白芷细胞外钙调素结合蛋白（ECBP21）cDNA克隆及鉴定
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摘要
目前，对植物Ca~(2+)信号系统各组分(Ca~(2+)、CaM和CaMBPs等)的研究不断取得更为深入的成果。在植物细胞内鉴定了许多CaM调节或结合的靶酶或靶蛋白(CaBMPs)，并证明它们在Ca~(2+)-CaM信号转导途径中发挥重要作用，参与了许多生命活动过程。我室多年的研究证实植物细胞外也普遍存在CaM，并且可能作为一种多功能肽类信使在植物生长发育过程中发挥重要作用。这已成为植物钙调素研究的最新进展之一。
我室在对植物细胞外CaM的研究过程中，发现细胞外还可能存在CaMBPs，并首次从白芷悬浮培养细胞外纯化了一种依赖Ca~(2+)的、分子量约为21kD的细胞外CaMBP(Tang等，1996)(定名为：ECBP21)。生理实验初步表明ECBP21影响白芷悬浮培养细胞的增殖(毛国红等，1999)。这些生物化学和生理学实验，仅为细胞外CaMBPs的存在及可能的功能提供了初步证据。为了进一步利用分子生物学手段证实细胞外ECBP21真实存在，并进一步研究其生物学功能，本实验从克隆基因入手进行了以下工作：
1．ECBP21全长cDNA克隆：将纯化的ECBP21蛋白经SDS—PAGE分离后电转移到PVDF膜上，氨基酸测序获得N-末端20个氨基酸序列；根据N-末端8个氨基酸序列合成兼并性引物，通过RT-PCR和5’-RACE方法，获得其cDNA序列。Northern blot分析表明克隆的cDNA序列为全长cDNA。ECBP21 cDNA全长947bp，编码216个氨基酸，序列分析显示其N端1-25氨基酸为信号肽，26-216氨基酸为成熟蛋白肽段，并且不具有跨膜结构域。将此cDNA成熟蛋白编码区核苷酸片断构建到pET-28b(+)表达载体，在大肠杆菌表达系统中诱导了该蛋白的表达，经CaM-gel overlay检测证实表达蛋白具有Ca~(2+)依赖的CaM结合特性，从而进一步证实了ECBP21为一种Ca~(2+)依赖的CaMBP。通过缺失表达分析实验证实CaMBD结构域位于ECBP21的羧基端。Southern blot检测表明白芷基因组中可能存在两个ECBP21基因拷贝。从而首次克隆并鉴定了一个细胞外CaMBP的全长cDNA。
2．ECBP21亚细胞定位：以重组ECBP21蛋白为抗原制备了特异性抗体。利用特异性抗体通过免疫金标定位研究表明ECBP21主要分布于细胞壁。进一步利用GFP荧光标记，通过瞬时表达方法研究显示转ECBP21-GFP
毛国红:白芷细胞外钙调素结合蛋白(ECBPZI)cDNA克隆及鉴定
融合基因细胞的细胞壁上有较强绿色荧光，而转缺失信号肤的
乙去乙岁苍介分户融合基因及贷尹基因的细胞中，细胞壁上无绿色荧光，表
明ECBP21是一种主动分泌到细胞外的分泌蛋白。由此进一步证实ECBP21
确为一种质外体多肤。本实验为细胞外CaMBPS的存在提供了直接证据。
3.ECBP21表达分析:Northern blot分析表明:在白芷悬浮培养细
胞生长周期中，ECBP21和CaM都为组成型表达，0一12天ECBP21表达量
逐渐增加，第12天达到最高，随后12一21天，ECBP21表达量稍有下降，
维持在一个比较稳定状态;而O一6天时，CaM表达量逐渐上升，第6一9
天达到最大表达量，随后，9一21天，CaM表达量有所下降。表明ECBP21
可能参与白芷悬浮培养细胞生长周期调控。热激、渗透、盐胁迫及JA处
理时，ECBPZI表达量下降，而6一BA、ABA及GA3处理时，不影响ECBPZI
的表达。推测ECBP21还可能与逆境胁迫相关。
4.初步开展了转基因研究:为了进一步确定ECBP21在植物生长发
育过程中的作用，构建了ECBP21植物表达二元载体，通过真空渗透法转
化了拟南芥并获得了转基因植株。表型观察仍在进行之中。
总之，通过上述研究我们首次克隆及鉴定了一种细胞外CaMBP
(EC即21) CDNA，并首次提供了细胞外存在CaMBPS的直接证据。本实验
结果也为利用分子生物学方法研究ECBP21的生物学功能奠定了基础，并
将有助于阐明胞外 CaM的作用机制及丰富植物质外体多肤的研究。
In recent years, Ca~(2+) signal system (Ca~(2+) , CaM, and CaMBPs) has been thoroughly researched. Many intracellular CaMBPs have been detected and studied. In our laboratory, a series of experiments has provided evidence that the presence of CaM in the apoplast area is universal, and extracellular calmodulin plays an important role in the plant growth and development as a polypeptide signal. This has become one of the latest progresses in plant CaM research work.
