1、綠色熒光蛋白基因在水稻細(xì)胞中的表達(dá)研究GFP簡(jiǎn)介T(mén)he 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.12 Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the p

2、rotein 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 renif

3、ormis) has a single major excitation peak at 498 nm.綠色螢光蛋白（green fluorescent protein），簡(jiǎn)稱(chēng)GFP，這種蛋白質(zhì)最早是由下村脩等人在1962年在維多利亞多管發(fā)光水母中發(fā)現(xiàn)。其基因所產(chǎn)生的蛋白質(zhì)，在藍(lán)色波長(zhǎng)范圍的光線激發(fā)下，會(huì)發(fā)出綠色螢光。這個(gè)發(fā)光的過(guò)程中還需要冷光蛋白質(zhì)水母素的幫助，且這個(gè)冷光蛋白質(zhì)與鈣離子(Ca2+)可產(chǎn)生交互作用。由維多利亞多管發(fā)光水母中發(fā)現(xiàn)的野生型綠色螢光蛋白，395nm和475nm分別是最大和次大的激發(fā)波長(zhǎng)，它的發(fā)射波長(zhǎng)的峰點(diǎn)是在509nm，在可見(jiàn)光綠光的范圍下是較弱的位置。由海腎(sea

4、 pansy)所得的綠色螢光蛋白，僅有在498nm有一個(gè)較高的激發(fā)峰點(diǎn)。GFP的性質(zhì)GFP是一種現(xiàn)成的熒光蛋白質(zhì)，因此它特別容易使用。大多數(shù)可以處理光的蛋白質(zhì)都利用外來(lái)的分子吸收和釋放光子。例如，我們眼睛里的視紫紅質(zhì)利用維生素來(lái)感光。這些“發(fā)光團(tuán)”必須是專(zhuān)門(mén)為了發(fā)光而生成的，并且被仔細(xì)地插入到該蛋白質(zhì)分子內(nèi)，不同的是，GFP控制光的部位是其自身的一部分，僅由氨基酸構(gòu)建而成，該部位含有一段三個(gè)氨基酸組成的特殊序列：絲氨酸酪氨酸甘氨酸（有時(shí)絲氨酸會(huì)被相似的蘇氨酸取代）。當(dāng)?shù)鞍踪|(zhì)鏈折疊時(shí)，這段短片段就被深埋在蛋白質(zhì)內(nèi)部，然后，發(fā)生一系列化學(xué)反應(yīng)：甘氨酸與絲氨酸之間形成化學(xué)鍵，生成一個(gè)新的閉合環(huán)，隨后

5、這個(gè)環(huán)會(huì)自動(dòng)脫水。最終，經(jīng)過(guò)大約一個(gè)小時(shí)的反應(yīng)，周?chē)h(huán)境中的的氧氣攻擊酪氨酸的一個(gè)化學(xué)鍵，形成一個(gè)新的雙鍵并合成熒光發(fā)色團(tuán)。由于GFP可以形成自己的發(fā)色團(tuán)，它非常適合于基因工程。你根本不必?fù)?dān)心操作任何奇怪的發(fā)色團(tuán)，你只需要利用遺傳學(xué)的方法操縱細(xì)胞合成GFP蛋白質(zhì)，GFP就會(huì)自動(dòng)折疊并開(kāi)始發(fā)光。GFP的性質(zhì)（續(xù)）GFP的用途GFP作為報(bào)告分子和細(xì)胞標(biāo)記最明顯的優(yōu)勢(shì)是無(wú)需底物或輔因子參與；無(wú)論在活細(xì)胞還是在完整的轉(zhuǎn)基因胚胎和動(dòng)物中，都能有效地監(jiān)測(cè)基因轉(zhuǎn)移的效率。但在這方面的應(yīng)用中，GFP最大的缺點(diǎn)就是沒(méi)有放大作用，它不能像酶一樣能通過(guò)加工無(wú)數(shù)的底物分子而將信號(hào)放大。所以一般都需強(qiáng)啟動(dòng)子以驅(qū)動(dòng)GFP

