坛紫菜泛素连接酶PhCUL1基因克隆与功能验证

The cDNA cloning and functional verification of the PhCUL1 gene from Pyropia haitanensis

中文关键词: 高温  坛紫菜  泛素连接酶  莱茵衣藻  转基因

英文关键词: High temperature  Pyropia haitanensis  Ubiquitin-protein ligase  Chlamydomonas reinhardtii  Transgenic

中文摘要:

      高温影响坛紫菜(Pyropia haitanensis)的产量和品质，是制约紫菜产业发展的主要因素。前期研究发现，高温胁迫下，坛紫菜泛素蛋白酶体系统相关基因显著上调表达，但其响应高温胁迫的分子功能还未知。本研究通过分子生物学、遗传学等技术手段对坛紫菜cullin E3连接酶基因(PhCUL1)的功能进行研究。利用PCR方法克隆了PhCUL1基因的全长，PhCUL1全长为2500 bp，开放阅读框(ORF)长度为2481 bp，该基因存在1个Cullin (407~618 aa)结构域和1个Cullin Nedd8 (754~821 aa)结构域，其中，Cullin Nedd8结构域为蛋白融合位点。进化树分析显示，PhCUL1在进化上与脐形紫菜(Porphyra umbilicalis)有较近的亲缘关系。qRT-PCR结果显示，PhCUL1基因被高温显著诱导。为进一步阐明PhCUL1的分子功能，将其转入莱茵衣藻(Chlamydomonas reinhardtii)进行功能验证，过表达PhCUL1株系比野生型更能耐受高温胁迫。同时，在高温33℃下处理3 h和6 h内，转基因植株的PhCUL1基因呈上调表达。这初步说明PhCUL1基因在坛紫菜响应高温胁迫过程中发挥着重要作用，其具体调控机制有待进一步研究。本研究有助于阐明坛紫菜泛素蛋白酶体系统响应高温胁迫的分子机制，为指导耐高温新品种选育提供理论依据。

英文摘要:

      Heat stress affects Pyropia haitanensis yield and quality, and is the main factor restricting the development of the Pyropia industry. Previous studies found that the genes related to the ubiquitin proteasome system (UPS) of P. haitanensis were significantly up-regulated under heat stress; however, its molecular function in response to high temperatures is unknown. Therefore, in this study, the function of the cullin E3 ligase gene (PhCUL1) was studied using molecular biology and genetics. The results showed that the full-length cDNA of PhCUL1 was 2,500 bp, including an open reading frame (ORF) of 2,481 bp. This gene has a Cullin domain (407~618 aa) and a Cullin Nedd8 domain (754~821 aa). The Cullin Nedd8 domain is a protein fusion site that confers PhCUL1 with E3 ligase activity. Phylogenetic analysis showed that PhCUL1 was clustered with P. umbilicalis. qRT-PCR results showed that the PhCUL1 gene was significantly induced by high temperature. In addition, a transgenic expression system of Chlamydomonas reinhardtii was constructed to verify the function of the PhCUL1 gene. Transgenic C. reinhardtii plants overexpressing PhCUL1 exhibited obvious tolerance to heat stress. The relative PhCUL1 expression in C. reinhardtii exhibited upregulation within 6 hours. These results indicated that PhCUL1 plays a crucial role in response to heat stress in P. haitanensis and its specific regulatory mechanism needs further study. This study provided a better understanding of the mechanisms of UPS in P. haitanensis in response to high temperature and a theoretical basis for the selection of new varieties with high temperature resistance.