杨树木质部特异启动子的序列分析和功能检测
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摘要
本研究以已分离得到的三个木质部纤维素合酶基因JCesAP,YCesAP和MDCesAP序列为基础,旨在筛选出具有启动子活性和木质部特异性的启动子。为利用其融合并启动杀虫基因只在木质部特异性表达,防治蛀干害虫(尤其是天牛类害虫)奠定了基础。木质部纤维素合酶基因(CesA基因)在植物的正常生长期仅在木质部特异性表达,受到外界压力诱导时可在韧皮部表达。本研究通过植物启动子及其顺式作用元件数据库对JCesAP,YCesAP和MDCesAP序列进行了启动子顺式作用原件的分析,并利用基因重组技术构建了pJCesAP-GUS和pYCesAP-GUS植物表达载体,连同已构建成功的pCesAP-GFP(其中CesAP分别为JcesAP,YCesAP和MDCesAP)和pMDCesAP-GUS植物表达载体,分别以GFP和GUS作为报告基因转化烟草,根据报告基因表达情况,对三个序列启动子活性及其木质部特异性进行检测。试验进一步以毛白杨雄株-D无菌苗为实验材料,采用农杆菌介导法将CesAP-GFP基因导入毛白杨雄株-D的基因组中,并获得了少量愈伤组织,对部分抗性愈伤组织进行了GFP报告基因活性的检测。本研究的主要结果如下:
1.启动子顺势作用元件分析结果表明,JCesAP、YCesAP和MDCesAP这三个序列均具有启动子的基本元件,木质部特异性元件和伤害诱导表达的元件。
2.成功构建了pJCesAP-GUS和pYCesAP-GUS植物表达载体(均以GUS作为报告基因)。此可读框架通过木质部特异性启动子启动(选择标记基因是新霉素磷酸转移酶(nptⅡ)基因,用Kan为筛选试剂),并将构建的表达载体电激转化导入根癌农杆菌(Agrobacterium tumefaciens) AGL-Ⅰ中。
3.确定烟草转化的头孢噻肟钠的抑菌浓度为400 mg/L。将植物表达载体pCesAP-GFP分别转入烟草中,均得到完整的烟草再生植株。经PCR检测,初步证明CesAP基因已整合到烟草的基因组中。GFP荧光检测结果表明,JCesAP,YCesAP和MDCesAP序列均诱导启动GFP蛋白的表达激发出黄绿色荧光,具有启动子的活性。
4.确定了毛白杨雄株-D转化的潮霉素临界筛选浓度为10 mg/L。将CesAP-GFP基因转入毛白杨中,多次继代筛选后获得了少量抗性愈伤组织,GFP荧光检测结果表明,与阴性对照和阳性对照相比,抗性愈伤组织只有部分部位激发出黄绿色荧光,证明JCesAP,YCesAP和MDCesAP序列除具有启动子活性外,还均具有组织特异性。
The main purpose of this paper is to detect the xylem-specific promoter in the three sequences of JCesAP,YCesAP and MDCesAP. If the promoters can be used to incorporate Coleoptera-resistance genes and made these genes to be only expressed in the xylem, this will provide scientific basis for controlling the stem-boring pests especially Cerambycidae. Xylem-specific cellulose synthase gene (CesA gene) is confined to developing xylem cells during plant normal growth only and induced in developing phloem fibers undergoing tension stress also. The cis-acting elements of JCesAP, YCesAP and MDCesAP were analyzed by the PLACE database, the plant expression vector pJCesAP-GUS and pYCesAP-GUS were constructed using DNA recombination technology separately, and then all the plant expression vectors including our lab preserving harboring CesAP-GFP/CesAP-GUS were transformed into tobacco using leaf-disk method. The promoter activity and reporter gene expression levels were tested. Selecting Populus tomentosa Stub-D as the experiment materials, CesAP-GFP gene were integrated into Populus tomentosa Stub-D by Agrobacterium-mediated transformation method. Some resistant calli were obtained and observed under fluorescence microscope. The main results were as follows:
1. The cis-acting elements of JCesAP, YCesAP and MDCesAP amplified respectively in earlier studies were analyzed by the PLACE database; the results showed that, the three sequences all had the core element of promoter and the elements related to xylem-specific and wound-defense functions.
2. The plant expression vector pJCesAP-GUS and pYCesAP-GUS were constructed successfully using GUS as reporter gene. Each gene was promoted with CesA promoter, containing nptⅡgene as a selective marker. The plant expression vectors were transformed into Agrobacterium tumefaciens strain AGL-Ⅰ.
