利用GFP标记对胞外钙调素定位的研究
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摘要
CaM作为一种重要的Ca~(2+)结合蛋白，参与细胞内许多生理过程的调控。多年来，我室一系列研究表明植物细胞外普遍存在CaM，并且细胞外CaM作为质外体多肽，发挥许多重要的生物学功能。近年来的研究表明，在植物中普遍存在钙调素亚型，它们在植物体中的表达和功能不尽相同。为了进一步利用分子生物学方法在基因水平上研究胞外CaM的存在，同时研究胞外CaM与CaM亚型之间的关系，本论文利用绿色荧光蛋白(GFP)作为报告基因研究了大豆CaM基因家族(SCaMs)的亚细胞定位。
首先，构建了含有SCaM5-GFP融合基因的中间载体(pUC-SCaM5-GFP)和双元载体(pGTV-SCaM5-GFP)，以及含有SCaM2-GFP融合基因的双元载体(pGTV-SCaM2-GFP)。将双元载体转化农杆菌菌株LBA4404。
随后，选用含有SCaM1-GFP、SCaM3-GFP、SCaM4-GFP、SCaM5-GFP融合基因以及仅含有GFP基因的农杆菌，采用叶盘转化法分别转化烟草(Nicotiana tobacco L．cv．NC89)，获得了转GFP、SCaM1-GFP，SCaM2-GFP，SCaM3-GFP、SCaM4-GFP、SCaM5-GFP六种基因的烟草植株，并对转基因植株进行了报告基因活性检测以及PCR、Southern-blot鉴定。
最后，在激光共聚焦显微镜下对转基因植株愈伤组织细胞进行观察。实验结果表明：转GFP的对照组细胞质壁分离后，细胞壁基本上没有检测到绿色荧光，绿色荧光主要集中在细胞内；转SCaM1-GFP、SCaM2-GFP、SCaM3-GFP的细胞质壁分离后，除在细胞内检测到绿色
河北师范大学硕士学位论文
荧光外，细胞壁上可以观察到较明显的绿色荧光;转SCcl几州代了厂尸、
SCaM万一GFI〕的细胞质壁分离后，细胞壁上基本没有检测到绿色荧光，绿
色荧光主要集中在细胞内。表明SCaMI、SCaMZ、SCaM3不仅存在于细
胞内，而且还存在于细胞外;而SCaM4、SCaMS可能仅存在于细胞内。
本实验利用分子生物学方法从基因水平上证实了细胞外CaM的存在，为
细胞外CaM的存在提供了更为确凿的证据;另外，由于SCaMI、SCaMZ、
SCaM3亚型特异性分泌到细胞外，表明细胞外CaM的存在可能具有亚
型的特异性。本实验中细胞外CaM亚型的确定为我室进一步研究质外体
CaM在植物体内(in viv(>)的生理功能以及细胞外CaM参与的信号转导途
径奠定了基础。
Calmodulin, as a versatile Ca~(2+) sensor, plays many important biological functions. Meanwhile, our previous experiments suggested that calmodulin exists in the apoplast of plant cells and has many physiological functions. Further more, we put forward a view that apoplast CaM maybe a multiple functional polypeptide signal in plants. In recent years, multiple genes of CaM isoforms have been found in many plants, which have some differences in expression and functions. In order to understand the apoplast CaM gene and the relationship between the apoplast CaM and CaM isoforms, we intended to get transgenic plants harboring soybean calmodulin isoform genes (SCaMs) fused to gfp as a reporter and study the subcellular localization of SCaMs.
First, SCaM5, one of the soybean calmodulin isoforms, was amplified by PCR and was inserted into pUC-GFP. Then the chimeric genes (SCaM2-GFP, SCaM5-GFP) were inserted into the binary vector pGTV.
The binary vectors which respectively contain the chimeric genes (SCaMl-GFP, SCaM2-GFP, SCaM3-GFP, SCaM4-GFP, SCaM5-GFP) were used to transform tobacco, and pGTV-GFP was used as control. The chimeric genes were transformed into tobacco plants via Agrobacterium tumefaciens. The transgenic tobacco harboring SCaMs genes were obtained and verified by reporter gene examination, PCR, Southern blot detection. Then we establish the transgenic callus. The plasmolyzed cells of transgenic callus were treated with 0.45M mannitol (PH 7.0) and observed using LSCM
(laser scanning confocal microscope).
