龙须菜琼胶合成相关基因的克隆及其表达调控的研究
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摘要
龙须菜(Gracilaria/Gracilariopsis lemaneiformis)是一种大型海洋红藻。隶属于红藻门(Rhodophyta)、红藻纲(Rhodophyceae)、杉藻目(Gigartinales)、江蓠科(Gracilariaceae)。龙须菜最重要的经济意义在于其产生琼胶。琼胶是藻类的一种特殊碳储存物质。作为主要的经济型产琼胶海藻,开展龙须菜琼胶合成相关基因的研究,具有重要的理论意义和经济价值。
UDP-葡萄糖焦磷酸化酶(UGPase)基因glugp是红藻琼胶代谢过程中的一个重要酶。我们采用RT-PCR方法获得glugp cDNA的部分序列,RACE方法获得其cDNA的3'端和5'端序列,本文从基因组DNA和cDNA的水平分析了该基因的特征。glugp cDNA序列全长为2200 bp,其中包含一个1485 bp-可翻译成494个氨基酸残基的完整开放阅读框(ORF)。该基因的氨基酸序列与其它物种的UGPase氨基酸序列相似性较高。rpsBLAST分析表明在105至400个氨基酸之间存在酶活性位点和底物结合位点,其中包含5个保守的赖氨酸残基。获得的5'UTR部分包含330个碱基,5'侧翼序列的翻译初始位点序列为GCTATG,与多数红藻的RCYATG的翻译起始特征一致。3'UTR部分包含385个碱基,在终止密码子UAG之后261个碱基处存在一个3'末端多聚A前的加尾信号：ATTATT。对glugp基因序列分析发现,该基因不含有内含子,它的DNA与cDNA序列一致。通过Southern杂交结果显示该基因是单拷贝的。
本实验研究了不同琼胶含量的藻株中glugp基因的表达情况。将同株的龙须菜从基部分为2份分别培养在正常和低盐诱导条件下,2周后分别提取样品琼胶和RNA。将RNA反转录为cDNA,然后利用荧光实时定量PCR的方法检测基因的表达水平,采用2-ΔΔCT法分析基因的相对表达量。结果表明这两组样品的琼胶含量差异不显著,且glugp基因的表达量间也不存在显著差异。同一海区中,龙须菜不同藻株的琼胶含量存在差异。选取了2组琼胶含量差异显著的野生藻株,分别提取每组中藻株样品的RNA并反转录为cDNA,利用荧光实时定量PCR的方法对glugp基因的表达情况进行分析。结果表明,glugp基因的表达与琼胶含量存在显著相关性,该基因在高琼胶组藻株中的表达量显著高于低琼胶组藻株。说明glugp是琼胶合成途径中的一种重要酶。glugp基因的表达量对琼胶含量高低具有指示作用,有作为龙须菜琼胶含量的指标而应用于产业化的潜能。
对龙须菜不同培养条件和环境因子影响下的琼胶含量及生化性状进行了研究,从而筛选出诱导琼胶高产的培养条件,为筛选琼胶合成相关基因奠定基础。对野生型龙须菜的雌配子体和四分孢子体的琼胶含量进行测定。结果表明,雌配子体的琼胶含量高于四分孢子体的琼胶含量。曾有研究报道过,江蓠属和石花菜属的产琼胶红藻在低盐度、氮限制和磷限制的培养条件下,琼胶含量增加。实验设置了低盐、氮限制、磷限制和正常这四种培养条件,提取各培养条件下藻体的琼胶并对各培养条件下藻体的藻红素含量和叶绿素a含量进行测定和分析,从而确定野生型龙须菜在这些不同培养条件下的琼胶含量变化和生化性状。实验结果表明,培养一周后,各条件下藻体琼胶含量差异不大,磷限制条件下藻体琼胶含量较低。培养3周后,低盐诱导对龙须菜琼胶含量的影响显著,低盐培养的藻体琼胶含量高于正常培养条件,且低盐培养时藻体藻红素含量正常。氮限制培养条件下藻体琼胶平均含量较高,但与正常对照之间的差异不显著,藻红蛋白和叶绿素a的含量均较低。磷限制条件对藻体琼胶含量的影响并不明显,且藻红蛋白和叶绿素a的含量与对照组相似。这些结果表明,低盐培养条件可诱导龙须菜琼胶含量增加,低盐诱导条件下差异表达的基因可能包含有琼胶合成过程的重要基因。这为构建抑制消减文库,筛选与琼胶合成相关的基因奠定了基础。
Gracilaria lemaneiformis is a red macroalga which belongs to Rhodophyta, Rhodophyceae, Gigartinales, Gracilariaceae. The most economical significance of G. lemaneiformis is the agar. The metabolism of agar is a major feature of red-algal physiology. As a main agarophyte, the research on the agar correlation genes of G. lemaneiformis is very important to the theoretic study and economic values.
