【作者】 白林含；

【导师】 杨志荣； 曹毅；

【作者基本信息】 四川大学 ， 遗传学， 2003， 博士

【摘要】 杜氏藻(Dunaliella)广泛分布于世界各地的盐湖和海水中，是一种单细胞绿藻，归属于绿藻纲(Chlorophyceae)、绿藻门(Chlorophyta)。杜氏藻属的野生生活环境最明显的特点是富含Na~+和Cl~-，尽管从各地分离到的野生型株系对NaCl的承受范围和最适生长浓度有所不同，但还是可以看出它们对NaCl有很大的适应浓度范围。因此我国常称杜氏藻为“盐藻”。盐藻中尤以Dunaliella. salina的盐适应能力最强，能够在0.1-饱和盐浓度范围内生存。 目前尽管从分子遗传学角度研究植物耐盐机制的工作日益受到重视，但由于高等植物和盐胁迫因素的组成及其相互作用十分复杂，要说清楚这个问题还有相当的距离。盐藻是真核生物，呈细胞结构，因此渗透调节机制相对简单，对耐盐机理研究可能是一种理想的材料。本文选择盐藻Dunaliella. salina作为研究材料，就盐藻的渗透调节力以及超微结构与渗透调节的关系作了详细的研究。并且对盐藻耐盐相关基因——烯醇酶基因进行了克隆和转基因功能鉴定。研究结果如下： 利用扫描电镜和透射电镜对盐藻的超微结构进行了观察：盐藻Dunaliella salina具有薄而不均匀的外被膜。鞭毛“9+2”结构。眼点位于细胞质中。内质网丰富。线粒体形状多样。叶绿体呈“杯形”。首次在杜氏藻中发现两个大型造粉粒。细胞核位于叶绿体凹窝中，核仁存在时期很短。在高渗震动下，细胞体积明显变小，胞内细胞器如叶绿体、线立体、高尔基体等的体积也相应变小，这些细胞器的膜部分融合至内质网膜中，使内质网的表面积增大。通过倒置显微镜观察生长在不同盐浓度，不同生长时期，以及经不同渗透压震动的盐藻， 四川大学博士学位论文发现其在O.IM一5.OMNaCI培养基中均能正常生长，但其生活状态及生长周期有所不同，其最适生长NaCI浓度为0.5一1.5M，还能适应各种高渗及低渗震动。 针对基因同源性克隆中存在的问题，采用改进简并PCR法对盐藻具抗逆功能的烯醇酶基因进行克隆。改进后的同源引物简并度从1000倍以上降低至100倍以下，利用RT一PCR成功地扩增出一条630bP的盐藻cDNA片段，该片段与其它物种的烯醇酶基因具有很高的相似性。在此基础上进行5’和3’RAcE获得了盐藻烯醇酶基因的全长cDNA，其长度为1734bp。 通过生物信息学的方法将所克隆的序列进行了分析，结果如下:BLASTh结果显示我们所克隆的基因的核酸序列与其它物种的烯醇酶基因的核酸序列有较高的相似性;其蛋白质读框包括479个氨基酸残基;将推导氨基酸序列进行BLASTx发现，该序列与其它物种烯醇酶的蛋白质序列具有高度同源性，具有烯醇酶基因的保守结构域，其中与衣藻烯醇酶基因具83%相同性和89%相似性。通过蛋白质理化性质分析及结构预测显示，我们所克隆的基因与已知的烯醇酶的性质及结构均具有高度相似性。系统发育分析表明，烯醇酶基因在发育过程中具有高度保守性，这一点与糖酵解途径的其它基因是一致的。盐藻烯醇酶基因的进化地位与衣藻相似，与高等植物相近。 对眼藻进行了不同的渗透震动:从1.5M NaCI到0.5M NaCI的低渗震动和从1.5M NaCI到3.0M NaCI的高渗震动。选择盐藻23SRNA作为内标基因，利用半定量Rl，一PCR对经不同渗透震动后(O一48小时)烯醇酶基因转录水平的表达量进行了测定。结果表明:烯醇酶基因经受渗透震动时，其表达量有明显的上调。在高渗震动下，烯醇酶基因的表达量随渗透震动时间而缓慢上调;在低渗震动下，烯醇酶基因的表达量在O一24小时有明显提高，24小时达最高值，然后缓慢降低，但表达量的绝对值仍明显高于未经渗透震动的对照组。说明烯醇酶基因为耐盐相关基因。 采用农杆菌转化系统转化模式植物烟草。以载体pcAMBIA2301为骨架引入pBnZI中的名刘￡基因，构建了简便、实用的中间载体pCA州田IA230lG。并将其中的一个gus基因用目的片段烯醇酶基因替换，构建了可以在植物中高效表达的载体pCAMBIA2301G一enolase，成功地将其转入根癌农杆菌EHA105中，为下一步进行转基因植物的研究作准备。 选用了农杆菌介导的烟草叶盘转化法进行基因转化。叶盘经农杆菌侵染以 四川大学博士学位论文后，在含卡那霉素的MSZ培养基上诱导丛生芽，从生芽在MS3培养基上诱导生根，生根率达到80%左右，最后将PCR阳性苗移栽到蛙石:沙(l:l)的培养介质中培养，得到转基因再生苗。 采用PCR、Southern杂交、Northern杂交对转基因再生苗进行了分子生物学鉴定。用PCR的方法对再生苗进行了初步筛选，转化率约为9%。用Southern杂交证实烯醇酶基因己整合到烟草基因组中，其拷贝数为2一4个。Northern杂交结果显示盐藻烯醇酶基因在烟草中能够正常转录表达。对转基因烟草进行耐盐功能鉴定发现其比阴性对照组的耐盐能力有所提高。更多还原