In the process of study for the extracellular CaM, the research in our lab indicates that CaMBPs also possibly exist in cell wall area. For the first time, an extracellular Ca~(2+)-dependent CaMBP (named: ECBP21) was purified from 0.1mol/L CaCl_(2) extracts of Angelica dahurica suspension-cultured cells (Tang et al. 1996). Furthermore, our preliminary evidence shows that the ECBP21 protein influences on proliferation of this cultured cell (Mao et al. 1999). By using methods of biochemistry and physiology, the above experiments only provided preliminary evidence for the existence and possible functions of the extracellular CaMBPs. In order to prove the real existence of the extracellular ECBP21 and its functions by means of molecular biology, we first cloned and identified the full-length cDNA for an extracellular CaMBP (ECBP21).
The purified protein was electrobloted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5 "RACE cloning. The cDNA contains 947 nucleotides that codes for a precursor protein of 216 amino acids. Sequence analysis shows that the N-terminal 1-25 amino acid sequence is a predicted signal peptide and other 26-216 amino acid sequence is a mature peptide which has no transmembrane domain. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E.coli BL21(DE3). A protein with relative molecular mass 21kD was expressed in E.coli. Using a biotinylated-CaM gel overlay technique, the expression protein has the ability to bind CaM in the presence of Ca~(2+). These
results further defined ECBP21is a Ca~(2+)-dependent CaM-binding protein. Following researches verified that the CaM-binding domain exists in the C-terminal of ECBP21. Southern blot analysis shows that there are two ECBP21 gene copies in genome.
Second, The ECBP21 was localized in the cell wall area by immuno-gold electron microscopy and by using GFP as a reporter. The results indicted that ECBP21 is a kind of secretive protein and ECBP21 is an apoplast polypeptide. These experiments provided the first and direct evidence for the existence of the extracellular CaMBPs.
Third, we examined the expression of ECBP21 dynamics during the incubation of Angelica suspension-cultured cells and stress treatments through Northern blot analysis. During the incubation of Angelica suspension-cultured cells, the expression of ECBP21 gene increased from 0 to 12th day, and it reached a peak on the twelfth day. While the expression of CaM gene increased from 0 to 9th day, and it reached a peak during the 6th-9th day. These results indicated that ECBP21 may involve in the regulation of cell growth. The stress treatments (such as: heat shock, osmotic and salt stresses) and JA (100uM) treatment repressed the expression of ECBP21 gene. They indicated that ECBP21 may have some relative with the response to stress treatments.
Further more, we constructed the binary vector containing ECBP21-GFP chimeric gene and got transgenic plants by using the Agrobacterium vacuum infiltration method. The phenotype observation is still in progress.