UDP-glucose pyrophosphorylase (UGPase) is a key enzyme in-agar metobiolism in red algae.Reverse transcription and PCR amplification were employed to obtain the part sequence of UGPase gene(glugp) of G.leimaneiformis. PCR amplification of the 3'and 5'end were performed using RACE. Putative glugp gene was characterized at the genomic DNA and cDNA levels. The gene has 2200 bp of cDNA, coding for 494 amino acids of ORF, involving 1485 bp. The deduced amino acid sequence showed significant similarity to the UGPase sequences of other species. RpsBLAST analysis indicated that UGPase has the conserved active sites between 105 and 400 amino acid, and it has five identified conserved lysine residues, that are at, or close to, the subtrate binding site.The gene have a 5'flanking region of 330 bp and a 3'flanking region of 385 bp. The 5'flanking sequence (GCTATG) conforms to the canonical sequence, RCYATG, at translation initiation sites in red algal genes so far characterized. The gene has its own special polyA signal, ATTATT, found 261 bp downstream of the stop codon site.The glugp devoid of introns and Southern blotting indicated that it has only one copy in G. lemaneiformis genome.
The relationship between gene expression level of glugp and the agar content in G.leimaneiformis were investigated. The same algae strains which divided into two parts from the holdfast were cultured in normal and low salinity conditions respectively. RNA of each sample was extracted and revere-transcribed into cDNA. Real time quantitative PCR technique was used to detect the gene expression of each sample.2'ΔΔCT data denoted for the comparative expression. The results showed that there were no statistically significant differences in the agar content of the two parts of algal samples, and glugp gene expressions of the two parts have no significant differences two. There are distinctnesses in agar content of wild type algal strains in G. lemaneiformis at the same sea area. The different algal strains with the biggest discrepancy on agar content were divided into two groups. RNA of each sample was extracted and revere-transcribed into cDNA. Real time quantitative PCR technique was used to detect the glugp expression of algal strains with different agar contents. 2-ΔΔCT data denoted for the comparative expression. The result revealed a highly correlation between glugp expression and agar contents. The expression level of glugp in high agar content strains is significantly higher than those in low, which may be applicable in indication of agar content and further algal cultivation.
The effect of some environmental factors and culture conditions on agar content and biochemical properties of G. leimaneiformis were studied in the laboratory. The selection of the high agar yielding culture condition is the foundation to screen differentially expressed genes involved in agar synthesis. The agar content of female gametophyte and tetrasporophyte of G. lemaneiformis were measured. The result shows that the agar content of algae is higer in tetrasporophyte than in female gametophyte. Agar content in Gracilaria and Gelidium has been shown to increase in nitrogen-limited cultures, phosphate-limited cultures and low salinity cultures. To study the differences in agar content, the same strain of G. leimaneiformis was treated in four conditions, including nitrogen-limited, phosphate-limited, low salinity and normal cultures. The normal culture condition is rich in nutrients. The contents of chlorophyll a and phycoerythrin in algae with different culture conditions were also detected. There were no dissimilarity in agar content after one week of experiments from nitrogen-limited, low salinity and normal culture conditions, but lower in phosphate-limited culture conditions. Increased agar productivity was observed in low salinity treatment after three weeks experiments. It was found that the most algae from the nitrogen limited condition contained higher levels of agar, but it not significantly different from the normal culture conditions. The contents of chlorophyll a and phycoerythrin were low nitrogen limited condition. Agar yielded was less dependent on the phosphate concentration after three weeks, no statistical differences in the content of chlorophyll a and phycoerythrin were observed between phosphate-limited condition and the normal.The result indicated that low salinity lead to an increase in agar content. The differentially expressed genes which induced by low salinity may be the key genes involved in the agar synthesis. The low salinity condition should be investigated as a favorable condition to construct suppressive subtractive hybridization library and obtain the related genes in agar synthesis.
