油茶Rubisco调控光合效应的机理研究

Camellia oleifera is one of the most important edible oil trees in China and produces high-quality vegetable oil. One of the key objectives in the current C. oleifera breeding and cultivation is to increase seed yield, thus high oil production per plant. Increasing plant photosynthetic efficiency is an important approach for improving crop yield. Ribulose-1,5-bisphosphate carboxylase(Rubisco)/oxygenase and its activase (RCA) exert significant roles in plant photosynthesis. This project is intended to study Rubisco and its RCA genes for improving C. oleifera photosynthetic efficiency. We will (1) isolate gene sequences of the rbcL and rbcS subunits for Rubisco and its RCA genes; (2) analyze the relationship between the levels of the subunits' expression and Rubisco activity; (3) study their functions by transforming them into the wild-type and deletion-mutant type of Arabidopsis thaliana,respectively;(4)determine the effects of overexpression and RNA inference on the levels of expression of rbcL,rbcS and RCA as well as Rubisco activity and photosynthesis in the transformed Arabidopsis seedlings; (5) elucidate the relationships among photosynthetic efficiency, Rubisco activity, and the physiological and biochemical responses of the elite C. oleifera trees; and (6) investigate the effects of external factors, including light, CO2, temperature, the application of of nitrogen, phosphorus, plant growth regulators,and nano-anatase catalyst, on the levels of transcription and translation of rbcL, rbcS and RCA, content and activity of Rubisco and photosynthetic efficiency; and thus determining the potential of the external and internal factors of Rubisco to photosynthesis rate, carbon accumulation, and its allocation of starch, protein and lipid. Thus far, there is little research on C. oleifera biology and its improvement, the execution of this projet will lay concerte foundations for C. oleifera production, and its germplasm collection and evaluation as well as breeding of high seed yield of C. oleifera cultivars through the improvement of their efficiency in photosynthesis.

油茶是我国特有的食用油料树种，其油品质优良。提高产油量是油茶育种和栽培的首要目标，而增加光合效率是提高油茶产量和品质的重要途径。光合作用中Rubisco(1,5-二磷酸核酮糖羧化酶/加氧酶)对调控植物净光合速率、提高产量具有重要作用。本研究从油茶中分离克隆Rubisco大小亚基等基因,通过油茶组织器官中的表达模式、拟南芥突变体中的功能验证、RNA干扰和过量表达等揭示Rubisco 的功能，进而阐明它与光合效能的关系；探索油茶无性系间的光合效能差异、Rubisco 的活性变化以及生理生化效应之间的关系；揭示环境因子、氮磷利用、生长调节剂和纳米催化剂等对油茶Rubisco活性和光合效能的影响；评估Rubisco对增加光合速率和碳积累的潜力，以及对淀粉、蛋白质的积累和油脂转化的影响。最终阐明油茶Rubisco调控光合效应的机理。本项目成果将为油茶良种创制和高效栽培提供重要的理论依据和技术基础。

油茶是我国重要的食用油料树种，茶油品质优良，提高产油量是油茶育种与栽培的首要目标。本研究从油茶获得Rubisco 大小亚基rbcL/rbcS和Rubisco 激酶(RCA) 基因的cDNA序列。其中，rbcL为1522 bp（编码区1425 bp），编码475 氨基酸。rbcS 为832 bp（编码区528 bp），编码176个氨基酸。前者与其它植物rbcL的氨基酸序列同源性较高，而后者与其它植物的同源性较低。通过油茶转录组测序获得3个RCA的cDNA基因，发现rbcL/rbcS中含有SSR重复序列和SNP位点，这对于鉴定油茶种质资源的光合差异具有重要意义。.qPCR分析揭示了rbcL/rbcS、RCA的表达模式，它们在成熟叶片转录表达量较幼嫩叶中多，在叶中表达均高于花和种子。这与净光合速率（PN）、气孔导度（gs）、蒸腾速率(E)、干种子产量、产油量等显著相关。分析了不同油茶品种PN、gs、 E以及羧化效率等的差异，揭示了它们与上述基因转录表达的关系。遮光后恢复光照导致PN、E等逐渐增加，rbcL转录表达下降，而rbcS 转录表达呈现“增加-下降-增加”趋势。随着光照时间增加，RCA表达量逐渐下降。随着温度升高，PN、E及这3个基因的转录表达逐渐增加。.在15和20 ℃不同N浓度处理油茶苗幼叶片中，PN、E、羧化效率（CE）均呈现相似趋势。PN、CE等显著相关。rbcL等3个基因转录表达与PN等光合作用参数变化趋势类似。中度浓度N处理叶片的PN、CE等及这3个基因的转录表达最高，而高浓度N抑制了上述参数和基因表达。.高温干旱伤害导致油茶叶片相对含水量（RWC）下降，浇水和降雨后，RWC增加，其变化依赖于基因型和叶片受伤害程度。单叶面积（LA）、单片叶鲜重（LFW）、叶绿素SPAD 指数均与RWC的变化类似，可溶性糖含量较雨后的高，而可溶性蛋白含量随着品种基因型、受害程度变化而变化。rbcL, rbcS转录表达随着胁迫程度增加而下降，降雨及浇水显著增加RCA的转录表达。表明其对水分较为敏感，可作为鉴定油茶耐旱性的重要指标。.总之，我们揭示了油茶Rubisco 相关基因的结构特点，分析其表达模式与光合效能的关系，阐述了环境因子等对光合效能、Rubisco的影响，筛选出种质鉴定的分子标记及调控光合效率的目标基因。本研究及正在进行实验的会达到本项目要求的预期目标。