6、基因在細(xì)胞內(nèi)足量的表達(dá)。也可用亞細(xì)胞分辨率的顯微成像系統(tǒng)檢測(cè)基因產(chǎn)物，靶入的基因被限制于一個(gè)細(xì)胞器內(nèi)，GFP的濃度則相對(duì)提高了許多倍。綠色熒光蛋白基因在水稻基因轉(zhuǎn)化中研究（續(xù)）1.2 Production of transgenic rice plants（生產(chǎn)轉(zhuǎn)基因大米植株）Calli（愈傷組織） were induced in MS medium ( Murashige and Skong, 1962) from mature embryos of japonica rice ( Oryza saliva L. ) cv. TNG67. Fifteen-day-old embryogeni

7、c 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 containing MS medium (Wang and Wu, 1995; Zhao et al, 1989), supplemented with 6 mg L-1 Bialaphos as the selective agent

8、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. Regenerated plants were transplanted into sterilized soil and grown in the greenho

9、use (30B day and 24 B night with a supplemented 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 bot

10、h 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.綠色熒光蛋白基因在水稻基因轉(zhuǎn)化中研究（續(xù)）1.3 Quantitative assay of GFP values in

11、transgenic rice plants（定量檢測(cè)綠色熒光蛋白在轉(zhuǎn)基因水稻植株中的表達(dá)值） To detect GFP value in R0 transgenic rice plants, a quantitative assay was carried out. The second leaf from the topof each two-month-old transgenic rice plant was collected and immediately frozen in liquid nitrogen. The leaves were ground in liquid ni

12、trogen 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 extraction buffer was found by preliminary experiments to give minimum autofluorescence of rice leaf ext

13、ractsr 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 fluoresce

14、nce units f g - 1 protein using a fluorometerr model SLM8000) after excitation at 385 nm and measure the emission maximum at 510 nm. Non-transformed tissues were used to estimate the autofluores-cence of the tissue. Protein concentration was determined according to Bradford (Bradford , 1976). Statis

15、tical analysis was carried out as described by Sokal and Rohlf (Sokal and Rohlf , 1969).綠色熒光蛋白基因在水稻基因轉(zhuǎn)化中研究（續(xù)）1.5 DNA-blot hybridization analysis of transgenic rice plants（轉(zhuǎn)基因大米植株的DNA印跡雜交分析）Genomic DNA from transgenic rice plants was prepared as described by Zhao et al. (1989). Eight micrograms of ge

16、nomic DNA were digested with restriction enzymes the DNA fragments were separated by electrophoresis through 1 % agarose gels and transferred to nylon membranes (Nytran , Schleicher & Schuell). Probe preparation , hybridization and detection were performed using the 32P-dCTP radioactive labeling sys

17、tem following the manufacturer s protocol .綠色熒光蛋白基因在水稻基因轉(zhuǎn)化中研（續(xù)）2.Results為了研宄比較GFP基因和GUS基因在水稻細(xì)胞中的瞬間表達(dá)，使用基因槍將質(zhì)粒pAct1，GFP和pActD分別或同時(shí)導(dǎo)入水稻胚性愈傷組織的細(xì)胞。在這兩種質(zhì)粒中，GFP基因和GUS基因的表達(dá)均由水稻Act啟動(dòng)子和NOS終止子控制。轉(zhuǎn)化后暗培養(yǎng)3 4 h，在藍(lán)光照射下觀察到用pAct1，GFP轉(zhuǎn)化的愈傷組織上開(kāi)始出現(xiàn)明亮的綠色熒光點(diǎn)。隨后間隔1 h的連續(xù)觀察中，發(fā)現(xiàn)表達(dá)GFP的愈傷組織數(shù)目及GFP綠色熒光點(diǎn)的數(shù)目和亮度都不斷增加。轉(zhuǎn)化后10 16 h，可在8

