小麦TaPHR1基因表达载体的构建及遗传转化研究
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摘要
充分挖掘作物高效利用营养元素的遗传潜力,培育养分高效利用的农作物新品种,对农业可持续发展具有重要意义。本研究以26份优良普通小麦主栽品种(系)为材料,对影响小麦成熟胚愈伤组织诱导、分化和转化的因素进行了试验,进行了磷高效基因TaPHR1高效表达载体的构建和遗传转化研究。获得的主要研究结果如下:
1、以植物表达载体pROK2-Ubi为基础,设计带有酶切位点KpnI和BamHI的一对引物,从载体pAC25上扩增到目的基因TaPHR1。酶切回收后与同样双酶切的表达载体pROK2-Ubi连接,获得新的高效表达载体pTaPHR1,并用冻融法将其导入根瘤农杆菌LB4404菌株;同时,进一步将表达载体pTaPHR1中的启动子Ubi换为TaPT2,构建了表达载体pTaPHR1-PT2,为农杆菌介导的磷高效基因遗传转化研究奠定了基础。
2、通过对小麦成熟胚进行愈伤组织诱导及分化再生研究,明确了小麦基因型、吸胀时间、诱愈培养基2,4-D浓度、分化培养基KT浓度等是影响小麦成熟胚愈伤组织形成和分化再生的重要因子。利用剥胚法(embryo-isolated method, EI)诱导小麦成熟胚产生愈伤组织,并进行分化再生培养,筛选出了成熟胚出愈率高、愈伤质量好、分化成苗率较高的济麦22等基因型材料,确定了适宜的吸胀时间(18h)、诱愈(2 mg·L- 1 2,4-D)、分化(5 mg·L- 1 KT)条件。
3、试验明确了侵染菌液浓度及侵染时间、高渗处理中蔗糖浓度、Ca2+离子处理、共培养中滤纸处理、共培养时间等因素,对小麦成熟胚愈伤组织遗传转化效率有较大影响。接种菌液浓度为OD600=0.6,浸染时间为60min时,抗性愈组率达到最大值4.08%;浸染前进行蔗糖高渗处理,当蔗糖浓度75 g·L- 1时抗性愈组率最高(济麦22,2.90%;泰农18,2.78%);侵染后20 mmol/L Ca2+处理,抗性愈组率达到最高(5.05%);共培养过程中添加滤纸片可以提高转化效率,抗性愈组率达到4.29%;共培养3d,可使抗性愈组率获得最高,达到4.52%。
4.利用构建的磷高效表达载体pTaPHR1和建立的小麦成熟胚遗传转化体系将磷高效基因TaPHR1转入济麦22,获得了可育的转基因植株,经PCR检测初步证明目的基因已转入其中。
It is important for agricultural sustainable development to fully exploit the crop genetic potential of nutrients utilize efficiency and cultivate new varieties of efficient nutrients use. In this research, gene TaPHR1 was used to construct efficient gene expression vector for genetic transformation system of mature embryos of common wheat by A.tumefacienen. At the same time, 26 wheat genotypes were used for factors research on wheat mature embryos callus induction and differentiation to further optimize the conditions of tissue culture of wheat mature embryos. The major results were as followed:
1. In this study, Triticum aestivun L. gene TaPHR1 was amplified using specific primers containing restriction enzyme site of KpnI and BamH1. PCR products and the binary expression vector pROK2-Ubi were digested by the corresponding restricted enzymes respectively, and linked directionally. The resulting vector with TaPHR1 was named pTaPHR1. The pTaRHR1 was further introduced into Agrobacterium tumefaciens strain LB4404. At the same time, new expression vector pTaRHR1-PT2 were constructed by changing the promoter Ubi of pTaRHR1 to promoter TaPT2. These vectors were used for phosphorus efficiency genetic transformation system of mature embryos of common wheat by A.tumefaciene.
2. Genotype, imbibition time, concentration of plant growth regulator and so on were important influence factors for the induction, differentiation and regeneration of wheat mature embryos callus. Some of excellent wheat material including Jimai22 and so on with the high efficiency of induction, differentiation and regeneration of mature embryos callus were selected out from 26 wheat genotypes via embryo-isolated method (EI). The optimal method of induction, differentiation and regeneration of mature embryos callus of these genotypes were determined. The optimized system for induction, differentiation and regeneration of wheat mature embryos callus was established.
3. Density of A. tumefaciens cell and inoculation time, density of sugar in osmotic treatment, density of Ca2+, putting filter paper, different co-culture time were important influence factors for transformation of wheat mature embryos. A. tumefaciens cell density, inoculation time and selection pressure of selection reagent were confirmed; At the sugar density of 75 g·L- 1, the frequency of resistant callus received the maximum rate to 2.90%(Jimai 22) and 2.78%(Tainong 18); At the Ca2+ density of 20 mmol/L, the frequency of resistant callus received the maximum rate to 5.05%; At the condition of putting filter paper, the frequency of resistant callus received the maximum rate to 4.29%; At the condition of training 3 d, the frequency of resistant callus received the maximum rate to 4.52%; The system optimized relatively of genetic transformation of mature embryo of wheat seed mediated by A. tumefacienens was further optimized.
