拟南芥LEAFY及龙眼LEAFY同源基因转化雪柑研究
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摘要
柑桔(Citrus L.)是当今世界最重要的水果之一,也是我国南方最重要的经济果树。柑桔是多年生木本果树,以往的常规育种由于存在童期长、遗传高度杂合性和多胚现象,使得柑桔育种工作困难重重。近年来,不断完善的转基因技术为我们提供了新的育种和品种改良手段。
目前,转基因作物的安全性问题受到极大关注。利用遗传转化技术改良柑桔品种首先要考虑生物安全性问题,使用安全的选择标记基因是有效方法之一。
本实验以福建传统优良甜橙品种雪柑(C. sinensis L.Osb.)为材料,在初步建立以PMI(Phospho-mannose-isomerase,磷酸甘露糖异构酶)基因为选择标记转基因体系基础上,进一步优化转化体系;同时将拟南芥LEAFY基因以及龙眼LEAFY同源基因LLFY分别导入雪柑,以期培育童期短的雪柑新品系并研究LLFY基因的功能;本实验还对农杆菌介导的“针刺”法进行了探索研究,为柑桔和木本植物的转基因研究提供了新的思路。主要研究结果如下:
1.以PMI基因为选择标记雪柑遗传转化体系的优化。通过对影响转化的各种因素的优化实验建立了雪柑以PMI基因/甘露糖选择标记系统的高效遗传转化体系。实验结果表明,在菌液OD600为0.6,1mg·L~(-1) 2,4-D预处理3h,农杆菌侵染30min,4d的共培养时间,选择压为甘露糖20g·L~(-1)的情况下大大提高了转化后的再生率,而在筛选培养基中加入2mg·L~(-1)BA+0.5mg·L~(-1)NAA的情况下有助于抗性大苗的筛选。
2. LFY和LLFY基因导入雪柑的研究。以优化的条件为转化体系,将拟南芥LEAFY基因以及龙眼LEAFY同源基因LLFY分别导入雪柑中,获得转基因植株。研究表明,抗性芽在附加20g·L~(-1)甘露糖和10g·L~(-1)蔗糖的壮苗培养基中能够有效快速淘汰假阳性抗性芽;通过CPR和PCR检测, LFY和LLFY基因的大苗转化率分别为:3.7%和7.4%;通过滤纸桥生根法对转基因芽进行了生根,获得完整植株并移栽成功。
3.农杆菌介导“针刺法”LFY基因转化雪柑的研究。以雪柑种子为外植体,以pC1301-PMI-LFY重组载体为转化载体,将蘸有农杆菌侵染液的接种针在种子胚的顶端分生组织部位刺2~3次,深度1mm左右,当实生苗成活后移栽。通过PCR检测和PCR-Southern杂交检测得到了1株转基因植株,转化率为4.35%。针刺法简便快速,在雪柑上能够成功应用,为木本植物的遗传转化提供了新的思路。
4.龙眼LFY同源基因LLFY和SLLFY植物表达载体的构建。将红核子龙眼LLFY基因插入载体pC2300-35S-OCS的35S启动子和OCS终止子之间,构建了pC2300-35S-LLFY-OCS植物表达载体;将四季蜜龙眼SLLFY基因插入载体pC2300-35S-OCS的35S启动子和OCS终止子之间,构建了pC2300-35S-SLLFY-OCS植物表达载体,为研究这两个基因的功能创造条件。
Citrus is one of the most important economic fruits in the world and the south China and also is a perennial and woody fruit tree characterized by long juvenile period, genetic heterozygosity and polyembryony, which made the breeding of this specie become difficult. In recent years, transgene technology had brought a novel way for the improvement of cultivars.
At the present time, more and more attention were paid on the issue of the biosafety of transgenic crops. The application of selective marker gene with biology safety in the genetic transformation was an effective solution.
