GFP绿色荧光蛋白在水稻中的表达

绿色荧光蛋白基因在水稻细胞中 的表达研究
GFP简介
The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues (26.9kDa) that exhibits bright green fluorescence when exposed to blue light.[1][2] Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower green portion of the visible spectrum. The GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm. 绿色萤光蛋白（green fluorescent protein），简称GFP，这种蛋白质最早是 由下村脩等人在1962年在维多利亚多管发光水母中发现。其基因所产生的蛋 白质，在蓝色波长范围的光线激发下，会发出绿色萤光。这个发光的过程中 还需要冷光蛋白质水母素的帮助，且这个冷光蛋白质与钙离子(Ca2+)可产生 交互作用。 由维多利亚多管发光水母中发现的野生型绿色萤光蛋白，395nm和475nm分 别是最大和次大的激发波长，它的发射波长的峰点是在509nm，在可见光绿 光的范围下是较弱的位置。由海肾(sea pansy)所得的绿色萤光蛋白，仅有在 498nm有一个较高的激发峰点。
绿色荧光蛋白基因在水稻基因转化中研究
1 Materials and Methods.（材料与方法） Methods. pJPM5 1.1 Construction of plasmids containing GFP gene in pJPM （构建含有GFP基因的pJPM 质粒） pJPM5 pJPM We used the pGHNC5 plasmid, reported by Allen et al. (1993, 1996), for the construction of pJPM5 ( Figure 1). Plasmid pJPMO was constructed by inserting a Klenow enzyme-filled EcoRI fragment, isolated from pBY505 (Wang and Wu, 1995; Zhao et al, 1989), which contains the Pin2 terminator, 35S promoter, bar gene and Nos termina­tor, into the Sma1 site of pGHNC5. Plasmid pJPM5 was constructed by inserting the rice actin1 promoter (McElroy et al, 1991) and a GFP gene isolated from pSBGXOO, into pJPMO. Plasmids pSBGXOO was constructed by Jukon Kim and Ray Wu ( unpublished results ).
绿色荧光蛋白基因在水稻基因转化中研究（续）
1.3 Quantitative assay of GFP values in transgenic rice plants（定量检测绿 （ 色荧光蛋白在转基因水稻植株中的表达值 蛋白在转基因水稻植株中的表达值） 色荧光蛋白在转基因水稻植株中的表达值） To detect GFP value in R0 transgenic rice plants, a quantitative assay was carried out. The second leaf from the top of each two-month-old transgenic rice plant was collected and immediately frozen in liquid nitrogen. The leaves were ground in liquid nitrogen and homogenized in ice - cold extraction buffer (100 mmol HEPES, pH 8.0, 10 mmol EDTA, 5 mmol DTT, 10% glycerol, 1% PVP, 25 fig mL-1 PMSF, 15 fig mL-1 leupeptin, 1% activated carbon). This ex-traction buffer was found by preliminary experiments to give minimum autofluorescence of rice leaf extractsr data not shown). After the first centrifugation (12 000 rpm for 15 min at 4B ) , the crude extract was centrifuged again (12 000 rpm for 20 min at 4B ) to further reduce insoluble particles which affect the quantification of fluorescence. Then the GFP values were quantified based on fluorescence units f g - 1 protein using a fluorometerr model SLM8000) after excitation at 385 nm and measure the emission maximum at 510 nm. Nontransformed tissues were used to estimate the autofluores-cence of the tissue. Protein concentration was determined according to Bradford (Bradford , 1976). Statistical analysis was carried out as described by Sokal and Rohlf (Sokal and Rohlf , 1969).
GFP的性质（续）
GFP的用途
GFP作为报告分子和细胞标记最明显的优势是无需底 物或辅因子参与；无论在活细胞还是在完整的转基因 胚胎和动物中，都能有效地监测基因转移的效率。 但在这方面的应用中，GFP最大的缺点就是没有放大 作用，它不能像酶一样能通过加工无数的底物分子而 将信号放大。所以一般都需强启动子以驱动GFP基因 在细胞内足量的表达。也可用亚细胞分辨率的显微成 像系统检测基因产物，靶入的基因被限制于一个细胞 器内，GFP的浓度种现成的荧光蛋白质，因此它特别容易使用。大多数 可以处理光的蛋白质都利用外来的分子吸收和释放光子。例如， 我们眼睛里的视紫红质利用维生素来感光。这些“发光团”必 须是专门为了发光而生成的，并且被仔细地插入到该蛋白质分 子内，不同的是，GFP控制光的部位是其自身的一部分，仅由 氨基酸构建而成，该部位含有一段三个氨基酸组成的特殊序列： 丝氨酸－酪氨酸－甘氨酸（有时丝氨酸会被相似的苏氨酸取 代）。当蛋白质链折叠时，这段短片段就被深埋在蛋白质内部， 然后，发生一系列化学反应：甘氨酸与丝氨酸之间形成化学键， 生成一个新的闭合环，随后这个环会自动脱水。最终，经过大 约一个小时的反应，周围环境中的的氧气攻击酪氨酸的一个化 学键，形成一个新的双键并合成荧光发色团。由于GFP可以形 成自己的发色团，它非常适合于基因工程。你根本不必担心操 作任何奇怪的发色团，你只需要利用遗传学的方法操纵细胞合 成GFP蛋白质，GFP就会自动折叠并开始发光。
绿色荧光蛋白基因在水稻基因转化中研究（续）
1.2 Production of transgenic rice plants（生产转基因大米植株） （生产转基因大米植株） Calli（愈伤组织） were induced in MS medium ( Murashige and Skong, 1962) from mature embryos of japonica rice ( Oryza saliva L. ) cv. TNG67. Fifteen-day-old embryogenic calli were bombarded with gold beads coated with either pJPM5, or with plasmid pSBG7OO according to the procedure of Cao et al. ( 1992) . Resistant calli were selected in plates contain-ing MS medium (Wang and Wu, 1995; Zhao et al, 1989), supplemented with 6 mg L-1 Bialaphos as the selective agent for 4 weeks (su bcultured every 2 weeks) . Resistant calli were transferred to plates containing MS regeneration media with 1 mg L-1 kinetin, 2mgL-1BAP, 0.25 mg L-1 NAA and 3 mgL-1 Bialaphos to regenerate into plants. Re-generated plants were transplanted into sterilized soil and grown in the greenhouse (30B day and 24 B night with a sup-plemented photoperiod of 10 h) . The presence of the transgenes in regenerated plants was first inferred by the herbicide resistance of the plants. To test for herbicide resistance, one leaf from each of the one-month-old transgenic rice plants was painted on both sides with 0. 25 % ( v/ v ) of the herbicide Basta ( containing 162 g L -1 glufosinate ammonium: Hoechst- Roussel Agri- Vet Co., Somerville, NJ) and 0.05% (v/v) Tween 20. One week later, the resistant or sensitive phenotypes were scored.