草酸氧化酶基因（OxO）转化大豆成熟胚的研究
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摘要
大豆是我国非常重要的油料作物之一,是豆科植物中最富含有营养又易于消化的食物。大豆菌核病对大豆的生长危害严重,菌核病在大豆的整个生育期都可以发病,并造成叶腐、苗枯、荚腐等症状。每次发病轻则减产20%-30%,严重时减产可达到50%-90%,甚至导致绝产。在农业生产上,人们主要采取化学防治、轮作等方式防治菌核病。但实际防治效果并不明显。因此,培育出高品质的大豆品种是十分必要的。然而,传统育种所需时间过长,对后代的表现预见性较差。因此随着基因工程技术的发展,转基因育种逐渐成为现阶段获得优良作物品种的重要方法。
众多研究表明,菌核病致病与核盘菌分泌的草酸毒素有着十分重要的关系。而草酸氧化酶(oxalate oxidase, OxO, E.C.1.2.3.4)则是最主要的草酸降解酶之一。草酸氧化酶主要存在于少数的禾谷类的植物中,因它能够使草酸降解为H202和C02,所以可以降解掉草酸的毒性,从而延缓病原真菌的侵染。因此我们可以将草酸氧化酶基因OxO转化进入大豆植株中,以期大豆植株获得对菌核病的抗性。
本研究以大豆成熟胚为主要研究对象,利用根癌农杆菌介导OxO基因遗传转化大豆成熟胚胚尖,并通过组织化学染色和PCR技术,初步鉴定目的基因已整合到目标材料中。在研究过程中,首先,我们建立了一种适合大豆成熟胚胚尖的再生体系,其次,我们系统分析了农杆菌浸染时间、共培养时间、恢复培养时间等因素对大豆遗传转化的影响,进而成功建立了一种适合大豆成熟胚胚尖遗传转化的体系。具体研究内容包括以下3个部分：
1.建立了一种适合于大豆成熟胚胚尖的再生体系
选取成熟光滑、表面无病斑的大豆种子→70%酒精消毒浸泡50s-1min→2%NaClO溶液浸泡大豆种子,期间不定期的摇晃20min→无菌水清洗4-5次→无菌水浸泡24h→剥取胚尖,将其接种于预培养基中培养2d→将外植体转接于含有3mg/L6-BA和0.05mg/L IBA的不定芽诱导培养基中→待不定芽生长至2-3cm时,将不定芽切下转接于含0.3mg/L IBA的生根培养基中→待不定根生长茁壮时,炼苗移栽。
2.建立了一种适合于大豆成熟胚胚尖外植体的遗传转化体系
大豆种子消毒后浸泡过夜→剥取胚尖,预培养基中培养2d→农杆菌LBA4404/3303.OxO活化、培养→用OD600=0.6一0.8ABS的浸染液浸染胚尖外植体20h,共培养基中暗培养5d→清洗两次外植体,恢复培养7d→筛选培养30d,每隔10d更换一次培养基→将经过筛选存活下来的抗性芽诱导生根→待不定根长至茁壮的时候,便可将其炼苗,移栽于营养土中。
3.转基因大豆苗的组织化学检测与鉴定
取经过筛选存活下来的大豆苗的新鲜叶片,分别进行GUS组织化学染色,gfd荧光显微观察及PCR检测。结果显示：有6株转化苗的叶片被染成了蓝色,且可以观察到绿色荧光,PCR也扩增出了与阳性对照相同的条带。结果表明外源基因OxO已成功整合至大豆基因组中。
Soybean, which is one of the most important oil crops in China and around the world, is a food of the most rich in nutrition and easy to digest in the all of Leguminous plants, and also is the most abundant and cheapest source of protein. Sclerotinia is a seriously harmful disease for soybean. It can harm soybean during the whole growth period of soybean, and cause a series of symptoms, such as leaves rot, seedling blight, pod rot. Sclerotinia can cause the reduction of soybean. And the reduction of soybean can reach20%to30%, and50%to90%when Sclerotinia is seriously for soybean, and even more have no harvests. For agricultural production, people take measures such as chemical control, cropping system to prevent Sclerotinia. But it is not obvious for the effect of prevention. So it is necessary for us to cultivate new soybean varieties of high-quality. However, the traditional breeding methods is time-consuming and uncertainty of the phenotype of offspring. With the development of the technology of genetic engineering, transgenic breeding has become an important method for the present stage to obtain excellent crop varieties.