The sweet orange cultivar of“Xuegan”(C. sinensis L.Osb.) was used as the material to optimize the genetic transformation system, based on the PMI (Phospho-mannose-isomerase) selection system established earlier. LEAFY gene of Arabidopsis and its ortholog LLFY in longan were both transformed into“Xuegan”genome for getting short juvenile varieties. Additionally, the exploring of the transformation using method of prickling mediated by Agrobacterium tumefaciens was also carried out, providing the new mehtod for the transgene of Citrus and woody trees. The main results were as follows:
1. Optimization of genetic transformation system of“Xuegan”using PMI selection marker. At the condition of 0.6 at OD600 of Agrobacterium mixture, explants pretreatment with 1mg·L~(-1) 2,4-D for 3 h, infection for 30 min, co-cultivation for 4d, 20g·L~(-1) mannose for selection, the regeneration rate was markedly increased after transformation. Supplement with 2mg·L~(-1) BA and 0.5mg·L~(-1) NAA on the media were benefit for the selection of resistant big plantlets.
2. Transformation of LFY and LLFY into“Xuegan”. Using the genetic transformation system optimized, LFY and LLFY were transformed into“Xuegan”. Transgentic plantlets were obtained. Medium for seedling strengthening were added with 20g·L~(-1) mannose and 10g·L~(-1) sucrose to screen resistant transgenic plantlets frequently and effectively. CPR and PCR positivity rates of resistant plantlets transformed with LFY and LLFY were 3.7% and 7.4% for the big ones, respectively, suggesting the target genes were successfully integrated into“Xuegan”genome. The transgenic plantlets were induced to root using method of filter paper bridge and transplanted.
3. Genetic transformation of“Xuegan”using method of prickling mediated by Agrobacterium tumefaciens was carried out. To inoculate A.tumefaciens into the embryonic apical meristerm of the soaked seeds, a region on the seed suiface where a shoot would later emerge was pierced twice up to a depth of about 1mm with a needle dipped in the A. tumefaciens inoculum.Germinated seedings were transplanted.One transgenic plantlet was obtained, which was confirmed by PCR assay and PCR-Southern blot, with the transformation rate of 4.35%, suggesting the feasibility of this method on the genetic transformation of“Xuegan”and providing the new method for the transgene of woody trees.
4. Construction of plant expression vector of homologous genes of LLFY and SLLFY in longan. LLFY gene of“Honghezi”and SLLFY gene of“Sijimi”were inserted into the pC2300-35S-OCS vector between 35S promoter and OCS terminator to produce the plant expression vectors of pC2300-35S-LLFY-OCS and pC2300-35S-SLLFY-OCS respectively for the research of the function of the two genes.
目前,转基因作物的安全性问题受到极大关注。利用遗传转化技术改良柑桔品种首先要考虑生物安全性问题,使用安全的选择标记基因是有效方法之一。
本实验以福建传统优良甜橙品种雪柑(C. sinensis L.Osb.)为材料,在初步建立以PMI(Phospho-mannose-isomerase,磷酸甘露糖异构酶)基因为选择标记转基因体系基础上,进一步优化转化体系;同时将拟南芥LEAFY基因以及龙眼LEAFY同源基因LLFY分别导入雪柑,以期培育童期短的雪柑新品系并研究LLFY基因的功能;本实验还对农杆菌介导的“针刺”法进行了探索研究,为柑桔和木本植物的转基因研究提供了新的思路。主要研究结果如下:
1.以PMI基因为选择标记雪柑遗传转化体系的优化。通过对影响转化的各种因素的优化实验建立了雪柑以PMI基因/甘露糖选择标记系统的高效遗传转化体系。实验结果表明,在菌液OD600为0.6,1mg·L~(-1) 2,4-D预处理3h,农杆菌侵染30min,4d的共培养时间,选择压为甘露糖20g·L~(-1)的情况下大大提高了转化后的再生率,而在筛选培养基中加入2mg·L~(-1)BA+0.5mg·L~(-1)NAA的情况下有助于抗性大苗的筛选。
2. LFY和LLFY基因导入雪柑的研究。以优化的条件为转化体系,将拟南芥LEAFY基因以及龙眼LEAFY同源基因LLFY分别导入雪柑中,获得转基因植株。研究表明,抗性芽在附加20g·L~(-1)甘露糖和10g·L~(-1)蔗糖的壮苗培养基中能够有效快速淘汰假阳性抗性芽;通过CPR和PCR检测, LFY和LLFY基因的大苗转化率分别为:3.7%和7.4%;通过滤纸桥生根法对转基因芽进行了生根,获得完整植株并移栽成功。
3.农杆菌介导“针刺法”LFY基因转化雪柑的研究。以雪柑种子为外植体,以pC1301-PMI-LFY重组载体为转化载体,将蘸有农杆菌侵染液的接种针在种子胚的顶端分生组织部位刺2~3次,深度1mm左右,当实生苗成活后移栽。通过PCR检测和PCR-Southern杂交检测得到了1株转基因植株,转化率为4.35%。针刺法简便快速,在雪柑上能够成功应用,为木本植物的遗传转化提供了新的思路。