3. Cefotaxime (CTX) critical concentration was 400 mg/L for the tobacco transformation. The plant expressed vector pCesAP-GFP was transformed to Agrobacterium tumefaciens strain AGL-I. GFP expression showed that, the green fluorescence was observed in all transgenic tobacco, it proved all the three sequences were of promoter activity.
4. Hygromycin (Hyg) critical concentration was 10 mg/L for the Populus tomentosa Stub-D transformation. pCesAP-GFP gene was transformed to Populus tomentosa Stub-D leaves. Through several transformations and selections, some resistant calli were obtained, green fluorescence was only observed in local tissues of the transgenic calli comparing to negative control and positive control, it proved that the three sequences not only obtain the promoter activities, but also tissue-specific.
1.启动子顺势作用元件分析结果表明,JCesAP、YCesAP和MDCesAP这三个序列均具有启动子的基本元件,木质部特异性元件和伤害诱导表达的元件。
2.成功构建了pJCesAP-GUS和pYCesAP-GUS植物表达载体(均以GUS作为报告基因)。此可读框架通过木质部特异性启动子启动(选择标记基因是新霉素磷酸转移酶(nptⅡ)基因,用Kan为筛选试剂),并将构建的表达载体电激转化导入根癌农杆菌(Agrobacterium tumefaciens) AGL-Ⅰ中。
3.确定烟草转化的头孢噻肟钠的抑菌浓度为400 mg/L。将植物表达载体pCesAP-GFP分别转入烟草中,均得到完整的烟草再生植株。经PCR检测,初步证明CesAP基因已整合到烟草的基因组中。GFP荧光检测结果表明,JCesAP,YCesAP和MDCesAP序列均诱导启动GFP蛋白的表达激发出黄绿色荧光,具有启动子的活性。
4.确定了毛白杨雄株-D转化的潮霉素临界筛选浓度为10 mg/L。将CesAP-GFP基因转入毛白杨中,多次继代筛选后获得了少量抗性愈伤组织,GFP荧光检测结果表明,与阴性对照和阳性对照相比,抗性愈伤组织只有部分部位激发出黄绿色荧光,证明JCesAP,YCesAP和MDCesAP序列除具有启动子活性外,还均具有组织特异性。
The main purpose of this paper is to detect the xylem-specific promoter in the three sequences of JCesAP,YCesAP and MDCesAP. If the promoters can be used to incorporate Coleoptera-resistance genes and made these genes to be only expressed in the xylem, this will provide scientific basis for controlling the stem-boring pests especially Cerambycidae. Xylem-specific cellulose synthase gene (CesA gene) is confined to developing xylem cells during plant normal growth only and induced in developing phloem fibers undergoing tension stress also. The cis-acting elements of JCesAP, YCesAP and MDCesAP were analyzed by the PLACE database, the plant expression vector pJCesAP-GUS and pYCesAP-GUS were constructed using DNA recombination technology separately, and then all the plant expression vectors including our lab preserving harboring CesAP-GFP/CesAP-GUS were transformed into tobacco using leaf-disk method. The promoter activity and reporter gene expression levels were tested. Selecting Populus tomentosa Stub-D as the experiment materials, CesAP-GFP gene were integrated into Populus tomentosa Stub-D by Agrobacterium-mediated transformation method. Some resistant calli were obtained and observed under fluorescence microscope. The main results were as follows:
1. The cis-acting elements of JCesAP, YCesAP and MDCesAP amplified respectively in earlier studies were analyzed by the PLACE database; the results showed that, the three sequences all had the core element of promoter and the elements related to xylem-specific and wound-defense functions.
2. The plant expression vector pJCesAP-GUS and pYCesAP-GUS were constructed successfully using GUS as reporter gene. Each gene was promoted with CesA promoter, containing nptⅡgene as a selective marker. The plant expression vectors were transformed into Agrobacterium tumefaciens strain AGL-Ⅰ.
3. Cefotaxime (CTX) critical concentration was 400 mg/L for the tobacco transformation. The plant expressed vector pCesAP-GFP was transformed to Agrobacterium tumefaciens strain AGL-I. GFP expression showed that, the green fluorescence was observed in all transgenic tobacco, it proved all the three sequences were of promoter activity.
4. Hygromycin (Hyg) critical concentration was 10 mg/L for the Populus tomentosa Stub-D transformation. pCesAP-GFP gene was transformed to Populus tomentosa Stub-D leaves. Through several transformations and selections, some resistant calli were obtained, green fluorescence was only observed in local tissues of the transgenic calli comparing to negative control and positive control, it proved that the three sequences not only obtain the promoter activities, but also tissue-specific.
引文
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