农杆菌介导菊苣遗传转化体系的建立及富硫氨基酸基因的导入和表达定位
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摘要
菊苣为菊科多年生草本植物,广泛分布于世界各地,是目前国内外广泛应用的主要饲草和蔬菜之一。菊苣体内含有多种重要的物质如倍半萜烯、内酯、内酯苷、黄酮类、香豆素、花色素甙、有机酸和细胞激肽类等,菊苣虽然营养丰富,但是蛋白质含量低。植物蛋白是包括人类在内的动物界所消耗蛋白质的主要来源。然而,作为日常蛋白质的主要来源,植物蛋白因缺乏某些人体必需氨基酸而营养不够平衡。一般来说,禾谷类蛋白质的赖氨酸含量较低；豆类和蔬菜蛋白质则缺乏含硫氨基酸,如蛋氨酸和半胱氨酸。含硫氨基酸在有效改善奶制品、肉类以及羊毛产量等方面起着决定性的作用,并可增加牲畜的重量。利用基因转化技术可以提高菊苣中含硫氨基酸的含量,改善菊苣品质。
本研究在建立菊苣高频再生体系和遗传转化体系的基础上,将构建的植物表达载体pCMBIA1302-γ-zein和pCMBIA1302-zeolin通过农杆菌介导法转入菊苣中,获得抗性植株,对抗性植株进行了分子检测和表达定位研究.表明外源基因已整合进菊苣基因组中,并得到表达,为菊苣分子育种提供了基础材料和理论依据。主要研究结果如下：
1、研究了培养基、基因型、外植体和激素等因素对菊苣再生的影响,建立了菊苣稳定高效再生体系：普那菊苣真叶在MS培养基中添加2.0mg/L 6-BA和0.2mg/L IBA的激素组合,能得到98.92%的愈伤诱导率,在相同培养基中分化率达到93.74%,平均每个外植体的分化芽数达到19.46个,分化芽在添加0.1 mg/L NAA的1/2 MS培养基中,生根率达到98.77%,炼苗后成活率达95%以上。
2、利用RAPD分子标记技术研究了体细胞无性系的遗传稳定性。从12条引物中筛选出2条RAPD引物对15个来自同一叶片一次和二次体细胞再生的植株和原供体植株DNA进行检测,结果表明菊苣一次直接体细胞再生后代没有体细胞无性系变异,二次再生植株中有1株发生了1条DNA多态性差异,说明本研究建立的经一次组织培养获得的再生植株遗传稳定,不易发生体细胞无性系变异,可用于遗传转化。
3、通过克隆载体pMD18-T的介导,将γ-zein和zeolin基因与质粒pCAMBIA1302相连,构建了以CaMV35S为启动子的植物表达载体pCM-γ-zein和pCM-zeolin,该载体含有hpt选择标记基因,并将该载体导入农杆菌LBA4404中,为遗传转化奠定了基础。
4、建立起菊苣遗传转化筛选系统：共培养结束后,转化体在无hyg的预培养基上延迟培养1-2d,在愈伤培养和芽分化阶段添加25mg/L hyg;抗性芽扩繁阶段添加15mg/L hyg,生根阶段添加10mg/L hyg。农杆菌介导转化时以头孢霉素(Cef)为抑菌剂,愈伤培养和分化阶段添加500mg/L,植株再生阶段添加250mg/L。
5、采用正交设计,利用叶盘转化法,对影响农杆菌介导菊苣遗传转化的预培养时间、农杆菌菌液浓度、浸染时间、共培养乙酰丁香酮(AS)浓度和共培养pH等因素进行了优化,以抗性率为指标,确定了农杆菌介导菊苣叶片转化的适宜条件为：预培养时间为2d,农杆菌悬浮液OD600值为0.4Abs,感染时间10min,共培养基pH值5.4～5.8,共培养时间3d,共培养基中添加100umol/L的AS,通过以上研究,构建了农杆菌介导菊苣叶片的遗传转化体系。用γ-zein和zeoin基因对该优化体系验证结果表明,两基因的Hyg抗性芽发生率平均为13.52%,比基本转化条件下的2.95%提高了3.58倍,证明了优化的参数能大幅度提高转化效率。
6、对获得的抗性植株进行PCR检测、PCR-Southern、Dot blot、Southern杂交分析、RT-PCR检测、离体叶片潮霉素抗性鉴定,表明外源目的基因随机整合进菊苣基因组中,并在RNA水平上得到了转录。
7、对转γ-zein基因的阳性植株进行了激光共聚焦显微镜定位观察,结果表明：转化的γ-zein基因主要在转基因菊苣表皮的细胞核中和根尖细胞膜上进行了表达。
8、本次实验共获得14株转γ-zein基因的阳性植株,阳性率为42.42%,转化率为5.6%,12株转zeolin基因的阳性植株,阳性率为46.15%,转化率为6.38%。
Chicory (Cichorium intybus L.) is a member of the compositae family and a perennial herb, widely distributed in the world. It is one of the major forage and vegetable widely utilized at home and abroad. C. intybus L.contains large numbers of pharmaceutically important phytochemicals such as sesquiterpene lactones, glycosides, flavonoids, coumarins, anthocyanins, organic acids,and cytokinins. C. intybus has rich nutrition, but protein content is very few in its body. Phytoprotein is main source of protein for human and haplo-stomach animal, but the plant nutrition is not overall such as low content lysine and tryptophane in seed protein of grain crop and lack of methionine and cysteine in pulse and greengrocery. Sulphur-containing amino acid (SAAs) plays an imimportant role in improving the milk and meat quality and wool yield and the weight of livestock. It can increase the content of SAAs in C. intybus L.and improve the quality by utilizing the genetic transformation technology.