Thought the above studies, we first cloned and identified an extracellular CaMBP (ECBP21) cDNA, and provided direct evidence for the existence of the extracellular CaMBPs. This work laid a foundation for elucidating the functions of ECBP21 by means of molecular biology. Further studies about ECBP21 may be benefit to elucidating the mechanism of apoplast calmodulin o
我室在对植物细胞外CaM的研究过程中，发现细胞外还可能存在CaMBPs，并首次从白芷悬浮培养细胞外纯化了一种依赖Ca~(2+)的、分子量约为21kD的细胞外CaMBP(Tang等，1996)(定名为：ECBP21)。生理实验初步表明ECBP21影响白芷悬浮培养细胞的增殖(毛国红等，1999)。这些生物化学和生理学实验，仅为细胞外CaMBPs的存在及可能的功能提供了初步证据。为了进一步利用分子生物学手段证实细胞外ECBP21真实存在，并进一步研究其生物学功能，本实验从克隆基因入手进行了以下工作：
1．ECBP21全长cDNA克隆：将纯化的ECBP21蛋白经SDS—PAGE分离后电转移到PVDF膜上，氨基酸测序获得N-末端20个氨基酸序列；根据N-末端8个氨基酸序列合成兼并性引物，通过RT-PCR和5’-RACE方法，获得其cDNA序列。Northern blot分析表明克隆的cDNA序列为全长cDNA。ECBP21 cDNA全长947bp，编码216个氨基酸，序列分析显示其N端1-25氨基酸为信号肽，26-216氨基酸为成熟蛋白肽段，并且不具有跨膜结构域。将此cDNA成熟蛋白编码区核苷酸片断构建到pET-28b(+)表达载体，在大肠杆菌表达系统中诱导了该蛋白的表达，经CaM-gel overlay检测证实表达蛋白具有Ca~(2+)依赖的CaM结合特性，从而进一步证实了ECBP21为一种Ca~(2+)依赖的CaMBP。通过缺失表达分析实验证实CaMBD结构域位于ECBP21的羧基端。Southern blot检测表明白芷基因组中可能存在两个ECBP21基因拷贝。从而首次克隆并鉴定了一个细胞外CaMBP的全长cDNA。
2．ECBP21亚细胞定位：以重组ECBP21蛋白为抗原制备了特异性抗体。利用特异性抗体通过免疫金标定位研究表明ECBP21主要分布于细胞壁。进一步利用GFP荧光标记，通过瞬时表达方法研究显示转ECBP21-GFP
毛国红:白芷细胞外钙调素结合蛋白(ECBPZI)cDNA克隆及鉴定
融合基因细胞的细胞壁上有较强绿色荧光，而转缺失信号肤的
乙去乙岁苍介分户融合基因及贷尹基因的细胞中，细胞壁上无绿色荧光，表
明ECBP21是一种主动分泌到细胞外的分泌蛋白。由此进一步证实ECBP21
确为一种质外体多肤。本实验为细胞外CaMBPS的存在提供了直接证据。
3.ECBP21表达分析:Northern blot分析表明:在白芷悬浮培养细
胞生长周期中，ECBP21和CaM都为组成型表达，0一12天ECBP21表达量
逐渐增加，第12天达到最高，随后12一21天，ECBP21表达量稍有下降，
维持在一个比较稳定状态;而O一6天时，CaM表达量逐渐上升，第6一9
天达到最大表达量，随后，9一21天，CaM表达量有所下降。表明ECBP21
可能参与白芷悬浮培养细胞生长周期调控。热激、渗透、盐胁迫及JA处
理时，ECBPZI表达量下降，而6一BA、ABA及GA3处理时，不影响ECBPZI
的表达。推测ECBP21还可能与逆境胁迫相关。
4.初步开展了转基因研究:为了进一步确定ECBP21在植物生长发
育过程中的作用，构建了ECBP21植物表达二元载体，通过真空渗透法转
化了拟南芥并获得了转基因植株。表型观察仍在进行之中。
总之，通过上述研究我们首次克隆及鉴定了一种细胞外CaMBP
(EC即21) CDNA，并首次提供了细胞外存在CaMBPS的直接证据。本实验
结果也为利用分子生物学方法研究ECBP21的生物学功能奠定了基础，并
将有助于阐明胞外 CaM的作用机制及丰富植物质外体多肤的研究。
In recent years, Ca~(2+) signal system (Ca~(2+) , CaM, and CaMBPs) has been thoroughly researched. Many intracellular CaMBPs have been detected and studied. In our laboratory, a series of experiments has provided evidence that the presence of CaM in the apoplast area is universal, and extracellular calmodulin plays an important role in the plant growth and development as a polypeptide signal. This has become one of the latest progresses in plant CaM research work.