UDP-葡萄糖焦磷酸化酶(UGPase)基因glugp是红藻琼胶代谢过程中的一个重要酶。我们采用RT-PCR方法获得glugp cDNA的部分序列,RACE方法获得其cDNA的3'端和5'端序列,本文从基因组DNA和cDNA的水平分析了该基因的特征。glugp cDNA序列全长为2200 bp,其中包含一个1485 bp-可翻译成494个氨基酸残基的完整开放阅读框(ORF)。该基因的氨基酸序列与其它物种的UGPase氨基酸序列相似性较高。rpsBLAST分析表明在105至400个氨基酸之间存在酶活性位点和底物结合位点,其中包含5个保守的赖氨酸残基。获得的5'UTR部分包含330个碱基,5'侧翼序列的翻译初始位点序列为GCTATG,与多数红藻的RCYATG的翻译起始特征一致。3'UTR部分包含385个碱基,在终止密码子UAG之后261个碱基处存在一个3'末端多聚A前的加尾信号：ATTATT。对glugp基因序列分析发现,该基因不含有内含子,它的DNA与cDNA序列一致。通过Southern杂交结果显示该基因是单拷贝的。
本实验研究了不同琼胶含量的藻株中glugp基因的表达情况。将同株的龙须菜从基部分为2份分别培养在正常和低盐诱导条件下,2周后分别提取样品琼胶和RNA。将RNA反转录为cDNA,然后利用荧光实时定量PCR的方法检测基因的表达水平,采用2-ΔΔCT法分析基因的相对表达量。结果表明这两组样品的琼胶含量差异不显著,且glugp基因的表达量间也不存在显著差异。同一海区中,龙须菜不同藻株的琼胶含量存在差异。选取了2组琼胶含量差异显著的野生藻株,分别提取每组中藻株样品的RNA并反转录为cDNA,利用荧光实时定量PCR的方法对glugp基因的表达情况进行分析。结果表明,glugp基因的表达与琼胶含量存在显著相关性,该基因在高琼胶组藻株中的表达量显著高于低琼胶组藻株。说明glugp是琼胶合成途径中的一种重要酶。glugp基因的表达量对琼胶含量高低具有指示作用,有作为龙须菜琼胶含量的指标而应用于产业化的潜能。
对龙须菜不同培养条件和环境因子影响下的琼胶含量及生化性状进行了研究,从而筛选出诱导琼胶高产的培养条件,为筛选琼胶合成相关基因奠定基础。对野生型龙须菜的雌配子体和四分孢子体的琼胶含量进行测定。结果表明,雌配子体的琼胶含量高于四分孢子体的琼胶含量。曾有研究报道过,江蓠属和石花菜属的产琼胶红藻在低盐度、氮限制和磷限制的培养条件下,琼胶含量增加。实验设置了低盐、氮限制、磷限制和正常这四种培养条件,提取各培养条件下藻体的琼胶并对各培养条件下藻体的藻红素含量和叶绿素a含量进行测定和分析,从而确定野生型龙须菜在这些不同培养条件下的琼胶含量变化和生化性状。实验结果表明,培养一周后,各条件下藻体琼胶含量差异不大,磷限制条件下藻体琼胶含量较低。培养3周后,低盐诱导对龙须菜琼胶含量的影响显著,低盐培养的藻体琼胶含量高于正常培养条件,且低盐培养时藻体藻红素含量正常。氮限制培养条件下藻体琼胶平均含量较高,但与正常对照之间的差异不显著,藻红蛋白和叶绿素a的含量均较低。磷限制条件对藻体琼胶含量的影响并不明显,且藻红蛋白和叶绿素a的含量与对照组相似。这些结果表明,低盐培养条件可诱导龙须菜琼胶含量增加,低盐诱导条件下差异表达的基因可能包含有琼胶合成过程的重要基因。这为构建抑制消减文库,筛选与琼胶合成相关的基因奠定了基础。
Gracilaria lemaneiformis is a red macroalga which belongs to Rhodophyta, Rhodophyceae, Gigartinales, Gracilariaceae. The most economical significance of G. lemaneiformis is the agar. The metabolism of agar is a major feature of red-algal physiology. As a main agarophyte, the research on the agar correlation genes of G. lemaneiformis is very important to the theoretic study and economic values.
