嗜热真菌Thermomyces lanuginosus丝/苏氨酸蛋白激酶的基因克隆和序列分析
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摘要
信号转导是外界同生物体或生物体细胞间的信息传递的重要手段。许多研究证实，生物信号在细胞内传递的基本和主要的方式就是蛋白激酶和蛋白磷酸酶催化的蛋白质可逆磷酸化。通过磷酸化和去磷酸化作用，蛋白质发生构型和构象的变化，从而活性受到调节，通过激酶级联信号传导途径将信号传导到细胞核，通过对转录因子和离子通道蛋白的调控而调节基因的表达。
Thermomyces lanuginosus是一类可以在50℃培养条件下正常生长的真菌。印度科学家Ray（1994）等发现在Pseudomonas syringae细胞膜上存在三个分子量不同的蛋白，这些蛋白在细菌感受外界温度变化时发生磷酸化作用，并猜测其中的一个是组氨酸蛋白激酶。本研究拟通过RT-PCR的方法结合RACE获得嗜热真菌的一个蛋白激酶基因，并研究其在信号传导中的作用。
本实验通过RT－PCR的方法获得了一个长1243bp的cDNA片断。编码一个长365个氨基酸的序列，长度和酵母的dsk1激酶相似，其推断的氨基酸和裂殖酵母dsk1激酶的同源性达到60％以上。利用DNAman软件进行分析发现，在推断的3'氨基酸序列中存在着Ⅵ、Ⅶ、Ⅷ、Ⅸ、Ⅹ和Ⅺ保守亚区。进行核苷酸比较发现和其他基因的核苷酸同源性很差（不到10％），同源区域长度不到40nt；GC％含量比其他物种要高（达到56％），表现出一定的物种特异性。对比其密码子偏好性，GC%含量达到53%，作者认为如此高的GC％含量可能是该真菌在50℃的较高温度下正常生长的遗传基础。随后通过5'RACE进行的5'末端的克隆，因为未知区域的长度过长而没有得到5'末端。利用Southern blot对cDNA片断进行拷贝数鉴定发现，在嗜热真菌T. lanuginosus的基因组里，该序列可能是单拷贝的。
本实验通过网络信息资源对已经得到的cDNA片断进行了二级结构疏水性区域、跨膜区域和功能域等基本性质进行了探讨，对其编码的蛋白质的三维结构进行了预测。Interpro程序分析认为，推断的氨基酸序列和PKA/PKG激酶相同的激酶磷酸化位点是39和254位氨基酸残基；和PKC
相同的磷酸化位点为61、124、215、229、253和353位氨基酸残基；和Casein 蛋白激酶II型激酶的相同的磷酸化位点为353位氨基酸残基。氨基酸2-270组成了类似激酶区域。经过PHD预测，我们可知此序列形成的主要功能域可以形成5个螺旋和5个β折叠。通过Swiss－pdbViewer程序观察到预测的蛋白质3D结构3个螺旋， 1个β折叠和3个β－strand。
通过对cDNA的C端的二级结构进行预测，发现其保守的功能区域具有和激酶类似的特征。
All living cells must constantly be interfered by all kinds of factors during their life cycle of proliferation, differentiation, development and death. These factors include not only the intercellular hormones but also extracellular light, temperature and water so on. All these request the cell to recognize and receive these signal factors correctly and respond to them as soon as possible to control the courses of progression and cellular activities. In this way, the receivers lie in the membrane of the cell recognizing the external ligand. Then the external signals are transducted into the nucleolus to regulate the gene expression and enzyme activity leading to the cell response. We can conclude that signal transduction plays an important role in the processes of environment-cell interaction.
A lot of studies have resulted that kinases and phosphatases are likely to be important mediators of signal transduction. Through phosphrylation and dephosphorylation, the activity and structure of protein changed. External signal are transducted into the cell nucleolus by the signal cascade. Protein kinase mediated phosphorylation regulates protein function, membrane channels and pumps and transcription factors by which changed the gene expression. Indian scientist Ray found in 1994 that three membrane proteins they isolated from Pseudomonas syringae which were phosphorylated in response to temperature changes and resulted that one of these proteins was possibly a histidine kinase. Thermomyces lanuginosus is a kind of heat-endurable fungus which can live normally at about 50℃.In order to probe the molecular mechanism of the characteristics of heat-enduration we have been trying to clone the gene of a protein kinase and looking for the molecules upstream and downstream of it. Using RT-PCR, one 1243bp cDNA fragment was obtained. Sequencing analysis showed that the cloned cDNA of T. lanuginosus was similar to that of fission yeast protein kinase dsk. Comparison results showed that the homogeneity of the 3'-deduced amino acids was above 60%. Six conservative amino acid subdomains which most ser/thr protein kinase contain can be found in the deduced amino acid sequence. Comparison of the necleotide sequence resulted that the homogeneity is not high(10%). Homogeneity domains is as short as 40nt. However, we can find that GC% of the cDNA is higher (about 56%), which showed an organism specificity. Looking into the preference codons of T. lanuginosus , we found the average value of GC% is about 53%. We surmise that such a high GC% value may be important factor for this kind of fungus to
live normally at about 50℃. We tried to obtain the 5'-end of the cDNA by 5'RACE(rapid amplification of cDNA ends). Unfortunately ,due to the complexity of 5'RACE and use of a single gene-specific primer(with the anchor primer),the length of 5'-end fragment we estimated is too long to be captured. We failed to isolate the 5'-ends of the cDNA. Southern blot analysis showed that T. lanuginosus genome may contain one homology of the cDNA fragment.