【Abstract】 Dunaliella, a single-celled green algae classified to Chlorophyceae of Chlorophyta, distributes throughout salt lakes and sea over the world. Dunaliella inhabits itself in the environment rich in Na+ and Cl-. Although wild types of Dunaliella screened from different environments presents distinct salt stress tolerance and optimal salt concentration, all of them are capable of living in the environment of a wide range of NaCl. Dunaliella. salina, has the greatest tolerance to salt stress, and can live in saturated salt solution. Accordingly, Dunaliella. salina is usually named as "Salt algae".Currently the study on the mechanism of plant’s tolerance to salt stress via molecular genetics is getting popular, there is still a long way, however, to decipher the secret due to the complexity of the factors influencing the salt stress in advanced plants. Dunaliella.salina, a single-celled eukaryotic organism, has a relatively simple mechanism of saturation regulation, therefore it is an ideal model organism to be used to study the mechanism of salt stress tolerance. In this paper, Dunaliella salina was used as the research material, and its ability to regulate the osmotic pressure, as well as the interaction of saturation regulation and ultramicro-structure of Dunaliella. salina have been fully examined. Furthermore, its enolase gene, related to salt stress toleration, has been cloned and identified for its function.Under Transmission Electron Microscope and Scanning Electron Microscope, Dunaliella salina had thin and asymmetric outer membrane, the flagellum of thetypical "9+2" structure, a mass of endoplasmic reticulum, mitochondria of various shapes, "cup"-shaped chloroplast, with the eyespot located in the plasm. Two big amyloplastids were observed in DunaUella.salina for the first time. Its nucleus was located in the concave of chloroplast and nucleolus occurs for a short period. Under high osmotic shock, the size of the cells obviously shrink, so did the chloroplasts, mitochondria and Golgi bodies, etc. The membranes of these organelles partially melt with that of endoplasmic reticulum, which makes its surface area increase. By using Inverted Microscope, it was observed that Dunaliella salina of different growth stages after the high osmotic shocks can live in the medium with NaCl concentration between 0.1M and 5.0M, but its growth status and period showed differently. The optimal concentration for the growth of Dunaliella salina was 0.5-1.5M, and this organism could stand a variety range of osmotic shock.Enolase gene, the anti-adversity gene of D. Salina, was cloned by modified degenerate PCR technique. The traditional homogene cloning method was modified on two aspects: first, homo-primers was designed at the sites with minimum degeneracy instead of maximum homology, Secondly, some "N" bases were replaced with inosine in degenerate codes, resulting hi the degeneracy reduced from over 1000 times to below 100 times. A pair of degenerate primers were designed as 5’ - GCI YTN CAR GAR TTY AGA T-3’ /5’ - CAT IAC NCC CCA NCC-3’, and a 630bp-long cDNA fragment of D. Salina was obtained, which had high similarity to the enolases from other species. A full-length enolase cDNA of 1734bp from D. Salina (GeneBank accession No. 245549) was acquired by 5’ RACE and 3’ RACE.The sequence of Enolase gene from D. Salina (DsENO gene) Was analyzed by using bioinformadcs. BLAST search was used to characterize the sequence feature. The nucleic acid sequence of DsENO gene had high similarity to that of other species. The DNA sequence had a complete ORF (open reading frame), which coded a protein of 479 amino acid residues. The protein sequence of enolase which contained the conserved domain, was homologue to enolase of other organisms. It showed 83% ideaity and 89% similarity compared to the enolase in Chlamydomonas reinhardtii. The physical and chemical property of enolase and the predicted three dimensional strucure revealed that DsENO protein was well matched to identified enolase. Multiple sequence alignments and syste更多还原