雨生红球藻质体球滴结构蛋白基因的克隆与原核表达

CLONING AND PROKARYOTIC EXPRESSION OF THE PLASTOGLOBULES PROTEIN GENE FROM HAEMATOCOCCUS PLUVIALIS

叶绿体或者有色体中的质体球滴结构(Plastoglobules)是多数植物的类胡萝卜素等次生代谢产物积累的场所,但在能大量积累虾青素的雨生红球藻中,这个结构一直没有得到确认。通过透射电子显微镜观察发现雨生红球藻的质体内确切存在plastoglobules结构;并通过RT-PCR结合RACE技术,从雨生红球藻cDNA文库中克隆到了与编码plastoglobules的结构蛋白(Plastoglobulin)具有高度同源性的基因序列全长,称做Hpgp基因;该基因的表达产物称之为雨生红球藻质体球滴蛋白(HPGP;Haematococcus plastoglobules pro-tein);并进一步利用原核表达系统将该编码基因进行原核诱导表达,用His-Tag蛋白分离纯化系统纯化到了目标蛋白,并用该His-Tag融合蛋白为抗原免疫实验兔,制备到了相应的一抗抗体,为下一步对该蛋白的功能阐明以及雨生红球藻的虾青素积累机制研究提供重要的基础。

Carotenoids were organized in plastoglobules located in the interthylakoid space of the chloroplast for many plants. However, the plastoglobule, a carotenoid deposit structure in chromoplast, was not clear in Haematococcus pluvialis yet up to date. In the present study, many osmiophilic globuli appeared in both the chromoplast and cytoplasm of 11. pluvialis under electron microscopy with a modified method. Those revealed clearly the presence of plastoglobules in the chloroplast of H. pluvialis. A full-length cDNA was isolated from H. pluvialis through a RT-PCR and RACE （rapid amplification of cDNA ends） method. And the completing cDNA encoded a protein which had high homology with the major component proteins of plastoglobules in many plants and was termed as HPGP （Haematococcus plastoglobules protein）. Furthermore, this cDNA was expressed in the prokaryotic expression system, and the resultant fusion proteins with His-Tags were purified by immobilized metal affinity chromatography （IMAC）. The polyclonal antibodies against the protein were raised in rabbits and the raw serum was collected without further purification. Those might provide an important basis for unraveling the function of the protein in the future.