金鱼PR55/B家族基因cDNA的克隆及表达分析
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摘要
蛋白质的可逆磷酸化修饰,是生物体内普遍存在的信号传导调节方式。蛋白磷酸酶行使的去磷酸化作用在调控细胞内各种生命过程中起了重要的作用。PP2A是主要的丝／苏氨酸蛋白磷酸酶之一,它参与DNA复制、转录、细胞周期调控、信号转导等生命活动。PR55／B家族是PP2A已知的调节亚基家族之一。
本文运用RACE和RT-PCR技术克隆得到金鱼PP2A调节亚基PR55／B家族中PR55δ,PR55β基因全长序列以及PR55γ基因编码区部分序列。结果显示PR55δ基因全长2163bp,其中编码区1347bp编码,推测编码448aa的序列。PR55β基因全长1940bp,其中编码区1332bp编码,推测编码443aa的序列。PR55γ基因部分cDNA长1218bp,编码的多肽共含405个氨基酸。通过序列分析发现PR55／B家族各亚基基因编码的蛋白与已知其他物种对应的蛋白质均有着很高的同源性。通过系统发育树的构建发现其系统发育关系与传统的分类基本一致,符合脊椎动物的进化规律,暗示了该家族基因在进化上和功能上的保守性。但是PR55δ和PR55β在氨基末端有显著差异,可能与各调节亚基功能,如决定底物特异性和细胞内定位有关。运用ExPASy和SMART等序列分析软件在PR55δ、PR55β、PR55γ三个基因推测的蛋白质中分别找到了WD40重复序列结构。PR55／B家族成员在WD40重复序列上的保守性使我们推测这一结构作为与PP2A结构亚基A相结合的区域,为PR55／B家族调节亚基参与PP2A三聚体的装配提供了支点。
在组织表达方面,金鱼PR55／B家族基因呈现明显的差异性表达。PR55δ基因的mRNA和蛋白水平都在各个组织中呈现广泛但不均一的表达。PR55β基因的mRNA和蛋白表达仅在大脑和心脏中检测到。PR55γ基因mRNA特异表达于在大脑和鳍中。其中,PR55β基因在心脏中的表达和PR55y基因在鳍中的表达是第一次报道。而且金鱼(低等脊椎动物)PR55／B在组织中的表达模式与小鼠(高等脊椎动物)有显著的差异。这也提示我们PR55β基因可能在鱼类(低等脊椎动物)心脏的发育和心脏组织行驶功能时具有非常重要的作用。而PR55γ基因在调控金鱼鳍的发育和功能方面也可能具有不可替代的作用。
在金鱼胚胎发育过程中,PR55δ基因的mRNA和蛋白以及PR55p的mRNA基因在胚胎发育的各个时期都有较高表达。但是,我们没有检测到PR55β基因在蛋白水平上的表达。PR55δ和PR55β基因在金鱼胚胎中的表达模式与小鼠胚胎中的表达模式又呈现了明显的差异,它们可能在调节不同物种的发育中有着不同的作用。PR55y基因的mRNA在胚胎发育早期没有表达,直到神经胚期才开始出现,随后呈现逐渐升高的表达模式。PR55γ基因从神经胚开始表达的特点让我们推测它在神经发育系统中的重要作用。
另外,我们设计构建PR55δ基因反义表达载体,通过显微注射到金鱼胚胎来抑制PR55δ基因的表达,观察其对胚胎发育的影响。结果表明,当PR55δ基因表达下调后,金鱼胚胎出现了不正常的组织器官分化。脑和眼的发育受到了影响,表现为无脑、脑发育缓慢、无眼、小眼等。这些结果证明了PP2A调节亚基直接控制着组织器官生成,PR55δ基因对于金鱼胚胎发育是至关重要的。
综上所述,我们的结果表明PR55／B家族在可能在金鱼不同组织和胚胎发育的不同时期过程中起着多种重要作用。
The reversible phosphorylation of proteins is an important posttranslational modification in eukaryotes. Phosphatases play a key role in various cellular process by dephosphorylation. Protein phosphatase 2A (PP2A) is one of the major serine/threonine-specific phosphatases and involved in many cellular processes such as cell cycle progression, DNA replication, transcription, and signal transduction. PR55/B family is one of identified regulatory B subunits of PP2A.
Using 3'-and 5'-RACE,PR55δ, PR55βand partial PR55γof PR55/B family of PP2A were cloned from goldfish. The full length of PR55δcDNA is 2163 bp, containing an open reading frame (ORF) of 1347 that encodes a deduced protein of 448 amino acids. Full-length PR55βconsists of 1940 bp with an ORF of 1332 nucleotides coding for a deduced protein of 443 amino acids. The partial PR55γcontains 1218 nucleotides, which encodes a deduced part protein of 405 amino acids. Sequence analysis showed that goldfishβ,δandγisoforms of PR55/B family show high identities with their counterparts in other species, thus suggesting the conservation of PR55/B family members through animal kingdom. The PR55/B phylogenetic tree based on the amino acid sequences from various species fits well with the traditional phylogenetic tree. Among the three isoforms of PR55/B family members, the N-termini were significantly divergent between PR55δand PR55β, suggesting that a short, divergent stretch of amino acids may confer specific targeting information. Amino acid sequence analysis through ExPASy and other program revealed that the WD 40 tandem repeats were conserved in PR55δ, PR55βand PR55γisoforms. It is predicted that this domain is important for their binding to the PP2A-A subunits.