Green fluorescence was found in the cell wall of transgenic SCaM1, SCaM2, SCaM3 callus. However there was no green fluorescence in the cell wall of transgenic SCaM4, SCaM5 callus. These results indicated that SCaMl , SCaM2 , SCaM3 can be secreted into the apoplast of plant cells, while SCaM4, SCaM5 don't exit in the apoplast of plant cells. This work laid a foundation for elucidating the in vivo functions of apoplast CaM by means of molecular biology.
首先，构建了含有SCaM5-GFP融合基因的中间载体(pUC-SCaM5-GFP)和双元载体(pGTV-SCaM5-GFP)，以及含有SCaM2-GFP融合基因的双元载体(pGTV-SCaM2-GFP)。将双元载体转化农杆菌菌株LBA4404。
随后，选用含有SCaM1-GFP、SCaM3-GFP、SCaM4-GFP、SCaM5-GFP融合基因以及仅含有GFP基因的农杆菌，采用叶盘转化法分别转化烟草(Nicotiana tobacco L．cv．NC89)，获得了转GFP、SCaM1-GFP，SCaM2-GFP，SCaM3-GFP、SCaM4-GFP、SCaM5-GFP六种基因的烟草植株，并对转基因植株进行了报告基因活性检测以及PCR、Southern-blot鉴定。
最后，在激光共聚焦显微镜下对转基因植株愈伤组织细胞进行观察。实验结果表明：转GFP的对照组细胞质壁分离后，细胞壁基本上没有检测到绿色荧光，绿色荧光主要集中在细胞内；转SCaM1-GFP、SCaM2-GFP、SCaM3-GFP的细胞质壁分离后，除在细胞内检测到绿色
河北师范大学硕士学位论文
荧光外，细胞壁上可以观察到较明显的绿色荧光;转SCcl几州代了厂尸、
SCaM万一GFI〕的细胞质壁分离后，细胞壁上基本没有检测到绿色荧光，绿
色荧光主要集中在细胞内。表明SCaMI、SCaMZ、SCaM3不仅存在于细
胞内，而且还存在于细胞外;而SCaM4、SCaMS可能仅存在于细胞内。
本实验利用分子生物学方法从基因水平上证实了细胞外CaM的存在，为
细胞外CaM的存在提供了更为确凿的证据;另外，由于SCaMI、SCaMZ、
SCaM3亚型特异性分泌到细胞外，表明细胞外CaM的存在可能具有亚
型的特异性。本实验中细胞外CaM亚型的确定为我室进一步研究质外体
CaM在植物体内(in viv(>)的生理功能以及细胞外CaM参与的信号转导途
径奠定了基础。
Calmodulin, as a versatile Ca~(2+) sensor, plays many important biological functions. Meanwhile, our previous experiments suggested that calmodulin exists in the apoplast of plant cells and has many physiological functions. Further more, we put forward a view that apoplast CaM maybe a multiple functional polypeptide signal in plants. In recent years, multiple genes of CaM isoforms have been found in many plants, which have some differences in expression and functions. In order to understand the apoplast CaM gene and the relationship between the apoplast CaM and CaM isoforms, we intended to get transgenic plants harboring soybean calmodulin isoform genes (SCaMs) fused to gfp as a reporter and study the subcellular localization of SCaMs.
First, SCaM5, one of the soybean calmodulin isoforms, was amplified by PCR and was inserted into pUC-GFP. Then the chimeric genes (SCaM2-GFP, SCaM5-GFP) were inserted into the binary vector pGTV.
The binary vectors which respectively contain the chimeric genes (SCaMl-GFP, SCaM2-GFP, SCaM3-GFP, SCaM4-GFP, SCaM5-GFP) were used to transform tobacco, and pGTV-GFP was used as control. The chimeric genes were transformed into tobacco plants via Agrobacterium tumefaciens. The transgenic tobacco harboring SCaMs genes were obtained and verified by reporter gene examination, PCR, Southern blot detection. Then we establish the transgenic callus. The plasmolyzed cells of transgenic callus were treated with 0.45M mannitol (PH 7.0) and observed using LSCM
(laser scanning confocal microscope).
Green fluorescence was found in the cell wall of transgenic SCaM1, SCaM2, SCaM3 callus. However there was no green fluorescence in the cell wall of transgenic SCaM4, SCaM5 callus. These results indicated that SCaMl , SCaM2 , SCaM3 can be secreted into the apoplast of plant cells, while SCaM4, SCaM5 don't exit in the apoplast of plant cells. This work laid a foundation for elucidating the in vivo functions of apoplast CaM by means of molecular biology.
引文
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