Based on the construction of high frequency regeneration system and genetic transformation system of chicory (C.intybus L.).γ-zein and zeolin gene were transformed into cnicory leaf disc via Agrobacterium infection transformation. The transformed plants were gained. Molecule detection and expression location were carried out. It was showed that the exogenous gene had integrated into the genome of chicory. Now the major results studied in the paper are showed as followings:
1. Effect of basal medium, genotype, explants, hormone type and concentration on regeneration of chicory were studied and a reliable and efficient regeneration system was established. The optimal regeneration medium for the euphylla of C.intybus L.cv.Puna was MS+6-2.0 mg/L BA +0.2mg/L IBA, with the max callus induction frequcy being 98.92% and the regeneration rate being 93.74%, the mean number of shoots per explan being 19.46.The regenerated shoots were transferred to 1/2 MS medium supplemented with 0.1mg/L NAA for rooting, and it was very easy to root and the rooting rate was 98.77% and survival rate was over 95%.
2. The genetic stability of regenerated plants was analyzed using RAPD markers. The results of RAPD amplification showed that the genetic stability of the regenerated plantlets with the first culturing was maintained, though slight variations were found with the second-culturing regeneration plant. This suggested that in vitro regeneration were a good rapid propagation way to obtain genetic stable descends of chicory.
3.Introduced by the cloning vector pMD18-T,γ-zein and zeolin gene and plasmid pCAMBIA1302 were joined, thus plant expression vector pCAM-γ-zein and pCAM-zeolin with selective marker hpt gene were constructed. The gene was controlled by CaMV35S promoter. The recombinant plasmid was transformed into Agrobacterium tumefaciens LBA4404 for genetic transformation.
4.Establishment of a genetic transformation selection system for chicory:Transformant was cultured on pre-medium with no hyg for 1-2d after the co-culture, then add 25mg/L hyg at callus cultivation and shoot regeneration stage, while 15mg/L hyg at enlarge propagation period and 10 mg/L hyg at rooting period. Cefotaxime (Cef) was used for the bacteriostasis in Agrobacterium infection transformation and it was 500mg/L at callus cultivation and regeneration stage while 250mg/L Cef at rooting stage.
5.Optimized protocols of Agrobacterium-mediated transformation for chicory and plant regeneration were developed by orthogonal design.The results indicated that the following conditions were outstanding for the improvement of transformation efficiency of chicory:Addition of 2mg/L6-BA and 0.2mg/LIBA to leaf Pre-culture medium, Pre-culture time of 2d, bacterial suspension OD600 of 0.4Abs, infection time of 10min, co-culture medium pH of 5.4-5.6, co-culture time of 3d and supplementation of 100umol/L acetosyringone to Agrobacterium co-culture medium.On the basis of the above studies, Agrobacterium-mediated transformation system of chicory euphyll has been set up. For verification the Agrobacterium-mediated transformation system,γ-zein gene and zeolin gene were incorporated into genomes of chicory, the result showed that the average resistant frequency of hyg was 13.52%.and it was 3.58 times more than that of basic transformation condition.
6. Resistant plants were detected by PCR assay. PCR-Southern、Dot blot、Southern hybridization, RT-PCR detection and analysis identification of hygromycin resistance of in vitro leaves, and the result showed that the exogenous gene had integrated into the genome of chicory and were transcribed on RNA level.