RT-PCR and Western blot analysis showed that PR55βgene could be only detected in brain and heart, whereas the PR55δwas expressed ubiquitously but non-uniformly in 9 tissues examined. And PR55γgene mRNA specifically expressed in brain and fin. Our demonstration of the PR55βmRNA in heart and the PR55γmRNA in fin provides the first evidence regarding their tissue-specific expression patterns. In addition, goldfish (lower vertebrates) and murine (higher vertebrates) displayed distinct difference in the tissue-specific expression patterns of PR55δ/β/γ. Our results suggest that PR55βmay play an important role in heart and PR55γin fin.
The PR55δis highly expressed at both mRNA and protein levels from early to later developmental stages of goldfish. The PR55βmRNA was also detected in goldfish embryo, but we rarely detected any PR55βprotein expression at the 12 different developmental stages examined. Distinct difference exists in the temporal expression patterns of the PR55δ&βbetween lower (goldfish) and higher vertebrates (murine), which suggests that PR55δ/βmay play different roles in regulating the developmental processes of the two types of vertebrates. The PR55γexpression was first detected at neurula stage, and then gradually enhanced. This expression pattern suggests that PR55γmay be important for development of the nervous system.
To explore PR55δfunction in regulating development, we expressed the anti-sense RNA from the exogenous PR55δcDNA which can block translation of the PR55δmRNA from the endogenous PR55δgene. When PR55δwas downregulated, the development of goldfish embryos displayed severe abnormality in organogenesis. We observed that during brain differentiation stage, the expression of the antisense PR55δRNA led to absence of a differentiated brain, while the expression of the vector (mock) had little effect on the brain development. In addition, the eye development was also interrupted, displaying microphthalmia in the embryos with reduced PR55δexpression. These results provide the first evidence that the regulatory subunit of PP2A directly controls organogenesis. Our demonstration that downregulation of PR55δby anti-sense RNA led to severe abnormality in the brain and eye development suggests that the specific PP2A activity contributed by PR55δregulatory subunit is crucial for development. In this case, the PP2A containing PR55δregulatory subunit may modulate a set of specific targets important for development that can't be dephosphorylated by PP2A with non-PR55δregulatory subunit.
Together, our results suggested that PR55/B family members have versatile functions during goldfish development and tissue homeostasis.
本文运用RACE和RT-PCR技术克隆得到金鱼PP2A调节亚基PR55／B家族中PR55δ,PR55β基因全长序列以及PR55γ基因编码区部分序列。结果显示PR55δ基因全长2163bp,其中编码区1347bp编码,推测编码448aa的序列。PR55β基因全长1940bp,其中编码区1332bp编码,推测编码443aa的序列。PR55γ基因部分cDNA长1218bp,编码的多肽共含405个氨基酸。通过序列分析发现PR55／B家族各亚基基因编码的蛋白与已知其他物种对应的蛋白质均有着很高的同源性。通过系统发育树的构建发现其系统发育关系与传统的分类基本一致,符合脊椎动物的进化规律,暗示了该家族基因在进化上和功能上的保守性。但是PR55δ和PR55β在氨基末端有显著差异,可能与各调节亚基功能,如决定底物特异性和细胞内定位有关。运用ExPASy和SMART等序列分析软件在PR55δ、PR55β、PR55γ三个基因推测的蛋白质中分别找到了WD40重复序列结构。PR55／B家族成员在WD40重复序列上的保守性使我们推测这一结构作为与PP2A结构亚基A相结合的区域,为PR55／B家族调节亚基参与PP2A三聚体的装配提供了支点。
在组织表达方面,金鱼PR55／B家族基因呈现明显的差异性表达。PR55δ基因的mRNA和蛋白水平都在各个组织中呈现广泛但不均一的表达。PR55β基因的mRNA和蛋白表达仅在大脑和心脏中检测到。PR55γ基因mRNA特异表达于在大脑和鳍中。其中,PR55β基因在心脏中的表达和PR55y基因在鳍中的表达是第一次报道。而且金鱼(低等脊椎动物)PR55／B在组织中的表达模式与小鼠(高等脊椎动物)有显著的差异。这也提示我们PR55β基因可能在鱼类(低等脊椎动物)心脏的发育和心脏组织行驶功能时具有非常重要的作用。而PR55γ基因在调控金鱼鳍的发育和功能方面也可能具有不可替代的作用。
在金鱼胚胎发育过程中,PR55δ基因的mRNA和蛋白以及PR55p的mRNA基因在胚胎发育的各个时期都有较高表达。但是,我们没有检测到PR55β基因在蛋白水平上的表达。PR55δ和PR55β基因在金鱼胚胎中的表达模式与小鼠胚胎中的表达模式又呈现了明显的差异,它们可能在调节不同物种的发育中有着不同的作用。PR55y基因的mRNA在胚胎发育早期没有表达,直到神经胚期才开始出现,随后呈现逐渐升高的表达模式。PR55γ基因从神经胚开始表达的特点让我们推测它在神经发育系统中的重要作用。
另外,我们设计构建PR55δ基因反义表达载体,通过显微注射到金鱼胚胎来抑制PR55δ基因的表达,观察其对胚胎发育的影响。结果表明,当PR55δ基因表达下调后,金鱼胚胎出现了不正常的组织器官分化。脑和眼的发育受到了影响,表现为无脑、脑发育缓慢、无眼、小眼等。这些结果证明了PP2A调节亚基直接控制着组织器官生成,PR55δ基因对于金鱼胚胎发育是至关重要的。
综上所述,我们的结果表明PR55／B家族在可能在金鱼不同组织和胚胎发育的不同时期过程中起着多种重要作用。
The reversible phosphorylation of proteins is an important posttranslational modification in eukaryotes. Phosphatases play a key role in various cellular process by dephosphorylation. Protein phosphatase 2A (PP2A) is one of the major serine/threonine-specific phosphatases and involved in many cellular processes such as cell cycle progression, DNA replication, transcription, and signal transduction. PR55/B family is one of identified regulatory B subunits of PP2A.
