高粱SbbHLH1调控木质素合成代谢机理的研究

Less severe pretreatment is needed for lower lignin biomass substrates in ethanol production,which could greatly increase digest efficiency of cellulase, and reduce the final production cost. Sorghum bmr mutant lines contain less lignin in leaves and stems. In our previous work, a sorghum bHLH transcription factor gene SbbHLH1 had been identified from a SSH library constructed with 13 bmr lines and BTx623. SbbHLH1 has transcriptional activation activity in yeast, and its overexpression in Arabidopsis greatly reduced the lignin content of leaves and stems. In the overexpression lines, many lignin biosynthesis genes from phenylpropanoid pathway were down-regulated, while several MYB and NAC trancription factor genes playing crucial regulatory performance in lignin biosynthesis were up-regulated. In this study, we propose to identify the DNA biding site of SbbHLH1 with the SELEX method, and further identify its target genes, to isolate the interacting protein of SbbHLH1 with the Y2H, BiFC and pull down assays, and then to confirm the roles of these targets genes and interacting proteins by transforming them into Arabidopsis with the aim to primarily understand the mechanisms of SbbHLH1 in regulating lignin biosynthesis as well as its relationship with cellulose, hemicellulose and anthocyanidin/flavonoids biosythesis. Effects of SbbHLH1 on development and the response to stress and hormones will be studied as well to know the role of SbbHLH1 in orchestrating lignin synthesis and the response to environmental cues. Finally, transformation of poplar with SbbHLH1 will be conducted to create new low-lignin energy plants for biofuel production.

高粱是重要的能源作物，但其过高的木质素含量导致其需要高强度的前期处理，同时降低了纤维素酶效率和乙醇产量，在生物能源生产中存在较大障碍。因此，降低其木质素含量成为研究热点。我们从13个木质素含量较低的高粱褐色中脉突变体与野生型BTx623 的抑制性消减杂交文库中，分离到一个转录因子基因SbbHLH1，其在SSH库出现频率最高，表达变化最显著，过表达明显降低了拟南芥茎的木质素含量，木质素合成代谢途径的多个关键基因显著下调表达，表明是木质素合成的负调控因子。本研究拟对SbbHLH1的作用机理进行研究：通过DNA结合位点分析，鉴别其直接作用的靶基因；通过酵母菌双杂交等方法筛选其互作蛋白；进而验证这些互作蛋白及靶基因的功能；探讨SbbHLH1参与木质素合成代谢的调控网络、信号传导途径及与纤维素、半纤维素素、花青素/类黄酮等次生代谢途径的关系；并通过杨树转化，评价其在能源植物木质素改良中的应用前景。

降低纤维素原料中的木质素含量有利于提高乙醇发酵效率，增加收益，降低生产成本。本项目从13个bmr突变体中分离克隆到转录因子基因SbbHLH1，过表达可降低茎中木质素的含量，其苯并烷途径多个基因的表达下调，多个重要的木质素合成转录调控因子MYB也发生上调或者下调。本研究通过SELEX技术识别SbbHLH1的DNA结合位点为CAC(A/G)TG,已经识别的作用靶基因包括AtPAL、At4CL、SbHLH1，其启动子的多个位点可与其结合。通过亲和层析-质谱技术，酵母菌双杂交及双分子荧光互补检验了50多个蛋白质与其的互作，结果表明，高粱的一个bHLH有较强的互作，拟南芥的TTG8有较弱的互作，而AtC3H互作中等强度，但得到烟草细胞内的双分子荧光互补的验证。过表达SbbHLH1-His-GFP的拟南芥茎中的荧光信号主要位于维管束的木质部，过表达纯系发育正常，纤维素和半纤维素含量上升，黄酮含量增加，花青素略有下调。而SbbHLH1与SbLim1杂种表现出更大幅度木质素含量的下降，木质素合成的AtPAL、At4Cl1、AtCCOAOMT也大幅下调。SbbHLH1转基因杨树均为嵌合体，阳性纸条木质素含量下降明显，多个木质素合成基因也同时下调，经发酵试验，证明低木质素杨树具有较高的纤维素转化率，显示其在能源植物木质素改良中的良好应用前景。