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题名: 小球藻南极株抗寒机理的研究
作者: 李惠英
答辩日期: 2007-03-29
导师: 徐旭东
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 小球藻南极株 ; 寒冷适应 ; 基因差异表达 ; 转基因衣藻
其他题名: Studies on the antifreeze mechanism of Chlorella vulgaris strain NJ-7
摘要: 小球藻(Chlorella vulgaris)属于绿藻门(Chlorophyta)绿藻纲(Chlorophyceae)绿球藻目(Chlorococcales)卵囊藻科(Oocystaceae)小球藻属(Chlorella),是单细胞真核藻类生物。本实验室从南极中山站附近分离纯化到一株小球藻(Chlorella vulgaris)NJ-7,该藻株能在4℃的低温下正常生长,同时也可在20℃的温度下生长。为了探讨其适应低温条件的分子机理,本研究分析了在两种温度下生长的小球藻NJ-7的基因差异表达情况,获得了一批在低温下特异表达基因的全长cDNA和基因组DNA序列,并比较了这些基因在小球藻NJ-7和小球藻的一个温带株UTEX 259中的表达模式差异。此外,将小球藻 NJ-7的两个抗冻蛋白基因转入莱茵衣藻(Chlamydomonas reinhardtii)中进行了表达。 本文的主要研究内容和结果分为三个部分: 第一,以小球藻 NJ-7为材料,利用抑制性差减杂交技术(SSH),构建了在4℃和20℃下生长的小球藻NJ-7相互差减的cDNA文库。经过两轮点杂交筛选,共获得20个在4℃特异表达或表达上调的cDNA片段。其中,与已知功能基因具有较高同源性的共有6种,分别是:与小球藻C-27中抗冻基因hiC6和hiC12同源的cDNA片段;与噻唑生物合成酶基因thi同源的cDNA片段;与热激蛋白基因hsp70同源的cDNA片段;与核糖体蛋白基因RPL10A及RPL18同源的cDNA片段。另外,还有14个在4℃特异表达的克隆,在GenBank中未发现与其显著同源的序列。经Northern blot 验证,这些基因片段在两种温度条件下的表达水平确实存在着显著的差异。除此之外,还得到了19个在4℃表达下调的cDNA片段。 第二,应用RACE技术,获得了5个与已知基因同源的基因全长cDNA序列;通过筛选基因组文库,进一步得到了这些基因的全长基因组DNA序列。分析表明,真核生物基因启动子中普遍存在的调控元件如TATA框、CAAT框等在这些基因上游并未出现,但这些基因的3’-UTR区均有典型的藻类加尾信号TGTAA。通过Northern blot的方法,比较这些基因在小球藻南极株NJ-7和温带株UTEX-259中诱导表达模式的差别,发现它们对低温的响应存在着明显的区别:在温带株中基因受低温诱导很快,而在南极株中受低温诱导极为缓慢。除与已知基因同源的基因外,还获得了4个新基因的cDNA全长序列。 第三,将获得的两个抗冻蛋白全长基因NJ7hiC6和NJ7hiC12装置莱茵衣藻 rbcL或atpA基因的启动子,构建了4个转基因载体。利用磨珠法分别转入莱茵衣藻cc-849,获得了高效表达外源抗冻蛋白的转化子。 本研究得到以下主要结论: (1)在4℃和20℃条件下,小球藻南极株NJ-7的抗冻蛋白基因hiC6和hiC12等存在表达上的显著差异; (2)小球藻NJ-7中基因的调控序列与绝大多数真核生物的基因存在区别,小球藻南极株基因表达对低温诱导的响应比温带株显著减慢; (3)莱茵衣藻可作为一种新型的生物反应器,生产外源抗冻蛋白。
英文摘要: Chlorella vulgaris is a unicellular green alga of the genus Chlorella, Oocystaceae, Chlorococcales, Chlorophyceae, Chlorophyta. C. vulgaris NJ-7 was found on the surface of rocks nearby the Zhongshan Station (China) of Antarctica, isolated and purified by our laboratory. Unlike most other C. vulgaris strains, it grows normally at 4℃ and above 20℃. To explore the molecular mechanism of its adaption to low temperature, I analyzed the differential expression of genes in C. vulgaris NJ-7 at the two temperatures. The complete cDNA and genomic DNA sequences were obtained for several genes up-regulated at the low temperature. The expression mode of these genes was compared in C. vulgaris NJ-7 and a temperate strain C. vulgaris UTEX259. Moreover, two antifreeze protein genes of C. vulgaris NJ-7 were expressed in Chlamydomonas reinhardtii. The main researches and results are described in three parts: (1) By using Suppression Subtractive Hybridization (SSH) technology, two subtractive cDNA libraries were constructed with C. vulgaris NJ-7 growing at 4℃ and 20℃. After two rounds of dot blot hybridizations, 20 cDNA fragments up-regulated at 4℃ were identified from 400 clones. Of these fragments, six were homologous to certain functional genes, including genes encoding two antifreeze proteins in C. vulgaris C-27 (hiC6, hiC12), thiazole biosynthetic enzyme (thi), hot shock protein (hsp70), ribosomal proteins L10A and L18. The other 14 cDNA fragments showed no significant similarity to any gene in GenBank and were presumptively novel genes. Northern blot hybridizations confirmed the differential expression of these cDNA fragments at the two temperatures. In addition, 19 cDNA were found to be down-regulated at 4℃. (2) Using Rapid Amplification of cDNA End (RACE) technology, complete cDNA sequences of 5 genes similar to known genes were obtained. By screening a genomic library, the complete genomic DNA sequences of these genes were also obtained. Analysis showed that these genes contained no TATA and CAAT boxes at 5’-UTR usually found in eukaryotic genes. However, all the genes contain the typical algal tailing signal sequence at 3’-UTR. Comparison of the expression of these genes in the two strains using Northern blot hybridization revealed their remarkable difference in response to low temperature. Genes were up-regulated by low temperature quickly in temperate strain, but much more slowly in the Antarctic strain. (3) Four plasmids were constructed in which antifreeze protein genes NJ7hiC6 and NJ7hiC12 were positioned downstream of rbcL or atpA promoters of Chlanmydomonas reinhardtii. Using the glass bead method, these plasmids were transferred into C. reinhardtii cc-849. Western blot analyses confirmed the high efficient expression of antifreeze proteins in C. reinhardtii cc-849. The main conclusions of this study are: (1) At 4℃ and 20℃, antifreeze protein genes NJ7hiC6 and NJ7hiC12 and other genes showed significant difference in expression in C. vulgaris NJ-7. (2) Transcriptional regulation sequences for genes in C. vulgaris NJ-7 were different from those in most other eukaryotes. The response of gene regulation to low temperature in the Antarctic strain was greatly reduced compared to that of the temperate strain. (3) C. reinhardtii cc-849 could be used as a novel type of bioreactor to produce exogenous antifreeze proteins for industry purpose.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/11950
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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Recommended Citation:
小球藻南极株抗寒机理的研究.李惠英[d].中国科学院水生生物研究所,2007.20-25
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