18、5%的愈傷組織塊上觀察到十幾個(gè)至上百個(gè)綠色熒光點(diǎn),各點(diǎn)的亮度變化很大,有的非常耀眼，有的則勉強(qiáng)可以看見(jiàn)。此后的觀察發(fā)現(xiàn)大部分的綠色熒光點(diǎn)逐漸開(kāi)始暗淡以至熄滅。培養(yǎng)至36 h，僅在20%的愈傷組織塊上發(fā)現(xiàn)1 5個(gè)綠色熒光點(diǎn)。其中有2個(gè)點(diǎn)在培養(yǎng)12h后仍能清楚地看見(jiàn)。作為對(duì)照，用pAct1-D單質(zhì)粒轉(zhuǎn)化的愈傷組織上一直沒(méi)有觀察到綠色熒光點(diǎn)。轉(zhuǎn)化后培養(yǎng)4 96 h之間，以4h的間隔各取10塊用0轉(zhuǎn)化的愈傷組織進(jìn)行組織化學(xué)染色，觀察表明培養(yǎng)4h的愈傷組織上開(kāi)始出現(xiàn)GUS基因表達(dá)產(chǎn)物與底物乂X-Glue反應(yīng)產(chǎn)生的藍(lán)點(diǎn)。轉(zhuǎn)化后40 48 1，藍(lán)點(diǎn)數(shù)目達(dá)到最多。雖然采取了固定步驟,仍然有少數(shù)藍(lán)點(diǎn)的面積較大。

19、綠色熒光蛋白基因在水稻基因轉(zhuǎn)化中研（續(xù)）為了研宄基因在水稻細(xì)胞中的穩(wěn)定表達(dá),進(jìn)行了 HPT基因和GFP基因的共轉(zhuǎn)化實(shí)驗(yàn)。使用熒光顯微鏡對(duì)篩選培養(yǎng)和再生培養(yǎng)過(guò)程中的植物材料進(jìn)行了跟蹤觀察，發(fā)現(xiàn)0 GFP基因瞬間表達(dá)結(jié)束后，沒(méi)有再觀察到GFP綠色熒光現(xiàn)象。共進(jìn)行了 3次轉(zhuǎn)化實(shí)驗(yàn)，使用潮霉素B篩選得到46 個(gè)轉(zhuǎn)化系的700多株可育的潮霉素抗性再生植株。分子雜交證明GFP基因在這些植株中的整合頻率達(dá)到64%，整合拷貝數(shù)目為1個(gè)至多個(gè)。對(duì)所有轉(zhuǎn)GFP基因當(dāng)代植株（包括白化苗)的葉片及根、由轉(zhuǎn)基因植株誘導(dǎo)的愈傷組織和部分子一代種子在光下及暗中的萌發(fā)過(guò)程進(jìn)行了觀察，均沒(méi)有發(fā)現(xiàn)綠色熒光現(xiàn)象。綠色熒光蛋白基因在

20、水稻基因轉(zhuǎn)化中研究（續(xù)）3.2 It is possible to quantify GFP expression by fluorescence measurement even though there is auto fluorescence somewhat in green parts of plant Some plants have auto fluorescence due to the exist of chlorophyll and other segments . The ability of auto-fluorescence is high or low, wheth

21、er it will affect the observation of GFP is a major concern issue for the application of gp as a reporter gene. Our result show that GFP has significant higher fluorescence signal compared with auto-fluorescence signal.綠色熒光蛋白基因在水稻基因轉(zhuǎn)化中研究（續(xù)）3.3 The results show that GFP is also an ideal reporter gene for the research of genetic transformation . In some aspects, it is better than GUS gene as reporter gene .GUS gene is a conventional reporter gene . It is used as an important tool in the res