Bioinformatics is pushed ahead quickly because of the internet development. Using the internet information to explore the distinction of life becomes one major research field. Human genome project was carried out and brought rapid growth to bioinformation. All these make it possible to use bioinformation for gene cloning, sequence analysis and protein function prediction. We used the bioinformation in internet to predict the transmembane domain, secondary structure and 3D structure for the deduced amino acid sequence of the cDNA fragment.
Thermomyces lanuginosus是一类可以在50℃培养条件下正常生长的真菌。印度科学家Ray（1994）等发现在Pseudomonas syringae细胞膜上存在三个分子量不同的蛋白，这些蛋白在细菌感受外界温度变化时发生磷酸化作用，并猜测其中的一个是组氨酸蛋白激酶。本研究拟通过RT-PCR的方法结合RACE获得嗜热真菌的一个蛋白激酶基因，并研究其在信号传导中的作用。
本实验通过RT－PCR的方法获得了一个长1243bp的cDNA片断。编码一个长365个氨基酸的序列，长度和酵母的dsk1激酶相似，其推断的氨基酸和裂殖酵母dsk1激酶的同源性达到60％以上。利用DNAman软件进行分析发现，在推断的3'氨基酸序列中存在着Ⅵ、Ⅶ、Ⅷ、Ⅸ、Ⅹ和Ⅺ保守亚区。进行核苷酸比较发现和其他基因的核苷酸同源性很差（不到10％），同源区域长度不到40nt；GC％含量比其他物种要高（达到56％），表现出一定的物种特异性。对比其密码子偏好性，GC%含量达到53%，作者认为如此高的GC％含量可能是该真菌在50℃的较高温度下正常生长的遗传基础。随后通过5'RACE进行的5'末端的克隆，因为未知区域的长度过长而没有得到5'末端。利用Southern blot对cDNA片断进行拷贝数鉴定发现，在嗜热真菌T. lanuginosus的基因组里，该序列可能是单拷贝的。
本实验通过网络信息资源对已经得到的cDNA片断进行了二级结构疏水性区域、跨膜区域和功能域等基本性质进行了探讨，对其编码的蛋白质的三维结构进行了预测。Interpro程序分析认为，推断的氨基酸序列和PKA/PKG激酶相同的激酶磷酸化位点是39和254位氨基酸残基；和PKC
相同的磷酸化位点为61、124、215、229、253和353位氨基酸残基；和Casein 蛋白激酶II型激酶的相同的磷酸化位点为353位氨基酸残基。氨基酸2-270组成了类似激酶区域。经过PHD预测，我们可知此序列形成的主要功能域可以形成5个螺旋和5个β折叠。通过Swiss－pdbViewer程序观察到预测的蛋白质3D结构3个螺旋， 1个β折叠和3个β－strand。
通过对cDNA的C端的二级结构进行预测，发现其保守的功能区域具有和激酶类似的特征。
All living cells must constantly be interfered by all kinds of factors during their life cycle of proliferation, differentiation, development and death. These factors include not only the intercellular hormones but also extracellular light, temperature and water so on. All these request the cell to recognize and receive these signal factors correctly and respond to them as soon as possible to control the courses of progression and cellular activities. In this way, the receivers lie in the membrane of the cell recognizing the external ligand. Then the external signals are transducted into the nucleolus to regulate the gene expression and enzyme activity leading to the cell response. We can conclude that signal transduction plays an important role in the processes of environment-cell interaction.
A lot of studies have resulted that kinases and phosphatases are likely to be important mediators of signal transduction. Through phosphrylation and dephosphorylation, the activity and structure of protein changed. External signal are transducted into the cell nucleolus by the signal cascade. Protein kinase mediated phosphorylation regulates protein function, membrane channels and pumps and transcription factors by which changed the gene expression. Indian scientist Ray found in 1994 that three membrane proteins they isolated from Pseudomonas syringae which were phosphorylated in response to temperature changes and resulted that one of these proteins was possibly a histidine kinase. Thermomyces lanuginosus is a kind of heat-endurable fungus which can live normally at about 50℃.In order to probe the molecular mechanism of the characteristics of heat-enduration we have been trying to clone the gene of a protein kinase and looking for the molecules upstream and downstream of it. Using RT-PCR, one 1243bp cDNA fragment was obtained. Sequencing analysis showed that the cloned cDNA of T. lanuginosus was similar to that of fission yeast protein kinase dsk. Comparison results showed that the homogeneity of the 3'-deduced amino acids was above 60%. Six conservative amino acid subdomains which most ser/thr protein kinase contain can be found in the deduced amino acid sequence. Comparison of the necleotide sequence resulted that the homogeneity is not high(10%). Homogeneity domains is as short as 40nt. However, we can find that GC% of the cDNA is higher (about 56%), which showed an organism specificity. Looking into the preference codons of T. lanuginosus , we found the average value of GC% is about 53%. We surmise that such a high GC% value may be important factor for this kind of fungus to
live normally at about 50℃. We tried to obtain the 5'-end of the cDNA by 5'RACE(rapid amplification of cDNA ends). Unfortunately ,due to the complexity of 5'RACE and use of a single gene-specific primer(with the anchor primer),the length of 5'-end fragment we estimated is too long to be captured. We failed to isolate the 5'-ends of the cDNA. Southern blot analysis showed that T. lanuginosus genome may contain one homology of the cDNA fragment.
Bioinformatics is pushed ahead quickly because of the internet development. Using the internet information to explore the distinction of life becomes one major research field. Human genome project was carried out and brought rapid growth to bioinformation. All these make it possible to use bioinformation for gene cloning, sequence analysis and protein function prediction. We used the bioinformation in internet to predict the transmembane domain, secondary structure and 3D structure for the deduced amino acid sequence of the cDNA fragment.
引文
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