农杆菌介导玉米幼胚遗传转化的研究
发布时间：2019-02-16 21:48
【摘要】：目的本研究以高粱HCN途径的关键酶基因CYP71E1为目的基因,以玉米幼胚为外植体,通过根癌农杆菌介导进行玉米遗传转化的研究。优化A188玉米再生体系,分析不同外植体大小、农杆菌浓度、侵染时间和热处理时间对玉米遗传转化效率的影响,同时采用GUS组织化学染色和PCR技术对转基因植株进行鉴定。利用高效率转化技术解决因生物和非生物胁迫带来玉米产量和品质降低的瓶颈,为提供玉米新品种的转化提供理论基础。方法利用聚合酶链式反应(PCR)从高粱中扩增出1854bp的CYP71E1基因全长。利用基于位点特异性重组的Gateway技术,将CYP71E1基因重组到表达载体(p MDC141)中,将构建成功的表达载体CYP71E1-p MDC141导入农杆菌EHA105菌株。通过优化外植体材料、诱导愈伤培养基、分化培养基、生根培养基,建立玉米再生体系。以携带CYP71E1-p MDC141(含GUS基因)质粒的农杆菌EHA105菌株侵染玉米优良自交系A188的幼胚,通过对影响农杆菌介导玉米幼胚的外植体大小、农杆菌浓度、侵染时间、热预处理时间进行优化,提高GUS基因的平均转化效率,同时对转基因植株进行PCR和GUS检测。结果1、利用基于位点特异性重组的Gateway技术,通过TOPO克隆将高粱HCN途径的关键酶基因CYP71E1克隆到入门载体p CRTM8/GW/TOPO?中,在LR克隆酶作用下将入门克隆重组到表达载体(p MDC141)中,成功构建了p MDC141-CYP71E1植物表达载体,导入农杆菌EHA105中。2、建立高效玉米再生体系,玉米幼胚大小为1.0~1.2mm时,愈伤诱导率最高,通过正交实验的方法优化诱导胚性愈伤培养基,由此得出诱导玉米A188愈伤组织的MS基础培养基中添加L-pro、2,4-D、Ag NO3三因素的最优组合为:L-pro700mg/L、2,4-D 2.0mg/L、Ag NO38 mg/L。分化培养基中玉米素浓度为5 mg/L时分化效率最高。生根培养基中IBA的最适浓度为2 mg/L。3、建立高效遗传转化体系,玉米幼胚大小为1.0~1.2mm,GUS的平均转化效率为35.3%,农杆菌菌液浓度为OD660nm为0.8,GUS的平均转化效率为46.8%,侵染时间为5min和43℃热预处理3 min,GUS的平均转化效率分别为46.4%和45.4%。因此最佳转化条件为:选择幼胚大小为1.0~1.2mm、菌液浓度OD660nm=0.8、侵染时间5min、43℃热预处理3 min。4、对获得的转基因植株进行PCR和GUS检测,获得12株转基因苗。结论1、Gateway技术与传统的克隆方法相比具有阳性克隆率高、快速、灵活等优点,通过Gateway技术成功构建p MDC141-CYP71E1表达载体。2、通过优化外植体材料、愈伤诱导培养基、分化培养基、生根培养基,建立了A188玉米幼胚再生体系。3、通过对影响农杆菌介导玉米幼胚的部分因素进行优化,最佳转化条件为:玉米幼胚大小为1.0~1.2mm、菌液浓度OD660nm=0.8、侵染时间5min、43℃热预处理3min。4、获得了高粱HCN途径的关键酶基因CYP71E1阳性植株。
[Abstract]:Objective to study the genetic transformation of maize by Agrobacterium tumefaciens mediated by the key enzyme gene CYP71E1 of sorghum HCN pathway and maize embryo as explant. The regeneration system of A188 maize was optimized, and the effects of different explant size, Agrobacterium concentration, infection time and heat treatment time on the genetic transformation efficiency of maize were analyzed. The transgenic plants were identified by GUS histochemical staining and PCR technique. The high efficiency transformation technology was used to solve the bottleneck of maize yield and quality reduction caused by biological and abiotic stress, which provided the theoretical basis for the transformation of new maize varieties. Methods the CYP71E1 gene of 1854bp was amplified from sorghum by polymerase chain reaction (PCR). Using the Gateway technique based on site-specific recombination, the CYP71E1 gene was recombined into the expression vector (p MDC141), and the successfully constructed expression vector CYP71E1-p MDC141 was introduced into Agrobacterium tumefaciens EHA105 strain. Maize regeneration system was established by optimizing explant material, inducing callus medium, differentiation medium and rooting medium. Agrobacterium tumefaciens (EHA105) carrying CYP71E1-p MDC141 (containing GUS gene) plasmid was used to infect immature embryos of maize inbred line A188. The effects of Agrobacterium tumefaciens on the size of explant, concentration of Agrobacterium tumefaciens and infection time were studied. The thermal pretreatment time was optimized to improve the average transformation efficiency of GUS gene, and PCR and GUS were used to detect the transgenic plants. Results 1. Using Gateway technique based on site-specific recombination, the key enzyme gene CYP71E1 of sorghum HCN pathway was cloned into the entry vector p CRTM8/GW/TOPO? by TOPO cloning. Under the action of LR cloning enzyme, the primer clone was recombined into the expression vector (p MDC141), and the plant expression vector of p MDC141-CYP71E1 was successfully constructed, which was introduced into Agrobacterium tumefaciens EHA105. 