UDP-glucose pyrophosphorylase (UGPase) is a key enzyme in-agar metobiolism in red algae.Reverse transcription and PCR amplification were employed to obtain the part sequence of UGPase gene(glugp) of G.leimaneiformis. PCR amplification of the 3'and 5'end were performed using RACE. Putative glugp gene was characterized at the genomic DNA and cDNA levels. The gene has 2200 bp of cDNA, coding for 494 amino acids of ORF, involving 1485 bp. The deduced amino acid sequence showed significant similarity to the UGPase sequences of other species. RpsBLAST analysis indicated that UGPase has the conserved active sites between 105 and 400 amino acid, and it has five identified conserved lysine residues, that are at, or close to, the subtrate binding site.The gene have a 5'flanking region of 330 bp and a 3'flanking region of 385 bp. The 5'flanking sequence (GCTATG) conforms to the canonical sequence, RCYATG, at translation initiation sites in red algal genes so far characterized. The gene has its own special polyA signal, ATTATT, found 261 bp downstream of the stop codon site.The glugp devoid of introns and Southern blotting indicated that it has only one copy in G. lemaneiformis genome.
The relationship between gene expression level of glugp and the agar content in G.leimaneiformis were investigated. The same algae strains which divided into two parts from the holdfast were cultured in normal and low salinity conditions respectively. RNA of each sample was extracted and revere-transcribed into cDNA. Real time quantitative PCR technique was used to detect the gene expression of each sample.2'ΔΔCT data denoted for the comparative expression. The results showed that there were no statistically significant differences in the agar content of the two parts of algal samples, and glugp gene expressions of the two parts have no significant differences two. There are distinctnesses in agar content of wild type algal strains in G. lemaneiformis at the same sea area. The different algal strains with the biggest discrepancy on agar content were divided into two groups. RNA of each sample was extracted and revere-transcribed into cDNA. Real time quantitative PCR technique was used to detect the glugp expression of algal strains with different agar contents. 2-ΔΔCT data denoted for the comparative expression. The result revealed a highly correlation between glugp expression and agar contents. The expression level of glugp in high agar content strains is significantly higher than those in low, which may be applicable in indication of agar content and further algal cultivation.
The effect of some environmental factors and culture conditions on agar content and biochemical properties of G. leimaneiformis were studied in the laboratory. The selection of the high agar yielding culture condition is the foundation to screen differentially expressed genes involved in agar synthesis. The agar content of female gametophyte and tetrasporophyte of G. lemaneiformis were measured. The result shows that the agar content of algae is higer in tetrasporophyte than in female gametophyte. Agar content in Gracilaria and Gelidium has been shown to increase in nitrogen-limited cultures, phosphate-limited cultures and low salinity cultures. To study the differences in agar content, the same strain of G. leimaneiformis was treated in four conditions, including nitrogen-limited, phosphate-limited, low salinity and normal cultures. The normal culture condition is rich in nutrients. The contents of chlorophyll a and phycoerythrin in algae with different culture conditions were also detected. There were no dissimilarity in agar content after one week of experiments from nitrogen-limited, low salinity and normal culture conditions, but lower in phosphate-limited culture conditions. Increased agar productivity was observed in low salinity treatment after three weeks experiments. It was found that the most algae from the nitrogen limited condition contained higher levels of agar, but it not significantly different from the normal culture conditions. The contents of chlorophyll a and phycoerythrin were low nitrogen limited condition. Agar yielded was less dependent on the phosphate concentration after three weeks, no statistical differences in the content of chlorophyll a and phycoerythrin were observed between phosphate-limited condition and the normal.The result indicated that low salinity lead to an increase in agar content. The differentially expressed genes which induced by low salinity may be the key genes involved in the agar synthesis. The low salinity condition should be investigated as a favorable condition to construct suppressive subtractive hybridization library and obtain the related genes in agar synthesis.
引文
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