Many researches indicate that there is an important relationship between Sclerotinia and oxalic acid which Sclerotinia scleroterum secrete. Oxalate oxidase (oxalate oxidase, OxO， E.C.1.2.3.4) is one of the most primary oxalic acid degradation enzyme. Oxalate oxidase mainly exists in the few cereal plants. It can degrade oxalic acid into H2O2and CO2to weaken the toxicity of oxalic acid, so that delay infection of pathogenic fungi. So we use oxalate oxidase gene (OxO) to transfect soybean plants, in order to get soybean plants which have high resistance to Sclerotinia.
In this study, we use OxO gene to transfect embryonic tips of mature soybeans mediated by Agrobacterium tumefaciens. First, we established a regeneration system for embryonic tips of soybean. Second, we discussed the influence factors of genetic transformation of soybean which are the time of transformation mediated by Agrobacterium tumefaciens, the time of co-culture and recovery-culture and so on. As a result, we established a transformation system that adapts embryonic tips of soybean. The main results were as follows:
1. Establishment of regeneration system for embryonic tips of soybean
Selected mature soybean seeds which are smooth and no lesion on surface→washed the soybean seeds with70%ethanol for50s-1min→soaked seeds in2%NaCIO for20min, we need shake irregularly→washed seeds with sterile water4～5times→soaked in sterile water for24h→stripped embryo tip as explants, and cultured on pre-incubation medium for2days→cultured the explants on shoot induction medium which contains3mg/L6-BA and0.05mg/L IBA→when the shoots were about2-3cm, cut the shoots and transferred them to rooting medium which contains0.3mg/L IBA→when the roots of shoots were strong, transferred them to nutritive soil.
2. Establishment of Agrobacterium-mediated transformation system for embryonic tips of soybean
Sterilized soybean seeds and soaked in sterile water for the whole night→stripped embryo tip as explants, and cultured on pre-incubation medium for2days→activated and cultured A. tumefaciens LBA4404/3304-OxO→infected embryo tips with infection solution whose OD600=0.6-0.8ABS for20h, then co-culture for5days in the darkness→washed explants2times, and transferred them to recovery-culture medium for7days→transferred explants to selection-culture medium for30days, and changed another fresh selection-culture medium for10days→selected the survival shoots to rooting medium→when the roots of shoots were strong, transferred them to nutritive soil.
3. Histochemical detection and identification of transgenic soybean plants
Cut fresh leaves of soybean plants which were survival from the selection-culture medium, to detection with GUS staining, observation of gfp fluorescence and PCR amplification. The results showed, there were6strains which were dyed blue, and can observe green fluorescence. And PCR amplification also showed that there were same band with positive control. All the results show that the foreign gene OxO has been successfully integrated into the soybean genome.
众多研究表明,菌核病致病与核盘菌分泌的草酸毒素有着十分重要的关系。而草酸氧化酶(oxalate oxidase, OxO, E.C.1.2.3.4)则是最主要的草酸降解酶之一。草酸氧化酶主要存在于少数的禾谷类的植物中,因它能够使草酸降解为H202和C02,所以可以降解掉草酸的毒性,从而延缓病原真菌的侵染。因此我们可以将草酸氧化酶基因OxO转化进入大豆植株中,以期大豆植株获得对菌核病的抗性。
本研究以大豆成熟胚为主要研究对象,利用根癌农杆菌介导OxO基因遗传转化大豆成熟胚胚尖,并通过组织化学染色和PCR技术,初步鉴定目的基因已整合到目标材料中。在研究过程中,首先,我们建立了一种适合大豆成熟胚胚尖的再生体系,其次,我们系统分析了农杆菌浸染时间、共培养时间、恢复培养时间等因素对大豆遗传转化的影响,进而成功建立了一种适合大豆成熟胚胚尖遗传转化的体系。具体研究内容包括以下3个部分：
1.建立了一种适合于大豆成熟胚胚尖的再生体系
选取成熟光滑、表面无病斑的大豆种子→70%酒精消毒浸泡50s-1min→2%NaClO溶液浸泡大豆种子,期间不定期的摇晃20min→无菌水清洗4-5次→无菌水浸泡24h→剥取胚尖,将其接种于预培养基中培养2d→将外植体转接于含有3mg/L6-BA和0.05mg/L IBA的不定芽诱导培养基中→待不定芽生长至2-3cm时,将不定芽切下转接于含0.3mg/L IBA的生根培养基中→待不定根生长茁壮时,炼苗移栽。
2.建立了一种适合于大豆成熟胚胚尖外植体的遗传转化体系
大豆种子消毒后浸泡过夜→剥取胚尖,预培养基中培养2d→农杆菌LBA4404/3303.OxO活化、培养→用OD600=0.6一0.8ABS的浸染液浸染胚尖外植体20h,共培养基中暗培养5d→清洗两次外植体,恢复培养7d→筛选培养30d,每隔10d更换一次培养基→将经过筛选存活下来的抗性芽诱导生根→待不定根长至茁壮的时候,便可将其炼苗,移栽于营养土中。
3.转基因大豆苗的组织化学检测与鉴定
取经过筛选存活下来的大豆苗的新鲜叶片,分别进行GUS组织化学染色,gfd荧光显微观察及PCR检测。结果显示：有6株转化苗的叶片被染成了蓝色,且可以观察到绿色荧光,PCR也扩增出了与阳性对照相同的条带。结果表明外源基因OxO已成功整合至大豆基因组中。