2. To establish an efficient maize regeneration system. The callus induction rate of maize immature embryos was the highest when the size of immature embryos was 1.0~1.2mm. The medium of embryogenic callus induction was optimized by orthogonal experiment, and the MS basic medium for inducing callus of maize A188 was obtained by adding L-proz2O4-D into the basic medium of inducing callus of maize A188. The optimal combination of three factors of Ag NO3 is: L-pro700mg / L 24-D 2.0mg / L NO38 mg/L. When the concentration of zeatin in differentiation medium was 5 mg/L, the differentiation efficiency was the highest. The optimal concentration of IBA in rooting medium was 2 mg/L.3, to establish an efficient genetic transformation system. The average transformation efficiency of maize immature embryo was 1.0 ~ 1.2mmGUS and the average transformation efficiency of Gus was 35.3m. The concentration of Agrobacterium tumefaciens was 0.8, and that of Agrobacterium tumefaciens was 0.8. The average conversion efficiency of GUS was 46.8%, and the average transformation efficiency of 5min and thermal pretreatment at 43 鈩，
本文编号：2424867
[Abstract]:Objective to study the genetic transformation of maize by Agrobacterium tumefaciens mediated by the key enzyme gene CYP71E1 of sorghum HCN pathway and maize embryo as explant. The regeneration system of A188 maize was optimized, and the effects of different explant size, Agrobacterium concentration, infection time and heat treatment time on the genetic transformation efficiency of maize were analyzed. The transgenic plants were identified by GUS histochemical staining and PCR technique. The high efficiency transformation technology was used to solve the bottleneck of maize yield and quality reduction caused by biological and abiotic stress, which provided the theoretical basis for the transformation of new maize varieties. Methods the CYP71E1 gene of 1854bp was amplified from sorghum by polymerase chain reaction (PCR). Using the Gateway technique based on site-specific recombination, the CYP71E1 gene was recombined into the expression vector (p MDC141), and the successfully constructed expression vector CYP71E1-p MDC141 was introduced into Agrobacterium tumefaciens EHA105 strain. Maize regeneration system was established by optimizing explant material, inducing callus medium, differentiation medium and rooting medium. Agrobacterium tumefaciens (EHA105) carrying CYP71E1-p MDC141 (containing GUS gene) plasmid was used to infect immature embryos of maize inbred line A188. The effects of Agrobacterium tumefaciens on the size of explant, concentration of Agrobacterium tumefaciens and infection time were studied. The thermal pretreatment time was optimized to improve the average transformation efficiency of GUS gene, and PCR and GUS were used to detect the transgenic plants. Results 1. Using Gateway technique based on site-specific recombination, the key enzyme gene CYP71E1 of sorghum HCN pathway was cloned into the entry vector p CRTM8/GW/TOPO? by TOPO cloning. Under the action of LR cloning enzyme, the primer clone was recombined into the expression vector (p MDC141), and the plant expression vector of p MDC141-CYP71E1 was successfully constructed, which was introduced into Agrobacterium tumefaciens EHA105. 2. To establish an efficient maize regeneration system. The callus induction rate of maize immature embryos was the highest when the size of immature embryos was 1.0~1.2mm. The medium of embryogenic callus induction was optimized by orthogonal experiment, and the MS basic medium for inducing callus of maize A188 was obtained by adding L-proz2O4-D into the basic medium of inducing callus of maize A188. The optimal combination of three factors of Ag NO3 is: L-pro700mg / L 24-D 2.0mg / L NO38 mg/L. When the concentration of zeatin in differentiation medium was 5 mg/L, the differentiation efficiency was the highest. The optimal concentration of IBA in rooting medium was 2 mg/L.3, to establish an efficient genetic transformation system. The average transformation efficiency of maize immature embryo was 1.0 ~ 1.2mmGUS and the average transformation efficiency of Gus was 35.3m. The concentration of Agrobacterium tumefaciens was 0.8, and that of Agrobacterium tumefaciens was 0.8. The average conversion efficiency of GUS was 46.8%, and the average transformation efficiency of 5min and thermal pretreatment at 43 鈩，
本文编号：2424867
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