竹根姜HCT基因在根茎木质素生物合成中的功能解析

Ginger (Zingiber officinale Rosc.cv.Zhugenjiang) is an important vegetable crop with excellent quality in Sichuan and Chongqing, and market demand is huge. The loss of edible quality of rhizome accelerated caused by lignin accumulation at the later stage of rhizome development. Although lignin biosynthetic pathway is relatively well characterized, it remains unclear the molecular mechanism how rhizome ripening modulates lignin biosynthesis. In our previous work, we screened out 26 genes encoding enzymes involved in lignin biosynthesis by RNA-seq analysis of rhizomes at different developmental stages in ginger. 3 HCT genes, regulating lignin monomer composition, were further selected. This project intends to take 'Zhugenjiang' ginger as materials, and explore the composition, distribution and accumulation of lignin during rhizome development by histochemistry staining and GC-MS. In addition, the correlation between ZoHCTs and lignin accumulation is going to be verified through expression pattern analysis of ZoHCTs by qRT-PCR and in situ hybridization. And thereby the key ZoHCT (or ZoHCTs) associated with lignin biosynthesis will be identified. Furthermore, enzymatic properties of ZoHCTs will be definited by enzyme activity analysis of recombinant protein. At last, the regulatory effects of ZoHCT(s) on lignin accumulation will be revealed through transgenic techniques. The results will clarify the function of HCT gene on lignin biosynthesis in ginger rhizome tissues, and also provide important gene resources for controlling lignin content by genetic engineering technology.

竹根姜是川渝地区特有的鲜食生姜品种，年市场需求量巨大。证据表明，竹根姜根茎生长发育后期木质素的积累导致其食用品质严重下降。尽管，木质素的生物合成调控已基本清楚，但是，根茎发育成熟介导木质素合成的分子调控机制尚不清楚。前期申请者通过竹根姜不同发育期根茎组织RNA-Seq分析，筛选出木质素合成相关酶基因26个，进一步获得调控木质素单体合成的关键酶HCT基因3个。基于此，本项目拟采用组织化学、GC-MS等方法探明竹根姜根茎发育过程木质素的组成、分布及积累特征；利用qRT-PCR和原位杂交技术分析ZoHCTs的表达模式，明晰其与木质素积累的相关性，进一步筛选木质素合成的关键ZoHCTs成员；通过重组蛋白活性分析明确ZoHCTs的酶学特性；通过转基因技术，揭示ZoHCTs对木质素积累的调控效应。研究结果可阐明HCT基因在竹根姜根茎木质素合成中的功能，也为通过基因工程手段调节木质素含量提供基因资源。

竹根姜是川渝地区的特色鲜食生姜品种，根茎发育后期木质化导致其食用品质急剧下降。迄今为止，木质素的生物合成调控在模式植物、作物及林木中取得了长足的进展，但在果蔬中的研究较少，生姜中尚未见报道。本项目采用组织化学、GC-MS、LC-MS/MS等方法检测了竹根姜根茎发育过程木质素及其前体物质和木质素单体含量，结果表明，随着直立根茎的发育成熟，木质素单体含量发生了显著的变化，紫丁香基木质素（S-木质素）逐渐增加，而愈创木基木质素（G-木质素）呈下降趋势，S/G比例显著增加导致生姜脆嫩品质下降。利用RNA-Seq 挖掘了调控木质素合成的候选酶基因（如HCT、CSE、CAD、COMT、LAC等）及NAC、MYB转录因子；利用qRT-PCR发现ZoHCT1/ZoHCT2基因表达随着根茎发育逐渐下降，原位杂交结果也表明ZoHCT1在幼嫩的姜芽中表达量最高，且主要定位于维管束中，这与木质素的积累显著相关。为进一步研究ZoHCT1/ZoHCT2的生物学功能，克隆其全长cDNA序列，将其构建到超表达载体上并转入本氏烟草中。对T1代阳性植株进行表型观察，发现：与野生型烟草相比，ZoHCT1转基因株系（45d苗龄）茎秆变细、株高及节间长度增加、叶片变小、侧枝提前出现、开花结实提前；而ZoHCT2转基因株系株高及节间长度缩短、叶片变大、营养生长旺盛、开花结实推迟；且超表达ZoHCT1/ZoHCT2显著提高了茎秆中木质素总量及G-、S-木质素单体的含量。利用大肠杆菌表达体系在体外诱导表达并纯化了ZoHCT1蛋白，将其加入含底物的反应液中进行色谱分析初步发现其具催化香豆酰CoA和莽草酸的能力，后续需要利用LC-MS/MS鉴定反应产物，明确其对底物的催化能力。研究结果初步阐明了ZoHCT1/ZoHCT2基因在竹根姜根茎木质素合成中的功能，可为通过基因工程手段调节木质素含量提供重要的基因资源。