4. TaPHR1 gene were transferred into Jimai 22 by high phosphorus efficient expression vector pTaPHR1 and fertile transgenic plants had been gotten. Molecular detection results by PCR analysis of transgenic wheat showed that the useful gene had been transferred successfully.
1、以植物表达载体pROK2-Ubi为基础,设计带有酶切位点KpnI和BamHI的一对引物,从载体pAC25上扩增到目的基因TaPHR1。酶切回收后与同样双酶切的表达载体pROK2-Ubi连接,获得新的高效表达载体pTaPHR1,并用冻融法将其导入根瘤农杆菌LB4404菌株;同时,进一步将表达载体pTaPHR1中的启动子Ubi换为TaPT2,构建了表达载体pTaPHR1-PT2,为农杆菌介导的磷高效基因遗传转化研究奠定了基础。
2、通过对小麦成熟胚进行愈伤组织诱导及分化再生研究,明确了小麦基因型、吸胀时间、诱愈培养基2,4-D浓度、分化培养基KT浓度等是影响小麦成熟胚愈伤组织形成和分化再生的重要因子。利用剥胚法(embryo-isolated method, EI)诱导小麦成熟胚产生愈伤组织,并进行分化再生培养,筛选出了成熟胚出愈率高、愈伤质量好、分化成苗率较高的济麦22等基因型材料,确定了适宜的吸胀时间(18h)、诱愈(2 mg·L- 1 2,4-D)、分化(5 mg·L- 1 KT)条件。
3、试验明确了侵染菌液浓度及侵染时间、高渗处理中蔗糖浓度、Ca2+离子处理、共培养中滤纸处理、共培养时间等因素,对小麦成熟胚愈伤组织遗传转化效率有较大影响。接种菌液浓度为OD600=0.6,浸染时间为60min时,抗性愈组率达到最大值4.08%;浸染前进行蔗糖高渗处理,当蔗糖浓度75 g·L- 1时抗性愈组率最高(济麦22,2.90%;泰农18,2.78%);侵染后20 mmol/L Ca2+处理,抗性愈组率达到最高(5.05%);共培养过程中添加滤纸片可以提高转化效率,抗性愈组率达到4.29%;共培养3d,可使抗性愈组率获得最高,达到4.52%。
4.利用构建的磷高效表达载体pTaPHR1和建立的小麦成熟胚遗传转化体系将磷高效基因TaPHR1转入济麦22,获得了可育的转基因植株,经PCR检测初步证明目的基因已转入其中。
It is important for agricultural sustainable development to fully exploit the crop genetic potential of nutrients utilize efficiency and cultivate new varieties of efficient nutrients use. In this research, gene TaPHR1 was used to construct efficient gene expression vector for genetic transformation system of mature embryos of common wheat by A.tumefacienen. At the same time, 26 wheat genotypes were used for factors research on wheat mature embryos callus induction and differentiation to further optimize the conditions of tissue culture of wheat mature embryos. The major results were as followed:
1. In this study, Triticum aestivun L. gene TaPHR1 was amplified using specific primers containing restriction enzyme site of KpnI and BamH1. PCR products and the binary expression vector pROK2-Ubi were digested by the corresponding restricted enzymes respectively, and linked directionally. The resulting vector with TaPHR1 was named pTaPHR1. The pTaRHR1 was further introduced into Agrobacterium tumefaciens strain LB4404. At the same time, new expression vector pTaRHR1-PT2 were constructed by changing the promoter Ubi of pTaRHR1 to promoter TaPT2. These vectors were used for phosphorus efficiency genetic transformation system of mature embryos of common wheat by A.tumefaciene.
2. Genotype, imbibition time, concentration of plant growth regulator and so on were important influence factors for the induction, differentiation and regeneration of wheat mature embryos callus. Some of excellent wheat material including Jimai22 and so on with the high efficiency of induction, differentiation and regeneration of mature embryos callus were selected out from 26 wheat genotypes via embryo-isolated method (EI). The optimal method of induction, differentiation and regeneration of mature embryos callus of these genotypes were determined. The optimized system for induction, differentiation and regeneration of wheat mature embryos callus was established.
3. Density of A. tumefaciens cell and inoculation time, density of sugar in osmotic treatment, density of Ca2+, putting filter paper, different co-culture time were important influence factors for transformation of wheat mature embryos. A. tumefaciens cell density, inoculation time and selection pressure of selection reagent were confirmed; At the sugar density of 75 g·L- 1, the frequency of resistant callus received the maximum rate to 2.90%(Jimai 22) and 2.78%(Tainong 18); At the Ca2+ density of 20 mmol/L, the frequency of resistant callus received the maximum rate to 5.05%; At the condition of putting filter paper, the frequency of resistant callus received the maximum rate to 4.29%; At the condition of training 3 d, the frequency of resistant callus received the maximum rate to 4.52%; The system optimized relatively of genetic transformation of mature embryo of wheat seed mediated by A. tumefacienens was further optimized.
4. TaPHR1 gene were transferred into Jimai 22 by high phosphorus efficient expression vector pTaPHR1 and fertile transgenic plants had been gotten. Molecular detection results by PCR analysis of transgenic wheat showed that the useful gene had been transferred successfully.
引文
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