酸，NO的浓度于诱导后6d达到最高，比对照组高6倍，而茉莉酸的产生则在诱导后8d出现高峰，产生量比对照组高7倍；而同时发现诱导子处理后，毛状根的生物量和总丹酚酸含量也相应升高，总丹酚酸含量最高达到83.87mg•g-1DW，比对照提高了34.6% (P＜0.05) 。采用荧光定量PCR技术，发现BDF09能明显诱导丹参毛状根酚酸类成分次生代谢相关酶基因的mRNA的表达，诱导9d时，PAL、CAH、4CL2、TAT、HPPR、HPPD的转录水平有显著增加，同时毛状根中丹酚酸的含量也有相同的增加趋势，因此，推测NO和茉莉酸在BDF09诱导白花丹参酚酸类成分合成中起着重要作用。为了证实这一假说，我们建立了BDF09诱导子与白花丹参毛状根共培养体系，发现加入NO 专一性淬灭剂cPTIO和茉莉酸阻断剂(IBU)不仅可以分别抑制BDF09诱导子引起的毛状根中NO和茉莉酸迸发，还都能部分阻断BDF09诱导子促进白花丹参毛状根总丹酚酸合成。添加NO供体(SNP)和茉莉酸都可引起白花丹参毛状根中总丹酚酸积累增加，但二者效果不同，暗示着NO和茉莉酸都是介导内生真菌BDF09诱导子促进白花丹参毛状根总丹酚酸合成的信号分子。BDF09诱导后，IBU不能抑制NO的产生，而加入cPTIO后可以抑制茉莉酸的产生，说明茉莉酸在内生真菌BDF09诱导丹酚酸形成途径中位于NO下游。同时添加NO的淬灭剂cPTIO和茉莉酸阻断剂IBU并不能完全抑制BDF09诱导子引起的白花丹参毛状根总丹酚酸积累增加，这表明内生真菌BDF09诱导子还可以通过其他方式促进白花丹参毛状根酚酸类成分合成。课题的开展，从新的视角揭示白花丹参毛状根中酚酸类物质的代谢调控机制，为白花丹参资源开发利用及酚酸类物质的规模化生产奠定理论基础。