Using 3'-and 5'-RACE,PR55δ, PR55βand partial PR55γof PR55/B family of PP2A were cloned from goldfish. The full length of PR55δcDNA is 2163 bp, containing an open reading frame (ORF) of 1347 that encodes a deduced protein of 448 amino acids. Full-length PR55βconsists of 1940 bp with an ORF of 1332 nucleotides coding for a deduced protein of 443 amino acids. The partial PR55γcontains 1218 nucleotides, which encodes a deduced part protein of 405 amino acids. Sequence analysis showed that goldfishβ,δandγisoforms of PR55/B family show high identities with their counterparts in other species, thus suggesting the conservation of PR55/B family members through animal kingdom. The PR55/B phylogenetic tree based on the amino acid sequences from various species fits well with the traditional phylogenetic tree. Among the three isoforms of PR55/B family members, the N-termini were significantly divergent between PR55δand PR55β, suggesting that a short, divergent stretch of amino acids may confer specific targeting information. Amino acid sequence analysis through ExPASy and other program revealed that the WD 40 tandem repeats were conserved in PR55δ, PR55βand PR55γisoforms. It is predicted that this domain is important for their binding to the PP2A-A subunits.
RT-PCR and Western blot analysis showed that PR55βgene could be only detected in brain and heart, whereas the PR55δwas expressed ubiquitously but non-uniformly in 9 tissues examined. And PR55γgene mRNA specifically expressed in brain and fin. Our demonstration of the PR55βmRNA in heart and the PR55γmRNA in fin provides the first evidence regarding their tissue-specific expression patterns. In addition, goldfish (lower vertebrates) and murine (higher vertebrates) displayed distinct difference in the tissue-specific expression patterns of PR55δ/β/γ. Our results suggest that PR55βmay play an important role in heart and PR55γin fin.
The PR55δis highly expressed at both mRNA and protein levels from early to later developmental stages of goldfish. The PR55βmRNA was also detected in goldfish embryo, but we rarely detected any PR55βprotein expression at the 12 different developmental stages examined. Distinct difference exists in the temporal expression patterns of the PR55δ&βbetween lower (goldfish) and higher vertebrates (murine), which suggests that PR55δ/βmay play different roles in regulating the developmental processes of the two types of vertebrates. The PR55γexpression was first detected at neurula stage, and then gradually enhanced. This expression pattern suggests that PR55γmay be important for development of the nervous system.
To explore PR55δfunction in regulating development, we expressed the anti-sense RNA from the exogenous PR55δcDNA which can block translation of the PR55δmRNA from the endogenous PR55δgene. When PR55δwas downregulated, the development of goldfish embryos displayed severe abnormality in organogenesis. We observed that during brain differentiation stage, the expression of the antisense PR55δRNA led to absence of a differentiated brain, while the expression of the vector (mock) had little effect on the brain development. In addition, the eye development was also interrupted, displaying microphthalmia in the embryos with reduced PR55δexpression. These results provide the first evidence that the regulatory subunit of PP2A directly controls organogenesis. Our demonstration that downregulation of PR55δby anti-sense RNA led to severe abnormality in the brain and eye development suggests that the specific PP2A activity contributed by PR55δregulatory subunit is crucial for development. In this case, the PP2A containing PR55δregulatory subunit may modulate a set of specific targets important for development that can't be dephosphorylated by PP2A with non-PR55δregulatory subunit.
Together, our results suggested that PR55/B family members have versatile functions during goldfish development and tissue homeostasis.
引文
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