花生种子特异表达载体构建与农杆菌介导基因转化技术体系的优化
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摘要
花生是世界范围内广泛栽培的油料与经济作物,是全球四大油料作物之一,也是我国单产、总产和出口创汇额最高的油料作物。利用转基因技术改良花生籽仁品质是花生分子育种研究的重要领域。本研究采用同源克隆技术从花生栽培品种丰花3号基因组中分离获得了两个种子特异表达启动子,并构建成了种子特异表达载体;研究了基因型、芽丛诱导培养基和芽丛成苗培养基三个因素对花生胚小叶再生效率的影响,获得了三因素最优组合的花生胚小叶离体再生体系;并对21个基因型花生进行了基因转化效率的比较研究,获得了转基因植株,PCR检测证明目的基因已整合到花生基因组中。为花生品质改良的分子育种研究奠定了基础。其主要结果如下:
1花生油体蛋白Oleosin基因启动子的克隆及其种子特异表达载体的构建
根据GeneBank上发表的花生Oleosin17.8基因的序列(EF695400)设计引物,用丰花3号基因组DNA为模板进行PCR扩增花生Oleosin启动子,琼脂糖凝胶电泳回收PCR扩增的目的片段,并与克隆载体pGM-T easy连接,获得重组质粒pT-OL1584,序列分析表明,获得的启动子序列长1584bp,与GenBank发表的Oleosin17.8启动子同源性为99.37%,除含有TATA、CAAT基本转录元件外,还有2个富含AT序列、2个RY重复序列元件、1个ACCCCA元件,2个TACACAT盒、5个CATG盒、5个E-Box、6个GATA-Box及14个DOF蛋白的AAAG基序等种子特异型启动子所具有的顺式作用元件。OL1584的GeneBank登录序列号为EU518466。重组子pT-OL1584用HindⅢ、BamHⅠ双酶切,并回收目的OL1584基因片断和切除35S启动子的pBI121载体DNA,经连接构建成种子特异表达载体pB-OL1584。并且在OL1584内部设计2个上游引物以重组质粒pT-OL1584为模板,进行PCR扩增,获得含Oleosin启动子5’端缺失片段的重组子pT-OL755和pT-OL375。
2.花生致敏蛋白基因Ara h1启动子的克隆及其种子特异表达载体的构建
参考王静(2005)和Olga M(2003)报道的花生Ara h1基因的序列设计引物,用丰花3号基因组DNA为模板进行PCR扩增花生Ara h1启动子,1.2%琼脂糖凝胶电泳回收PCR扩增的目的片段,并与克隆载体pGM-T easy连接,获得重组质粒pT-Ah1968,序列分析表明,Ah1968(GeneBank登录号为EU526898)启动子序列长1968bp,与王静发表的序列同源性为99.14%,与Olga M发表的序列同源性为98.27%.Ah1968除含有基本转录元件TATA、CAAT外,还含有3个AT-Rich、2个RY元件、3个CATG、1个ACCCCA、2个E-Box、2个TACACAT类似序列、2个TAACACA、10个GATA-Box和13个AAAG(DOF蛋白结合基序)等种子特异型启动子所具有的顺式作用元件。重组子pT-Ah1968和质粒pBI121分别用HindⅢ、BamHⅠ双酶切,并回收目的基因片断(约1716bp)和切除35S启动子的pBI121载体DNA,经连接构建成种子特异表达载体pB-Ah1716。在Ah1968内部设计3个上游引物以重组质粒pT-Ah1968为模板,进行PCR扩增,获得了Ara h1启动子5’端缺失片段的重组子pT-Ah1300、pT-Ah1000和pT-Ah500。
3花生胚小叶离体再生技术体系的优化
(1)不同品种适宜芽丛诱导培养基的筛选
小花生品种丰花2号适宜的芽丛诱导培养基为MSB + 3.0mg/L BA + 0.7mg/L NAA,芽丛诱导率为81.49%,再生率为94.39%;大花生品种丰花3号适宜的芽丛诱导培养基为MSB + 4.5mg/L BA + 0.2mg/L NAA,芽丛诱导率为72.33%,再生率为10.70%。
(2)不同品种适宜芽丛成苗培养基的筛选
丰花2号适宜的芽丛成苗培养基为MSB+ 2.5mg/L BA,芽丛出苗率、再生率分别为:172.00%和122.10%;丰花3号适宜的芽丛成苗培养基为MSB+ 5.0mg/L KIN,芽丛出苗率、再生率分别为:33.33%、4.03%。
不同基因型之间的成苗芽丛百分率、芽丛出苗率和植株再生率差异显著,基因型不同是造成花生再生率差异的主要因素。以胚小叶为外植体获取较高植株再生率的三因素最佳组合为:花生品种丰花2号、芽丛诱导培养基为MSB + 3.0mg/L BA + 0.7mg/L NAA,芽丛成苗培养基为MSB+ 2.5mg/L BA。可作为花生基因转化良好的受体系统。
4不同基因型花生基因转化效率的比较
以花生胚小叶为外植体,用携带有γ-维生素E甲基转移酶基因(γ-tmt)和筛选标记基因bar的植物表达载体(质粒为pGBVE ,根癌农杆菌为GV3101),对花生21个基因型进行了基因转化。结果表明:农杆菌侵染后再生率较高的10个基因型为:丰花2号、02P181、丰花3号、不结瘤系、丰花1号、白沙1016、丰花6号、05D651、海花1号、蓬莱一窝猴;基因转化率较高的5个基因型为:丰花2号、丰花1号、丰花3号、白沙1016、02P181,丰花2号的基因转化效率最高2.67%,获得的转基因植株经PCR检测呈阳性。
Peanut (Arachis hypogaea L.), the world’s fourth largest oilseed crop, is the oil and economic crop grown worldwide, also is the oil crop that having highest single yield、total yield and export income in our country. So improving peanut trait by genetic transformation is the important field of molecular breeding. Two seed-specific promoters were isolated from genome DNA of Fenghua3 by homology cloning, then seed-specific expression vectors were constructed; the effect of genotype、medium for the inducement of buds and medium for the elongation of buds to regeneration frequency were researched, regeneration system was Optimized in three factors; Genetic transformation frequencies of 21 genotypes were compared; Genetic transformation seeding were obtained, and identified by PCR. All these establish basics for molecular breeding of improving peanut quality. The main results were as follows:
1 Cloning of the Oleosin promotor from peanut and constructing of its seed- specific expression vector
