【作者】 刘滨；

【导师】 张学成；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2008， 博士

【摘要】 本论文克隆了9种鲽形目鱼类的生长激素基因cDNA序列并运用PAUP软件进行了分子系统进化树分析,构建了四种不同类型的酿酒酵母（Saccharomycescerevisiae）表达牙鲆生长激素的载体,并研究了牙鲆生长激素基因在酿酒酵母中的表达情况。从7种重要鲽形目经济鱼类——高眼鲽（Cleisthenes herzensteini）、黄盖鲽（Limanda yokohamae）、木叶鲽（Pleuronichthys Cornutus）、宽体舌鳎（Cynoglossusrobustus）、石鲽（Platichthys bicoloratus）和条鳎（Zebrias zebra）、夏鲆（Paralichthysdentatus）的脑垂体中提取mRNA,并采用RT-PCR方法,根据已克隆得到的鲆鲽鱼类生长激素基因的保守性序列设计特异性引物,克隆了含开放阅读框的生长激素（Growth Hormone,GH）cDNA序列,测序结果显示,高眼鲽、黄盖鲽、木叶鲽、宽体舌鳎、石鲽、条鳎和夏鲆的GH cDNA长度依次为479 bp、564 bp、519 bp、440 bp、564 bp、440 bp和522 bp,编码140-170个氨基酸的成熟GH多肽片段;从漠斑牙鲆（Paralichthys lethostigma）和大菱鲆（Scophthalmus maximus）脑垂体中提取mRNA,利用SMART-RACE技术建立脑垂体cDNA文库,并从该文库中克隆出大菱鲆和漠斑牙鲆生长激素基因cDNA全长序列。测序结果表明,克隆的大菱鲆GH cDNA序列全长为876 bp,包括108 bp的5’UTR和174 bp的3’UTR序列,开放阅读框全长591 bp,编码由197氨基酸残基组成的生长激素成熟肽序列,漠斑牙鲆GH cDNA序列全长为902 bp,包括131 bp的5’UTR和198 bp的3’UTR序列,开放阅读框全长570 bp,编码由190氨基酸残基组成的生长激素成熟肽序列。运用PAUP软件对15种鲽形目鱼类与另外7种不同种属鱼类进行了分子系统进化树分析,结果与根据传统的形态学和生化特征分类进化地位基本一致,在15种鲽形目鱼类中只有大菱鲆和塞内加尔鳎各自单独形成一个分支且与鲆科和鲽科鱼类相距较远,其中塞内加尔鳎等种类的分类地位与根据线粒体DNA序列分析的系统发生模式基本吻合。说明生长激素基因可以有效地用于研究鲽形目等硬骨鱼类的亲缘关系及分类地位。通过将牙鲆生长激素基因,插入酿酒酵母（Saccharomyces cerevisiae）表面展示载体pYD1中,构建了非整合型载体pYD-GH,并在此基础上构建了三种整合型载体:适合于细胞外诱导表达的pYD-E2、细胞内诱导表达的pYD-E4和细胞内组成表达的pYD-EP4。将载体转化至酿酒酵母菌株EBY100中,得到转牙鲆生长激素酿酒酵母,对牙鲆生长激素在酿酒酵母中的表达和生物学活性进行了研究。非整合型载体pYD-GH无法在酵母细胞的传代中稳定存在而整合型载体pYD-E2、pYD-E4和pYD-EP4经过30代后的阳性检出率为100%,证明通过同源重组目的基因已经稳定整合到酵母基因组。对细胞外诱导表达的pYD-GH和pYD-E2转化酵母进行荧光标记,荧光显微镜和流式细胞仪分析外源基因表达情况,结果显示半乳糖诱导6小时的转化酵母即具有最高的荧光强度而12小时的转化酵母仍具有较高的荧光强度。经过计算,牙鲆生长激素在GAL为启动子的胞外表达工程菌E2中的表达量为1.02‰,在GAL为启动子的胞内表达工程菌E4中的表达量为1.49‰,而以PGK为启动子的胞内表达工程菌EP4的表达量为1.93‰。对表达了牙鲆生长激素的酿酒酵母进行生物活性检测,发现E2、E4和EP4在促进牙鲆生长、提高饵料转化率方面均有显著的作用,即使添加量仅有0.5%,在7周时间里体重增长分别比商品饲料对照组提高了27.16%、26.76%和31.21%,饵料转化率比对照组分别提高22.75%、22.34%和26.15%,随着酿酒酵母添加量的增加,作用更加显著,当酿酒酵母添加量达到1%,转基因酿酒酵母（E2、E4、EP4）组鱼的体重增长分别比商品饲料对照组提高了47.29%、46.87%和50.24%,饵料转化率比对照组分别提高30.65%、30.46%和32.07%。最后为评价转牙鲆生长激素基因酵母的安全性,将转基因酵母灌胃昆明种小鼠进行急性毒性试验、传统致畸试验、骨髓微核和精子畸形试验,急性毒性实验结果显示转牙鲆生长激素基因的酿酒酵母对小鼠均不具有毒性(LD₅₀＞10g/kg)。另外在传统致畸试验、骨髓微核和精子畸形试验和小鼠组织器官影响实验中三种转基因酿酒酵母的三个剂量组（2.0g/kg、5.0g/kg及10.0g/kg）与对照组比较都没有显著性差异（P＞0.05）。证明转牙鲆生长因子基因酿酒酵母对小鼠不会产生致畸和致突变作用,可能是一种安全的转基因饲料添加剂。更多还原