Soybean, which is one of the most important oil crops in China and around the world, is a food of the most rich in nutrition and easy to digest in the all of Leguminous plants, and also is the most abundant and cheapest source of protein. Sclerotinia is a seriously harmful disease for soybean. It can harm soybean during the whole growth period of soybean, and cause a series of symptoms, such as leaves rot, seedling blight, pod rot. Sclerotinia can cause the reduction of soybean. And the reduction of soybean can reach20%to30%, and50%to90%when Sclerotinia is seriously for soybean, and even more have no harvests. For agricultural production, people take measures such as chemical control, cropping system to prevent Sclerotinia. But it is not obvious for the effect of prevention. So it is necessary for us to cultivate new soybean varieties of high-quality. However, the traditional breeding methods is time-consuming and uncertainty of the phenotype of offspring. With the development of the technology of genetic engineering, transgenic breeding has become an important method for the present stage to obtain excellent crop varieties.
Many researches indicate that there is an important relationship between Sclerotinia and oxalic acid which Sclerotinia scleroterum secrete. Oxalate oxidase (oxalate oxidase, OxO， E.C.1.2.3.4) is one of the most primary oxalic acid degradation enzyme. Oxalate oxidase mainly exists in the few cereal plants. It can degrade oxalic acid into H2O2and CO2to weaken the toxicity of oxalic acid, so that delay infection of pathogenic fungi. So we use oxalate oxidase gene (OxO) to transfect soybean plants, in order to get soybean plants which have high resistance to Sclerotinia.
In this study, we use OxO gene to transfect embryonic tips of mature soybeans mediated by Agrobacterium tumefaciens. First, we established a regeneration system for embryonic tips of soybean. Second, we discussed the influence factors of genetic transformation of soybean which are the time of transformation mediated by Agrobacterium tumefaciens, the time of co-culture and recovery-culture and so on. As a result, we established a transformation system that adapts embryonic tips of soybean. The main results were as follows:
1. Establishment of regeneration system for embryonic tips of soybean
Selected mature soybean seeds which are smooth and no lesion on surface→washed the soybean seeds with70%ethanol for50s-1min→soaked seeds in2%NaCIO for20min, we need shake irregularly→washed seeds with sterile water4～5times→soaked in sterile water for24h→stripped embryo tip as explants, and cultured on pre-incubation medium for2days→cultured the explants on shoot induction medium which contains3mg/L6-BA and0.05mg/L IBA→when the shoots were about2-3cm, cut the shoots and transferred them to rooting medium which contains0.3mg/L IBA→when the roots of shoots were strong, transferred them to nutritive soil.
2. Establishment of Agrobacterium-mediated transformation system for embryonic tips of soybean
Sterilized soybean seeds and soaked in sterile water for the whole night→stripped embryo tip as explants, and cultured on pre-incubation medium for2days→activated and cultured A. tumefaciens LBA4404/3304-OxO→infected embryo tips with infection solution whose OD600=0.6-0.8ABS for20h, then co-culture for5days in the darkness→washed explants2times, and transferred them to recovery-culture medium for7days→transferred explants to selection-culture medium for30days, and changed another fresh selection-culture medium for10days→selected the survival shoots to rooting medium→when the roots of shoots were strong, transferred them to nutritive soil.
3. Histochemical detection and identification of transgenic soybean plants
Cut fresh leaves of soybean plants which were survival from the selection-culture medium, to detection with GUS staining, observation of gfp fluorescence and PCR amplification. The results showed, there were6strains which were dyed blue, and can observe green fluorescence. And PCR amplification also showed that there were same band with positive control. All the results show that the foreign gene OxO has been successfully integrated into the soybean genome.
引文
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