巨尾桉抗寒因子CSD1分子调控网络解析

CSD1 is the major superoxide detoxifying enzyme inside cytoplasm catalyzing dismutation of superoxide into oxygen and H2O2. CCS1 is the chaperon, which delivers copper ions to the CSD1 apo-protein.MiR398 binds to the CSD1 and CCS1 transcripts and catalyzes their degradation. However, the relationship between miR398-CSD1-CCS1 under temperature stress is unknown. Cloning of CCS1 and miR398 gene from Eucalyptus and construction of plant expression vector by bidirectional promoter vector, which were sense CSD1 and CCS1, sense CSD1 and anti-sense CCS1, sense CSD1 and miR398, sense CSD1 and anti-sense miR398, transformed to Eucalyptus grandis×E.ophylla by agrobacterium-mediated. The eucalyptus taken sense CSD1 with CCS1 and sense CSD1 with anti-sense CCS1 were used to analysis the two activation pathways of CSD1 identified in many species by two-dimensional gel electrophoresis. The eucalyptus with sense CSD1 and miR398 fusion and sense CSD1 and anti-sense miR398 fusion were used to analyzed whether the accession specific variation depends on miR398 regulation, whether the variation in CSD1 correlates with accession specific regulation of the transcript levels of the other main copper-binding proteins CCS1. SPL7 (for SQUAMOSA promoter binding protein- like7) is essential for this response of miR398.The regulatory mechanism of SPL7-miR398-CCS-CSD1 in eucalyptus derived from realtime PCR in seven kinds of transgenic plants.

本项目旨在借助植物双向启动子载体，将与铜锌超氧化物岐化酶1相关的激活因子CCS，miR398的正义和反义基因以两两组合的方式构建并通过叶盘法转化获得转基因巨尾桉。在转基因桉树中研究CSD1的激活途径，通过铜分子伴侣CCS在桉树中的大量表达和基因沉默的两种极端转基因桉树，使用双向蛋白质电泳技术研究CCS表达量降低时桉树的CSD1的另外一条激活途径，分析其中可能存在的调控蛋白。以miR398表达异常的转基因桉树为对象，研究低温胁迫下miR398低表达与CSD1的调控关系，高温状态下miR398超表达与CSD1的调控关系。将已有的7种转基因桉树，通过实时定量PCR技术分析探讨在不同温度条件下转录因子SPL7-miR398-CCS-CSD1的关系。通过以上研究结果绘制CSD1在低温胁迫下的分子调控网络。

本项目的研究目标是探索巨尾桉CuZnSOD1基因在低温下的激活机理，通过克隆巨尾桉的CuZnSOD1、CCS、GR基因，通过基因的原核表达确定基因功能，qPCR研究其在巨尾桉中的表达特性。获得CuZnSOD1和CuZnSOD1+反义4CL1基因的两种转基因桉树18株，筛选到了两株优质植株，一株是组培苗耐受-13.1℃5h没有表现萎蔫现象，寒害后100d观察无白化现象完全恢复，木质素含量与对照一致的p40号转基因桉树；另一株是组培苗耐受-13.1℃5h茎尖表现萎蔫现象，寒害后100d观察无白化现象，完全恢复且全株硫酸木质素含量比对照植株降低了56%的P41号植株。两株优质转基因桉树具有较好的应用前景。通过qPCR监测桉树CuZnSOD1、CCS、GR基因在常温下、4℃36h和48h的表达模式发现，CuZnSOD1表现为先升高后降低，在4℃36h的表达量相比常温mRNA表达量升高了约3.6倍；CCS在低温胁迫下mRNA表达量先降低后升高，在4℃36h的表达量比常温下降低了约5.3倍，GR在低温胁迫下mRNA一直在降低。综合研究结果表明 CCS可能在常温和低温胁迫48h之后的铜离子转运中起主要作用，但是在桉树低温胁迫前48h的CuZnSOD1的激活中GSH可能参与了非CCS激活途径，但是不排除有其他蛋白因子参与非CCS激活途径的可能。