【Abstract】 In this paper,Growth hormone cDNAs of 9 Pleuronectiformes fishes were cloned and the phylogenetic analysis via these sequences was performed by PAUP software.Four vectors expressing flounder GH in Saccharomyces cerevisiae were constructed and studies on the transgenetic yeast were made.The mRNA of pituitary glands from seven important economic Pleuronectiformes fishes was extracted,the cDNA sequences of GH(Growth Hormone) gene were then isolated from Cleisthenes herzensteini,Limanda yokohamae,Pleuronichthys Cornutus,Cynoglossus robustus,Platichthys bicoloratus, Zebrias zebra and Paralichthys dentatus with RT-PCR method by using designed specific primers based on the reported GH cDNA sequences of Pleuronectiformes. The results of sequencing showed that lengths of the above cDNAs are as follows: 479 bp,564 bp,519 bp,440 bp,564 bp,440 bp and 522 bp.The GH cDNA sequences encode mature polypeptide of about 140 to 170 amino acid.The full-length growth hormone cDNAs of Paralichthys lethostigma and Scophthalmus maximus were cloned by Switching Mechanism At 5’ end of the RNA Transcript (SMART) RACE technology.The GH full-length cDNA of Scophthalmus maximus is 876 nucleotides long,codes for a polypeptide of 197 amino acids and the GH full-length cDNA of Paralichthys lethostigma is 902 nucleotides long,codes for a polypeptide of 190 amino acids.The deduced GH amino acid sequences of 15 Pleuronectiformes and 7 outgroup species were used for the phylogenetic analysis, which was performed by PAUP software with the maximum parsimony method.The result was consistent with morphology of Pleuronectiformes on the whole.In the topology founded,Scophthalmus maximus and Solea senegalensis formed an independent branch each other and showed far relationship with species of Bothidae and Pleuronectidae,the phylogenic position of Solea senegalensis is accorded with the reported result of analysis made by mitochondrial genome.It seems reasonable to speculate that GHs can be effectively used in analyzing genetic relationship and taxonomic status of Pleuronectiformes and other species of teleostean.The non-integrated vector pYD-GH was constructed by inserting fGH gene into yeast surface display vector pYD1.And three integration vector,E2 used for expressing on cell surface by induce,E4 used for expressing in cell by induce and EP4 used for expressing in cell not by induce.The Vectors were transformed into Saccharomyces cerevisiae strain EBY100 to expressing flounder growth hormone, then expression and biological activity of heterologous protein were studied.The non-integrated vector pYD-GH in yeast cells can not be passaged in the presence of stable,but the positive rate of integration vector pYD-E2,pYD-E4 and pYD-EP4 after 30 generations was 100%,which means fGH gene had been integrated into the yeast genome through homologous recombination.The fluorescence microscopy and flow cytometry analysis of fluorescent marked pYD-GH and pYD-E2 showed that the yeast has the highest intensity of the fluorescence after galactose induced 6 hours and after induced 12 hours the yeast still has higher fluorescence intensity.After calculation,the expression amount of yeast strain E2 with GAL promoter for extracellular expression of flounder growth hormone was 1.02‰,the expression amount of E4 with GAL promoter for intracellular expression was 1.49‰,and the expression amount of EP4 with PGK promoter for intracellular expression was 1.93‰.The detection of biological activity showed that E2,E4 and EP4 were all in prominent role of promoting growth of flounder and conversion rate of food.Even if the addition was only 0.5%,weight of flounder increased 27.16%,26.76%and 31.21%more than the control group in the seven weeks study,conversion rate of food also increased 22.75%,22.34%and 26.15 more than the control group.When addition of Saccharomyces cerevisiae was up to 1%,the weight of fish feed with transgenic Saccharomyces cerevisiae(E2,E4,EP4) increased 47.29%,46.87%and 50.24% more than the control group,conversion rate of food also increased 30.65%,30.46% and 32.07%more than the control group. Finally,acute toxicity test,the traditional teratogenic test,bone marrow and sperm abnormality micronucleus test were performed on KM mice by the oral route for evaluating safety of transgenic Saccharomyces cerevisiae.Acute toxicity test results showed that transgenic Saccharomyces cerevisiae had no toxic to mice (LD50＞10g/kg).In the traditional teratogenic test,bone marrow and sperm abnormality micronucleus test and experiments of impact in mice organs,three yeast strains of the three-dose(2.0 g/kg,5.0g/kg and 10.0 g/kg) compared to the control group were not significantly different(P＞0.05).These results suggest that Saccharomyces cerevisiae transformed with flounder growth hormone does not produce teratogenic and mutagenic effects in mice,and may be a safe transgenic feed additives.更多还原