工业大麻响应铅胁迫的差异蛋白质组学研究

Pb pollution seriouly endangers the ecological envrionment and human health. Industrrial hemp has good effect of tolerance and accumulation of Pb. Research on the molecular mechanism of the response of hemp under Pb pollution is one of the effective ways to solve the Pb pollution. Our previous study have already established the platform of proteome analysis system in our previous study. In this project, one high-accumulation of Pb and Pb Sensitive cultivars of hemp will be seleced from alternative 15 cultivars of hemp by treatment of high Pb concentration. One treatment is from hemp treated with high Pb concentration, and the other treatment is from treated with no Pb (CK) at rapid growth period. With the sensitive period of poison symptom appeared in the sensitive Pb cultivar as sampling time, iTRAQ-based proteome analysis will be used to analyze the differential protein responding to Pb stress from various organs (stem phloem, reciprocal 3-5 fully expanded leaf) in the two cultivars selected by pervious research. Base on the known data of hemp genome and the leaf transcriptome data from our previous research, using comparative bioinformatics method, about 50 proteins responding to Pb stress are expected to be found. Among them, the gene expression patterns of about 20 of these proteins will be verified by qRT-PCR, and the Pb-resistance test will be analyzed according to the these proteins of SSR markers in the alternative 15 cultivars. This project will provide theoretical reference and technical support for efficient absorption, transport and accumulation of Pb in hemp

重金属Pb污染严重危害生态环境和人类健康，工业大麻是耐受和富集Pb效果良好的作物之一，研究大麻响应Pb污染的分子机制是利用大麻解决土壤Pb污染的理论基础。本项目通过高浓度Pb处理从15个大麻品种中筛选高富集Pb和Pb敏感型大麻品种各一个，根据已建立蛋白组学研究平台，在已选择的两个品种快速生长期，进行高浓度Pb处理，无Pb处理为对照。以Pb敏感型的Pb毒害敏感期为取样时间点，通过iTRAQ蛋白质组学技术分析该时期两品种不同器官（茎中韧皮部、倒3-5完全展开叶）响应Pb胁迫的差异蛋白。参考大麻基因组和本实验室的转录组数据，进行生物信息学分析，解析高Pb胁迫大麻的差异蛋白响应机制。预期发掘与Pb富集密切相关蛋白约50个，qRT-PCR分析其中约20个密切蛋白基因表达模式，根据这些蛋白对应基因的SSR标记验证备选15个大麻品种的Pb抗性，为大麻Pb高效吸收、转运和富集研究提供理论参考和技术支持。

重金属Pb污染严重危害生态环境和人类健康，工业大麻是耐受和富集Pb效果良好的作物之一，研究大麻响应Pb污染的分子机制是利用大麻解决土壤Pb污染的理论基础。本项目完成对国内11个代表性大麻品种进行耐Pb性评价，并获得萌发期和快速生长期耐Pb型品种“巴马火麻”，Pb敏感型品种“云麻1号”；完成可以使用于蛋白组学研究得工业大麻蛋白样品制备体系优化，即蛋白水化液为：5M Urea, 2M Thiourea, 1% Triton X-100, 50mM Tris-Hcl(PH 8.8), 1%DTT，蛋白水化后加入4倍体积100%预冷丙酮进行蛋白纯化。完成耐Pb型品种“巴马火麻”，Pb敏感型品种“云麻1号”快速生长期叶片差异蛋白组学分析，结合SWATH技术，生物信息学分析，在SwissProt/UniProt分类库成功匹配2131个蛋白，其中差异蛋白（≥1.5倍）BM 和Y1分别为63和372，BM和Y1上调蛋白分别为39、231，分别占所有蛋白61.9%、62.1%，对两个品种差异蛋白功能分析，得到差异蛋白功能可分成14类，比较两品种差异蛋白功能得到与大麻Pb耐性和富集相关得关键蛋白20个，得到大麻适应Pb胁迫的内在分子机制为主要通过提高ATP代谢，加强呼吸作用、光吸收和光能递，促进细胞内N、C同化，清除活性氧，调控气孔发育及关闭，促进叶片水分和CO2交换，促进胞间物质运输，护未折叠蛋白发生聚合及降解错误折叠的蛋白，加强ATP、叶绿体跨膜运输等来适应Pb胁迫。实验结果可为未来大麻Pb耐性和富集品种选育，遗传改良提供重要的品种资源、参考蛋白和基因信息。