【作者】 李芳；

【导师】 周涵韬；

【作者基本信息】 厦门大学 ， 发育生物学， 2008， 硕士

【摘要】 植物受盐碱和水分胁迫时，细胞质中积累大量有机渗透调节剂，如甘露醇、海藻糖、甜菜碱和脯氨酸等，而将细胞质中的无机渗透调节剂（主要是K⁺）挤向液泡，使胞质与细胞（液泡）外环境维持渗透平衡，这样就避免了细胞质高浓度无机离子对酶和代谢的毒害。除维持细胞的正常膨压外，甜菜碱还具有无毒渗透保护剂的作用，能够稳定复杂蛋白质高级结构，从而使得许多代谢过程中的重要酶类在渗透胁迫下继续保持活性。甜菜碱是以胆碱为底物经两步酶催化氧化生成，催化第二步反应的是甜菜碱醛脱氢酶（betaine aldehyde dehydrogenase，BADH）。甜菜碱脱氢酶是一个限速酶，甜菜碱的增加与它的活性有关，因此BADH基因的有效表达可增强植物的耐盐性。自1981年从高等植物中首次分离得到BADH基因后，对BADH基因的研究报告陆续有报道。目前，已经先后从大肠杆菌、酵母以及多种高等植物中克隆到BADH基因的cDNA。据推测，单子叶植物的BADH基因可能位于微体中，而双子叶植物BADH基因位于叶绿体中。本文对红树植物BADH基因的克隆与分析进行了初步的探索，主要结果如下：1、采用CTAB法提取白骨壤总RNA，经1．0％琼脂糖凝胶电泳和OD₂₆₀与OD₂₈₀的检测，结果显示，28S rRNA条带亮度是18S rRNA条带亮度2倍以上，而且OD₂₆₀／OD₂₈₀值非常接近2．0，表明我们提取的白骨壤总RNA完整性较好，基本上没有发生降解；纯度较高，所含杂质很少，可用于后续实验中RT-PCR的进行。2、以提取的白骨壤总RNA为模板，用反转录酶反转录生成cDNA，利用简并引物，通过递减PCR（Touch down PCR）技术扩增出约250bp的BADH基因核心片段。经过比对，它和已报道的白骨壤BADH基因同源性高达80％。3、根据扩增得到的白骨壤BADH基因片段设计引物，分别进行3’RACE和5’RACE，扩增白骨壤BADH基因未知的3’端和5’端，并分别得到约350bp（3’RACE）和1．0kb（5’RACE）的特异性片段。测序和比对后，证实我们成功地扩增到了白骨壤BADH基因的3’端和5’端。4、在以上实验基础上，重新设计白骨壤BADH基因特异性引物，从白骨壤总RNA反转录生成的cDNA中扩增得到一条约1．5kb的特异性条带。经过测序和比对后，进一步确定成功克隆到白骨壤的BADH基因。BADH基因编码区全长1509bp，编码502个氨基酸。对BADH基因的分析发现，它与已报道的白骨壤BADH基因的同源性均在90％以上。5、用T4连接酶将pGBKT7和BADH基因连接起来，转入酵母AH109中，在含NaCl梯度浓度的SD-Trp培养基中生长，通过测量生长曲线发现，重组酵母AH109（pGBKT7-BADH）对NaCl的耐受度由原有的9％提高到14％，表明从白骨壤中扩增得到的BADH基因在酵母AH109中能有效转录并翻译出蛋白质，具有生物学活性。更多还原

【Abstract】 Cell accumulates organic osmotic regulators in the cytoplast （Mannitol, trehalose, betaine and praline, for example）, while inorganic osmotic regulators in the vacuole from the cytoplast （one and most of them is K⁺）, when the plant was stressed by salt or water. That will keep osmotic balance between the cytoplast and outsides （vacuole）, avoiding the ion poison to the enzymes and metabolize when high concentration ion in the cytoplast. Beside keeping the cell normal swell press, the glycinebetaine（thereafter GB） can be a innocuity osmoprotectant and stablish the high structure of complex proteins. So the main enzymes in metabolize may keep activity under osmotic stress.In plants, it has reported that GB is synthesized by two steps. The second step is catalyzed by betaine aldehyde dehydrogenase （BADH）. Betaine aldehyde dehydrogenase is a rate-limiting enzyme, and the effective expression of BADH gene will enhance the plant’s salt-tolerance for referring to the betaine’s increase. Researches about BADH gene were reported after the BADH gene isolated for the first time from high plant in 1981. cDNAs of BADH gene were cloned from E.coli, yeast and kinds of high plant. Monocotyledon’s BADH genes are located on microsome and dicotyledon’s BADH genes are located on choloroplast.This study aimed to explore the clone and analysis of BADH gene of mangrove. Main results are followed:1. The total RNA was extracted from Avicennia marina using CTAB, tested by 1.0% agrose gel electrophoresis and OD₂₆₀、OD₂₈₀. The strength of 28S rRNA was over twice to the 18S rRNA from the electrophoresis. The value of OD₂₆₀/OD₂₈₀ was very close to 2.0. It indicated that the total RNA we extracted had good integrality and high purity for non-degradation and low impurity.2. We got the cDNA with RNase from the extracted total RNA. A fragment about 250bp was amplified using a pair of degenerate primers by touch down PCR. It had a 80% homology to the reported BADH gene of Avicennia marina.3. Based on the fragment above, we designed primers and processed 3’RACE and 5’RACE respectively to amplified the unknown 3’ terminus and 5’ terminus of BADE gene. About 350bp（3’RACE） and 1.5kb（5’RACE） special fragments was amplified. Results of sequencing and analysis indicated that we obtained successfully the 3’ RACE and 5’ RACE of BADE gene.4. A pair of gene special primers was redesigned and a 1.5kb fragment was amplified from the cDNA reversed from the total RNA. The fragment was confirmed after sequencing and analysis. The coding section of BADE gene was 1509bp and coded a polypeptide of 502 amino acids. Analysis of BADE gene indicated high homology to other BADE gene, and over 90% homology to the reported Avicennia marina BADE gene.5. The BADE gene was ligated to pGBKT7 with T4 Ligase, and the recombined plasmid was transformed to Saccharomyces cerevisiae AH109. The tolerance to NaCl of recombined Saccharomyces cerevisiae AH109（pGBKT7-BADH） enhanced to 14% from 9%. It showed the protein transcriped and translated in recombined Saccharomyces cerevisiae AH109（pGBKT7-BADH） has biological activity.更多还原