杨树类黄酮合成途径下游关键酶基因的功能鉴定及其转录调控研究

Poplar (Populus spp.) is a widespread forest tree with significant economic and ecological importance worldwide but the species is susceptible to different fungal diseases. Flavonoids occur in many plant species and play an important role in defense against herbivores and pathogens. Condensed tannins are the polyphenolic compounds synthesized via ?avonoid biosynthetic pathway. Condensed tannins also act as potential antioxidants with bene?cial effects for human health by protecting against free radical-mediated injury and cardiovascular diseases. The increase of condensed tannins accumulation in forage crops can protect ruminants against pasture bloat. Additionally, condensed tannins also contribute to the taste of numerous fruits and beverages, such as fruit juices, tea and wine. Thus, it is important to determine the mechanisms of flavonoid biosynthesis in planta.The early steps of flavonoid biosynthetic pathway of condensed tannins is considered to be catalyzed by leucoanthocyanidin reductase (LAR), converting leucocyanidin to catechin. Leucoanthocyanidin is also the substrate for anthocyanidin synthase (ANS) to produce anthocyanidin, and then converted to the other condensed tannin precursors, epicatechins, by the catalyzation of anthocyanidin reductase (ANR). Many questions related to the biosynthetic mechanism of condensed tannins in plants still remain unclear. With the completion of the P. trichocarpa genome sequencing, a wide range of genomic and genetic resources are now available for this species, thus Populus has been selected as a model legume for biochemical, genetic and genomic studies. The overall objective of this proposal is to investigate functions of PtrLARs,PtrANRs and PtrMYB006 in biosynthesis of flavonoids in Populus. To accomplish this research, we will firstly isolate PtrLARs,PtrANRs and PtrMYB006 as well as their promoters from poplar. And then we will analyze the sequences of these genes, and investigate their expression profiles at different tissues of Populus. Furthermore, the enzyme activity of PtrLAR and PtrANR will be determined via in vitro assay. On the another hand, transcription activition of PtrMYB006 will be analyzed by yeast hybridization and its function of transcriptional regulation involved in flavonoid biosynthetic pathway will charactered in transgenic poplar plants. Finally, we will figure out the molecular control of biosynthesis of flavonoids in transgenic poplar plants. Transgenic plants will be further evaluated for resistance to infection by pathogenic fungi and insects.

杨树是我国重要的经济林木，但大规模种植导致病虫害的危害日益严重，杨树抗病虫害育种已迫在眉睫。类黄酮物质是植物中广泛存在的一类次生代谢产物，其在植物防御体系中扮演重要角色。因此，搞清类黄酮生物合成途径及其调控机制对于提高植物抗病虫害能力具有十分重要的意义。为此，本项目在已克隆杨树类黄酮合成途径下游关键酶基因PtrANR、PtrLAR及其转录因子PtrMYB006基础上，拟：①采用qRT-PCR和启动子分析明确其组织表达特性; ②蛋白体外表达鉴定关键酶的代谢活性; ③通过拟南芥转化和遗传互补试验解析关键酶的催化功能; ④在转基因杨树中考察其对该途径上其它结构基因表达水平的影响;⑤酵母单杂交试验搞清PtrMYB006转录活性;⑥EMSA分析阐明PtrMYB006对结构基因的转录调控作用; ⑦病虫接种试验评价转基因毛白杨的病虫害抗性。最终筛选出抗病虫害的转基因杨树材料，为将来杨树抗性育种奠定基础。

杨树是我国重要的经济林木之一，但大规模种植导致病虫害的危害日益严重。而类黄酮物质是植物中广泛存在的一类次生代谢产物，其在植物防御体系中扮演重要角色。为此，本项目以杨树为材料，已克隆杨树类黄酮合成途径下游关键酶基因PtoLAR1、PtoLAR3、PtoANR和 PtoMYB6基础上，采用qRT-PCR和启动子分析显示，PtoANR、PtoLAR1和PtoLAR3在根中表达量最高，其次是茎、成熟叶片，在幼叶和叶柄中含量较低。PtoMYB6在毛白杨幼叶中表达最高，其次是根、茎和韧皮部，在成熟叶片中表达量较低。通过拟南芥遗传互补试验证明，杨树PtoANR1基因具有拟南芥ANR类似的功能。酵母杂交试验和亚细胞定位分析显示PtoMYB6是一个定位于细胞核的有转录激活因子。超量表达PtoANR1、PtoLAR1/3和PtoMYB6均导致转基因植株中花青素和缩合单宁含量显著增加，显示这些基因均参与了花青素和单宁的生物合成。而超量表达PtoMYB6导致转基因株系中类黄酮关键酶基因的表达量均明显提高。EMSA分析显示PtoMYB6可通过直接结合类黄酮合成关键酶基因启动子调控单宁和花青素的合成。转超表达PtrLAR3基因杨树的抗病分析表明，超表达PtrLAR3能有效抑制黑斑病菌丝的生长，从而提高转基因杨树对真菌病的抗性。本项目的研究初步搞清了杨树的类黄酮生物合成途径及其转录调控机制，对于提高杨树抗病害能力具有十分重要的意义。