4.龙眼LFY同源基因LLFY和SLLFY植物表达载体的构建。将红核子龙眼LLFY基因插入载体pC2300-35S-OCS的35S启动子和OCS终止子之间,构建了pC2300-35S-LLFY-OCS植物表达载体;将四季蜜龙眼SLLFY基因插入载体pC2300-35S-OCS的35S启动子和OCS终止子之间,构建了pC2300-35S-SLLFY-OCS植物表达载体,为研究这两个基因的功能创造条件。
Citrus is one of the most important economic fruits in the world and the south China and also is a perennial and woody fruit tree characterized by long juvenile period, genetic heterozygosity and polyembryony, which made the breeding of this specie become difficult. In recent years, transgene technology had brought a novel way for the improvement of cultivars.
At the present time, more and more attention were paid on the issue of the biosafety of transgenic crops. The application of selective marker gene with biology safety in the genetic transformation was an effective solution.
The sweet orange cultivar of“Xuegan”(C. sinensis L.Osb.) was used as the material to optimize the genetic transformation system, based on the PMI (Phospho-mannose-isomerase) selection system established earlier. LEAFY gene of Arabidopsis and its ortholog LLFY in longan were both transformed into“Xuegan”genome for getting short juvenile varieties. Additionally, the exploring of the transformation using method of prickling mediated by Agrobacterium tumefaciens was also carried out, providing the new mehtod for the transgene of Citrus and woody trees. The main results were as follows:
1. Optimization of genetic transformation system of“Xuegan”using PMI selection marker. At the condition of 0.6 at OD600 of Agrobacterium mixture, explants pretreatment with 1mg·L~(-1) 2,4-D for 3 h, infection for 30 min, co-cultivation for 4d, 20g·L~(-1) mannose for selection, the regeneration rate was markedly increased after transformation. Supplement with 2mg·L~(-1) BA and 0.5mg·L~(-1) NAA on the media were benefit for the selection of resistant big plantlets.
2. Transformation of LFY and LLFY into“Xuegan”. Using the genetic transformation system optimized, LFY and LLFY were transformed into“Xuegan”. Transgentic plantlets were obtained. Medium for seedling strengthening were added with 20g·L~(-1) mannose and 10g·L~(-1) sucrose to screen resistant transgenic plantlets frequently and effectively. CPR and PCR positivity rates of resistant plantlets transformed with LFY and LLFY were 3.7% and 7.4% for the big ones, respectively, suggesting the target genes were successfully integrated into“Xuegan”genome. The transgenic plantlets were induced to root using method of filter paper bridge and transplanted.
3. Genetic transformation of“Xuegan”using method of prickling mediated by Agrobacterium tumefaciens was carried out. To inoculate A.tumefaciens into the embryonic apical meristerm of the soaked seeds, a region on the seed suiface where a shoot would later emerge was pierced twice up to a depth of about 1mm with a needle dipped in the A. tumefaciens inoculum.Germinated seedings were transplanted.One transgenic plantlet was obtained, which was confirmed by PCR assay and PCR-Southern blot, with the transformation rate of 4.35%, suggesting the feasibility of this method on the genetic transformation of“Xuegan”and providing the new method for the transgene of woody trees.
4. Construction of plant expression vector of homologous genes of LLFY and SLLFY in longan. LLFY gene of“Honghezi”and SLLFY gene of“Sijimi”were inserted into the pC2300-35S-OCS vector between 35S promoter and OCS terminator to produce the plant expression vectors of pC2300-35S-LLFY-OCS and pC2300-35S-SLLFY-OCS respectively for the research of the function of the two genes.
引文
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