基于转录组学与蛋白质组学的青海野生沙棘抗旱分子机制的研究

Located in dry and arid region, the environment of Qinghai province is vulnerable with severe soil loss in China. The wild Sea buckthorn distribute in Qinghai province has high nutritive value and strong tolerance to drought, it is an important member to develop ecological economic forest and play important role in soil loss control. Drought resistance of Sea buckthorn is the process interact with multi gene, multi protein and multi signal molecule. Until now, there is little known about the molecular mechanism of Sea buckthorn under drought stress, and the data available is very shortage. Therefore, on the basis of our previous studies, the cellular structure of root, stem and leaf and the ultrastructure of leaf epidermis and cell under drought stress in different time of wild Sea buckthorn in Qinghai were studied by TEM and SEM; construct the transcriptome database by RNA-seq technology, screen and annotate different express genes, obtain full-length sequences of these different express genes by RACE technology, and analyze their species, structure and function, then invest the temporal and spatial expression mode and their function played in drought tolerance of different express genes by real-time PCR technology; screen the different protein under drought stress by iTRAQ technology, then comprehensive analysis the drought resistance gene and protein, definite the new genes and proteins that participate in the process of drought resistance. Based on the above research, the drought resistance mechanism of Sea buckthorn will cleared from cell, subcellular and molecular level, All of which will lay the foundation for the further study of new genes’ function and signal regulation mechanism of Sea buckthorn.

青海生态环境脆弱，水土流失严重。青海省内分布的野生沙棘抗旱性强且活性成分高，是治理水土流失、发展生态经济林的重要资源。沙棘抗旱是多基因、多蛋白与多信号分子作用的过程。目前国内外关于沙棘抗旱分子机制的研究仅限于申请人前期单一基因的研究，未见从转录组和蛋白质组学进行研究的报道。因此项目在前期研究的基础上采用TEM、SEM等方法明确干旱胁迫不同时间青海野生沙棘根、茎、叶的细胞形态结构、叶表面及细胞内超微结构；采用RNA-seq筛选沙棘抗旱差异基因，RACE延伸差异基因的5'-和3'-末端，拼接全长序列，实时荧光定量PCR明确干旱胁迫前后沙棘差异基因的时空表达特性；采用iTRAQ筛选沙棘抗旱差异蛋白；综合分析抗旱基因与蛋白的种类、结构与功能，明确有哪些新基因与新蛋白参与沙棘抗旱的过程。项目将从细胞、亚细胞、分子水平全面揭示沙棘抗旱机制，可为沙棘抗旱新基因功能与信号调控的研究奠定基础。

本研究以中国沙棘为对象，对其进行干旱胁迫后测定了抗旱生理指标，同时对不同程度干旱胁迫程度下的中国沙棘叶片进行了显微结构和超微结构的观察；在此基础上采用转录组测序（RNA-seq）技术对干旱胁迫处理前后的青海野生中国沙棘（又名沙棘）进行研究，获得了中国沙棘干旱胁迫差异表达基因，并通过生物信息学方法对差异表达基因的种类、结构与功能进行综合分析，明确了有哪些新基因参与了中国沙棘响应干旱胁迫的过程；cDNA末端快速扩增（RACE）技术延伸中国沙棘干旱相关基因CMO基因和AQP基因的5'-末端和3'-末端未知序列，拼接获得了两个基因的全长序列；此外通过全长转录组测序技术获得了13,271个中国沙棘抗旱相关基因的全长序列，其中与沙棘抗旱显著相关的基因548个，另外包括277个未在7大数据库中匹配上的基因，可能是未知的新基因，其中有93个新基因在不同程度干旱胁迫的中国沙棘中出现差异表达，可能是与沙棘抗旱功能密切相关的基因；通过iTRAQ对沙棘中干旱胁迫差异蛋白进行了筛选，并与转录组测序数据进行关联分析，鉴定出20个与差异基因表达趋势相同的差异蛋白，鉴定出7个与差异基因表达趋势相反的差异蛋白；通过生物信息学分析方法对获得的14个沙棘抗旱相关基因以及13个在沙棘中发现的未知的新基因进行了结构分析和预测；通过实时荧光定量PCR（Real-Time PCR）技术对53个抗旱相关基因（包括28个渗透调节物及保护酶合成相关基因，14个NAC转录因子基因及11个HB转录因子基因）中国沙棘抗旱关键基因在干旱胁迫不同时间的中国沙棘根、茎、叶中的时空表达特性进行了研究，明确了其表达模式，为后续沙棘抗旱关键基因的抗旱功能奠定了基础。.本研究的结果从分子水平研究中国沙棘的抗旱机制，研究结果将为进一步开展沙棘响应干旱胁迫过程中信号分子调控、新基因功能等分子机制的研究奠定了基础，同时也为植物转基因抗旱新品种的培育提供了丰富的基因资源。