蓝藻与绿藻类胡萝卜素合成酶基因的比较基因组学及代谢调控研究

【作者】 梁成伟；

【导师】 秦松；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2007， 博士

【摘要】 类胡萝卜素在生物体内具有重要的生理功能,其中虾青素是一类高附加值值的类胡萝卜素。本文通过比较基因组学和实验手段,探索了类胡萝卜素相关基因的转录调控,为类胡萝卜素的代谢工程奠定的了基础。1、对已测序的18种蓝藻的类胡萝卜素合成基因进行了比较基因组学研究,发现除了Gloeobacter violaceus PCC 7421的类胡萝卜素合成途径是细菌型外,其余的蓝藻类胡萝卜合成途径均属于植物型。序列比对发现蓝藻中参与合成途径上游的酶基因在进化中保守性较高。研究还发现,一些类胡萝卜素合成酶在结构和功能上存在趋同或趋异进化,揭示它们进化上的多样性。2、通过比较基因组学分析,发现在集胞藻Synechocystis sp.PCC6803等几种蓝藻中,没有典型的番茄红素环化酶基因的同源基因,而存在着与绿硫细菌中的γ-carotene环化酶基因相似的基因,例如集胞藻中的sll0147和sll0659。但是研究结果显示sll0147和sll0659的突变对番茄红素环化过程影响不明显,提示在这些蓝藻中可能有其他基因参与环化过程,而sll0659基因可能参与了细胞的分裂过程。3、从经济绿藻雨生红球藻中克隆了参与类胡萝卜素合成途径的八氢番茄红素合成酶基因(psy)和八氢番茄红素脱氢酶基因(pds)的cDNA和基因组序列。通过基因组步移的方法克隆了这两个基因的5’侧翼序列,以本实验室建立的lacZ为报告基因的瞬间表达体系研究了这两个基因上游区域的启动子活性。4、通过ABA、N饥饿和高光诱导雨生红球藻积累虾青素,利用半定量RT-PCR方法分析了在相同环境因子诱导下雨生红球藻中类胡萝卜素合成基因的表达调控模式,结果显示在虾青素积累过程中,除了番茄红素环化酶基因变化不明显,其他一些基因均存在明显的转录上调。本研究首次利用比较基因组学的方法分析了类胡萝卜素基因的进化,发现大部分蓝藻的类胡萝卜合成途径属于植物型,一些类胡萝卜合成酶存在结构和功能的多样性。同时分离了雨生红球藻中类胡萝卜素相关基因八氢番茄红素合成酶基因(psy)和八氢番茄红素脱氢酶基因(pds)的5’上游侧翼序列,验证了它们的启动子功能,研究了雨生红球藻虾青素合成酶基因在相同环境因子诱导下的表达调控模式。本文将基础研究与实验验证相结合,为下一步研究类胡萝卜素合成的代谢工程提供了线索。更多还原

【Abstract】 Caroteniods have many important physiological functions in organisms. Especially, astaxanthin has a high antioxidant activity, an anti-cancer activity, and other biological activities. This paper aims at the comparative genomics of the cyanobacterial carotenoid biosynthetic pathway. In addition, psy and pds were cloned and regulatory sequences were isolated and characterized in Haematococcus pluvialis. And also, expression patterns of crt gene were studied from Haematococcus pluvialis.Genome-wide screening of crt genes based on the genome-sequencing project provided us a new and comprehensive insight into the cyanobacterial carotenoid biosynthetic pathway. It is interesting to find that nearly all the cyanobacteria share quite similar pathway to synthesizeβ-carotene except for Gloeobacter violaceus PCC 7421 which is bacterial-type. The enzymes involved in the upstream pathway are more conserved than the subsequent ones. In addition, many carotenoid synthesis enzymes exhibit diversity in structure and function.Results of comparative genomics show there was not only no detectable crtL-e- but also no crtL-b-like lycopene cyclase gene in the genomes of Synechocystis sp. PCC6803 et al. However, there are homologous genes (sll0147 and sll0659,et al.) to a new type lycopene cyclase cruA from C. tepidum in these species. By genetical transformation system of Synechocystis sp.PCC6803, sll0147 and sll0659 which are similar to cruA were characterized. Full segregants of the sll0147 deletion in Synechocystis were not obtained, implying that this gene is essential for survival. The results of HPLC reveal these two enzymes have no lycopene cyclase activtity. However, the transmission electron micrographs showed that one major effect of the deletion is the abnormal phenotype, which suggests that sll0659 affects the cell division directly or indirectly.Phytoene synthease gene (psy) and phytoene desaturase gene (pds) were cloned from H. pluvialis. In addition, their 5’-flanking upstream sequences are obtained using adaptor-primer PCR method. The transient expressions of lacZ driven by psy 297bp and pds 210bp 5’-flanking sequence show that these sequences contain transcription regulatory sequences.In order to study the expression regulation, H.pluvialis was reduced by ABA, N starvation and high light. The expression patterns of crt genes were analysis by semi RT-PCR. The results showed all of genes were up-regulated except for lcy.This is the first report on the comparative genomics of carotenoid synthetic pathway of cyanobacteria and isolation of genomic genes and 5’flanking regions of psy and pds from H. pluvialis. This research provides a foundation for further study to finding new crt gene and the metabolic regulation of carotnoids.更多还原