7.γ-zein gene was expressed in cell nucleus of leaf epidermis cell and cell membrane of root tip of transgenic chicory by confocal microscopy localization
8. 14 positive plants withγ-zein gene and 12 positive plant with zeolin were obtained by this experiment,the positive plant frequency of transformationγ-zein gene and zeolin gene was 42.42% and 46.15% based on numbers of PCR, respectively. The averaged transformation frequency of two genes was 5.6% and 6.38%, respectively, based on numbers of resistant frequency and PCR of regenerated transgenic plants.
本研究在建立菊苣高频再生体系和遗传转化体系的基础上,将构建的植物表达载体pCMBIA1302-γ-zein和pCMBIA1302-zeolin通过农杆菌介导法转入菊苣中,获得抗性植株,对抗性植株进行了分子检测和表达定位研究.表明外源基因已整合进菊苣基因组中,并得到表达,为菊苣分子育种提供了基础材料和理论依据。主要研究结果如下：
1、研究了培养基、基因型、外植体和激素等因素对菊苣再生的影响,建立了菊苣稳定高效再生体系：普那菊苣真叶在MS培养基中添加2.0mg/L 6-BA和0.2mg/L IBA的激素组合,能得到98.92%的愈伤诱导率,在相同培养基中分化率达到93.74%,平均每个外植体的分化芽数达到19.46个,分化芽在添加0.1 mg/L NAA的1/2 MS培养基中,生根率达到98.77%,炼苗后成活率达95%以上。
2、利用RAPD分子标记技术研究了体细胞无性系的遗传稳定性。从12条引物中筛选出2条RAPD引物对15个来自同一叶片一次和二次体细胞再生的植株和原供体植株DNA进行检测,结果表明菊苣一次直接体细胞再生后代没有体细胞无性系变异,二次再生植株中有1株发生了1条DNA多态性差异,说明本研究建立的经一次组织培养获得的再生植株遗传稳定,不易发生体细胞无性系变异,可用于遗传转化。
3、通过克隆载体pMD18-T的介导,将γ-zein和zeolin基因与质粒pCAMBIA1302相连,构建了以CaMV35S为启动子的植物表达载体pCM-γ-zein和pCM-zeolin,该载体含有hpt选择标记基因,并将该载体导入农杆菌LBA4404中,为遗传转化奠定了基础。
4、建立起菊苣遗传转化筛选系统：共培养结束后,转化体在无hyg的预培养基上延迟培养1-2d,在愈伤培养和芽分化阶段添加25mg/L hyg;抗性芽扩繁阶段添加15mg/L hyg,生根阶段添加10mg/L hyg。农杆菌介导转化时以头孢霉素(Cef)为抑菌剂,愈伤培养和分化阶段添加500mg/L,植株再生阶段添加250mg/L。
5、采用正交设计,利用叶盘转化法,对影响农杆菌介导菊苣遗传转化的预培养时间、农杆菌菌液浓度、浸染时间、共培养乙酰丁香酮(AS)浓度和共培养pH等因素进行了优化,以抗性率为指标,确定了农杆菌介导菊苣叶片转化的适宜条件为：预培养时间为2d,农杆菌悬浮液OD600值为0.4Abs,感染时间10min,共培养基pH值5.4～5.8,共培养时间3d,共培养基中添加100umol/L的AS,通过以上研究,构建了农杆菌介导菊苣叶片的遗传转化体系。用γ-zein和zeoin基因对该优化体系验证结果表明,两基因的Hyg抗性芽发生率平均为13.52%,比基本转化条件下的2.95%提高了3.58倍,证明了优化的参数能大幅度提高转化效率。
6、对获得的抗性植株进行PCR检测、PCR-Southern、Dot blot、Southern杂交分析、RT-PCR检测、离体叶片潮霉素抗性鉴定,表明外源目的基因随机整合进菊苣基因组中,并在RNA水平上得到了转录。
7、对转γ-zein基因的阳性植株进行了激光共聚焦显微镜定位观察,结果表明：转化的γ-zein基因主要在转基因菊苣表皮的细胞核中和根尖细胞膜上进行了表达。
8、本次实验共获得14株转γ-zein基因的阳性植株,阳性率为42.42%,转化率为5.6%,12株转zeolin基因的阳性植株,阳性率为46.15%,转化率为6.38%。
Chicory (Cichorium intybus L.) is a member of the compositae family and a perennial herb, widely distributed in the world. It is one of the major forage and vegetable widely utilized at home and abroad. C. intybus L.contains large numbers of pharmaceutically important phytochemicals such as sesquiterpene lactones, glycosides, flavonoids, coumarins, anthocyanins, organic acids,and cytokinins. C. intybus has rich nutrition, but protein content is very few in its body. Phytoprotein is main source of protein for human and haplo-stomach animal, but the plant nutrition is not overall such as low content lysine and tryptophane in seed protein of grain crop and lack of methionine and cysteine in pulse and greengrocery. Sulphur-containing amino acid (SAAs) plays an imimportant role in improving the milk and meat quality and wool yield and the weight of livestock. It can increase the content of SAAs in C. intybus L.and improve the quality by utilizing the genetic transformation technology.