In the process of study for the extracellular CaM, the research in our lab indicates that CaMBPs also possibly exist in cell wall area. For the first time, an extracellular Ca~(2+)-dependent CaMBP (named: ECBP21) was purified from 0.1mol/L CaCl_(2) extracts of Angelica dahurica suspension-cultured cells (Tang et al. 1996). Furthermore, our preliminary evidence shows that the ECBP21 protein influences on proliferation of this cultured cell (Mao et al. 1999). By using methods of biochemistry and physiology, the above experiments only provided preliminary evidence for the existence and possible functions of the extracellular CaMBPs. In order to prove the real existence of the extracellular ECBP21 and its functions by means of molecular biology, we first cloned and identified the full-length cDNA for an extracellular CaMBP (ECBP21).
The purified protein was electrobloted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5 "RACE cloning. The cDNA contains 947 nucleotides that codes for a precursor protein of 216 amino acids. Sequence analysis shows that the N-terminal 1-25 amino acid sequence is a predicted signal peptide and other 26-216 amino acid sequence is a mature peptide which has no transmembrane domain. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E.coli BL21(DE3). A protein with relative molecular mass 21kD was expressed in E.coli. Using a biotinylated-CaM gel overlay technique, the expression protein has the ability to bind CaM in the presence of Ca~(2+). These
results further defined ECBP21is a Ca~(2+)-dependent CaM-binding protein. Following researches verified that the CaM-binding domain exists in the C-terminal of ECBP21. Southern blot analysis shows that there are two ECBP21 gene copies in genome.
Second, The ECBP21 was localized in the cell wall area by immuno-gold electron microscopy and by using GFP as a reporter. The results indicted that ECBP21 is a kind of secretive protein and ECBP21 is an apoplast polypeptide. These experiments provided the first and direct evidence for the existence of the extracellular CaMBPs.
Third, we examined the expression of ECBP21 dynamics during the incubation of Angelica suspension-cultured cells and stress treatments through Northern blot analysis. During the incubation of Angelica suspension-cultured cells, the expression of ECBP21 gene increased from 0 to 12th day, and it reached a peak on the twelfth day. While the expression of CaM gene increased from 0 to 9th day, and it reached a peak during the 6th-9th day. These results indicated that ECBP21 may involve in the regulation of cell growth. The stress treatments (such as: heat shock, osmotic and salt stresses) and JA (100uM) treatment repressed the expression of ECBP21 gene. They indicated that ECBP21 may have some relative with the response to stress treatments.
Further more, we constructed the binary vector containing ECBP21-GFP chimeric gene and got transgenic plants by using the Agrobacterium vacuum infiltration method. The phenotype observation is still in progress.
Thought the above studies, we first cloned and identified an extracellular CaMBP (ECBP21) cDNA, and provided direct evidence for the existence of the extracellular CaMBPs. This work laid a foundation for elucidating the functions of ECBP21 by means of molecular biology. Further studies about ECBP21 may be benefit to elucidating the mechanism of apoplast calmodulin o
引文
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