抗逆基因LcLycE、LcF3H及衰老相关基因SAG172的功能研究

发布时间：2019-03-25 21:02

【摘要】：日益增多的人口导致粮食需求量增多,而植物的生长发育与生长环境密切相关,逆境胁迫及早衰严重影响植物正常的生长发育,降低植物的存活率,严重制约了农作物的产量。植物生长过程中产生丰富的次生代谢产物,如酚类和萜类等化合物,是植物自身进化的一种应对胁迫因素的防御机制。类胡萝卜素和黄酮类化合物有助于逆境胁迫条件下活性氧的消除,对植物抗逆有重要的调节作用。本研究从枸杞中分离了类胡萝卜素代谢途径中的LYCE基因和黄酮类化合物代谢途径中上游基因F3H,并命名为LcLycE和LcF3H。颜色互补实验证明LcLycE能够催化phytoene生成δ-carotene。qPCR结果显示LcLycE基因表达水平受冷胁迫诱导,表明LcLycE基因可能参与调控耐寒胁迫。将LcLycE基因在拟南芥中过表达,发现转基因植物中Lutein含量升高,光氧化损伤减弱,植物耐冷性提高。薄层层析实验证实LcF3H能够催化NAR生成DHK。qPCR结果显示LcF3H基因表达受干旱胁迫诱导,表明LcF3H可能参与调控耐旱胁迫。过表达LcF3H基因促进了转基因烟草的flavan-3-ols水平,并且促进了转基因植物的抗氧化系统,有效清除干早胁迫产生的ROS,最终提高了植物的耐早性。叶片的衰老是叶片发育过程的最后一个阶段,伴随营养物质从源组织向库组织的转移,叶片的早衰制约果实的成熟和产量。因此,植物衰老相关机理的研究对调节植物生长发育和提高产量具有重要的理论和实践意义。本研究在拟南芥中发现了一个衰老相关LRR-RLK基因SAG172。sag172突变体表现叶片晚衰,叶片失水减慢;而诱导过表达SAG172基因的植物叶片提前衰老,叶片失水加快,ABA诱导的气孔敏感性降低,说明SAG172通过调节气孔运动,加速叶片失水,进而促进叶片衰老。qPCR与GUS报告基因检测结果显示SAG172的表达受衰老和ABA诱导,并且诱导表达趋势与AtNAP一致,推测AtNAP与S4G172存在调控关系。酵母单杂及体内GUS酶活实验证实SAG172受AtNAP转录因子直接调控。当用激素处理离体叶片时发现,sag172突变体叶片对ABA敏感,而对JA、ETH、SA表现不敏感。qPCR结果显示,sag172突变体中ABA诱导的标记基因表达量有所抑制,说明SAG172基因通过ABA信号通路调控叶片衰老。综上,本研究对LcLycE、LcF3H和SAG172基因生理功能的分析为植物抗逆、抗衰老、提高产量的相关分子育种研究提供了良好的科学依据。
[Abstract]:The growing population leads to the increase of food demand, and the growth and development of plants are closely related to the growth environment. Stress and early decline seriously affect the normal growth and development of plants, reduce the survival rate of plants, and seriously restrict the yield of crops. The production of abundant secondary metabolites such as phenols and terpenoids during plant growth is a defense mechanism of plant self-evolution against stress factors. Carotenoids and flavonoids contribute to the elimination of reactive oxygen species (Ros) under stress and play an important role in regulating stress resistance of plants. In this study, the LYCE gene in carotenoid metabolism pathway and the upstream gene F3H in flavonoids metabolism pathway were isolated from Lycium barbarum L. and named as LcLycE and LcF3H.. Color complementation experiments showed that LcLycE could catalyze the production of 未-carotene.qPCR from phytoene. The results showed that the expression level of LcLycE gene was induced by cold stress, suggesting that LcLycE gene might be involved in regulation of cold stress. The over-expression of LcLycE gene in Arabidopsis showed that the content of Lutein in transgenic plants increased, the photooxidative damage weakened, and the cold tolerance of transgenic plants increased. Thin layer chromatography (TLC) showed that LcF3H could catalyze the production of DHK.qPCR from NAR. The results showed that the expression of LcF3H gene was induced by drought stress, suggesting that LcF3H might be involved in the regulation of drought tolerance. Over-expression of LcF3H gene promoted the level of flavan-3-ols in transgenic tobacco and promoted the antioxidant system of transgenic plants, which effectively eliminated the ROS, produced by dry-early stress and finally improved the early tolerance of transgenic plants. Leaf senescence is the last stage of leaf development. Along with the transfer of nutrients from the source tissue to the sink tissue, the premature senescence of the leaves restricts the ripening and yield of the fruit. Therefore, the study of plant senescence-related mechanism has important theoretical and practical significance for regulating plant growth and development and increasing yield. In this study, a SAG172.sag172 mutant of senescence-related LRR-RLK gene was found in Arabidopsis thaliana, which showed late leaf senescence and decreased leaf water loss. However, the over-expression of SAG172 gene induced leaf senescence, accelerated leaf water loss and decreased stomatal sensitivity induced by ABA, indicating that SAG172 could accelerate leaf water loss by regulating stomatal movement. The results of qPCR and GUS reporter gene analysis showed that the expression of SAG172 was induced by senescence and ABA, and the induced expression trend was consistent with that of AtNAP. It was suggested that there was a regulatory relationship between AtNAP and S4G172. Single hybrid and in vivo GUS activity assay confirmed that SAG172 was directly regulated by AtNAP transcription factors. When the leaves were treated with hormone, it was found that the leaves of sag172 mutants were sensitive to ABA, but insensitive to JA,ETH,SA. The results of qPCR showed that the expression of marker genes induced by ABA in sag172 mutants was inhibited. These results suggest that SAG172 gene regulates leaf senescence through ABA signaling pathway. In conclusion, the analysis of physiological functions of LcLycE,LcF3H and SAG172 genes provides a good scientific basis for molecular breeding of stress resistance, senescence resistance and yield improvement in plants.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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