Based on the construction of high frequency regeneration system and genetic transformation system of chicory (C.intybus L.).γ-zein and zeolin gene were transformed into cnicory leaf disc via Agrobacterium infection transformation. The transformed plants were gained. Molecule detection and expression location were carried out. It was showed that the exogenous gene had integrated into the genome of chicory. Now the major results studied in the paper are showed as followings:
1. Effect of basal medium, genotype, explants, hormone type and concentration on regeneration of chicory were studied and a reliable and efficient regeneration system was established. The optimal regeneration medium for the euphylla of C.intybus L.cv.Puna was MS+6-2.0 mg/L BA +0.2mg/L IBA, with the max callus induction frequcy being 98.92% and the regeneration rate being 93.74%, the mean number of shoots per explan being 19.46.The regenerated shoots were transferred to 1/2 MS medium supplemented with 0.1mg/L NAA for rooting, and it was very easy to root and the rooting rate was 98.77% and survival rate was over 95%.
2. The genetic stability of regenerated plants was analyzed using RAPD markers. The results of RAPD amplification showed that the genetic stability of the regenerated plantlets with the first culturing was maintained, though slight variations were found with the second-culturing regeneration plant. This suggested that in vitro regeneration were a good rapid propagation way to obtain genetic stable descends of chicory.
3.Introduced by the cloning vector pMD18-T,γ-zein and zeolin gene and plasmid pCAMBIA1302 were joined, thus plant expression vector pCAM-γ-zein and pCAM-zeolin with selective marker hpt gene were constructed. The gene was controlled by CaMV35S promoter. The recombinant plasmid was transformed into Agrobacterium tumefaciens LBA4404 for genetic transformation.
4.Establishment of a genetic transformation selection system for chicory:Transformant was cultured on pre-medium with no hyg for 1-2d after the co-culture, then add 25mg/L hyg at callus cultivation and shoot regeneration stage, while 15mg/L hyg at enlarge propagation period and 10 mg/L hyg at rooting period. Cefotaxime (Cef) was used for the bacteriostasis in Agrobacterium infection transformation and it was 500mg/L at callus cultivation and regeneration stage while 250mg/L Cef at rooting stage.
5.Optimized protocols of Agrobacterium-mediated transformation for chicory and plant regeneration were developed by orthogonal design.The results indicated that the following conditions were outstanding for the improvement of transformation efficiency of chicory:Addition of 2mg/L6-BA and 0.2mg/LIBA to leaf Pre-culture medium, Pre-culture time of 2d, bacterial suspension OD600 of 0.4Abs, infection time of 10min, co-culture medium pH of 5.4-5.6, co-culture time of 3d and supplementation of 100umol/L acetosyringone to Agrobacterium co-culture medium.On the basis of the above studies, Agrobacterium-mediated transformation system of chicory euphyll has been set up. For verification the Agrobacterium-mediated transformation system,γ-zein gene and zeolin gene were incorporated into genomes of chicory, the result showed that the average resistant frequency of hyg was 13.52%.and it was 3.58 times more than that of basic transformation condition.
6. Resistant plants were detected by PCR assay. PCR-Southern、Dot blot、Southern hybridization, RT-PCR detection and analysis identification of hygromycin resistance of in vitro leaves, and the result showed that the exogenous gene had integrated into the genome of chicory and were transcribed on RNA level.
7.γ-zein gene was expressed in cell nucleus of leaf epidermis cell and cell membrane of root tip of transgenic chicory by confocal microscopy localization
8. 14 positive plants withγ-zein gene and 12 positive plant with zeolin were obtained by this experiment,the positive plant frequency of transformationγ-zein gene and zeolin gene was 42.42% and 46.15% based on numbers of PCR, respectively. The averaged transformation frequency of two genes was 5.6% and 6.38%, respectively, based on numbers of resistant frequency and PCR of regenerated transgenic plants.
引文
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