The primers were designed according to the published sequence of Oleosin17.8 gene on GenBank (EF695400). Extracting genome DNA from peanut Fenghua3, the fragment of promotor OL1584 was obtained by PCR. The OL1584 was cloned into pGM-T easy vector for sequencing and named pT-OL1584. Sequencing analysis indicated that the inserted fragment was showing 99.37% homology to the reported sequence Oleosin17.8 .And the accession number of sequence OL1584 in GeneBank is EU518466. TATA、CAAT、two AT-Rich elemengts、two RY elements、five CATG boxes、five E-Boxes、six GATA-Boxes two TACACAT elements and fourteen AAAG boxes are existed in OL1584. The recombinant plasmid pT-OL1584 and pBI121 were digested by HindⅢand BamHⅠ, the aimed 1584bp fragment and the longer of pBI121 resected 35S gene were recycled, and ligated by T4 DNA Ligase.The result proved that OL1584 was ligated into the normal vector of pBI121. And the seed-specific expression vector pB-OL1584 was constructed. Then the OL1584 5’deletion plasmids pT-OL755 and pT-OL375 were constructed by PCR with pT-OL1584 for template and different up-primers.
2 Cloning of the Ara h1 promotor from peanut and constructing of its seed-specific expression vector
The primers were designed according to the published sequence of Ara h1 gene (WANG jing, 2005; Olga M, 2003). Extracting genome DNA from peanut Fenghua3, the fragment of promotor Ah1968 was obtained by PCR. Then Ah1968 was cloned into pGM-T easy vector for sequencing and named pT-Ah1968. Sequencing analysis indicated that the inserted fragment was showing 99.14% homology to the reported sequence by WANG Jing(2005), 98.27% homology to the reported sequence by Olga M(2003).And the accession number of sequence Ah1968 in GeneBank is EU526898.TATA、CAAT、three AT enhancers、two RY elements、three CATG boxes、two E-Boxes、ten GATA-Boxes two TACACAT elements and thirteen AAAG boxes are existed in Ah1968. The recombinant plasmid pT-Ah1968 and pBI121 were digested by HindⅢand BamHⅠ, the aimed 1716bp fragment and the longer of pBI121 resected 35S gene were recycled and ligated by T4 DNA Ligase.The result proved that Ah1716 was inserted into the normal vector of pBI121. Then the seed-specific expression vector pB-Ah1716 was constructed. The Ah1968 5’deletion plasmids pT-Ah1300、pT-Ah1000 and pT-Ah500 were constructed by PCR with pT-Ah1968 for template and different up-primers .
3 Optimization of the Regeneration Technique from independent Leaflet of Peanut
(1)Study on the suitable medium for the inducement of buds
The experimental results indicated that the suitable medium for the inducement of buds were MSB + 3.0mg/L BA + 0.7mg/LNAA for Fenghua2, frequencies of buds inducement and regeneration are 81.49% and 94.39% respectively. MSB + 4.5mg/L BA + 0.2mg/L NAA is suitable for Fenghua3, frequencies of buds inducement and regeneration are 72.33% and 10.70% respectively.
(2)Study on the suitable medium for the elongation of buds
The experimental results indicated that the best media for the elongation of buds were MSB+ 2.5mg/L BA for fenghua2, the rate of seeding in buds and regeneration were 172.00% and 122.10% respectively. And MSB+ 5.0mg/L KIN is suitable for Fenghua3, the rate of seeding in buds and regeneration were 33.33% and 4.03% respectively.
Conclusion:There are significant differences on bud differentiation and plant regeneration between the two genotypes. Genotype is the main element of regeneration difference. The lesser change of Plant hormone deepness is not enough to offset the regeneration difference of genotypes. The best combination of three factors for higher regeneration frequency: Fenghua2, buds inducement media is MSB + 3.0mg/L BA + 0.7mg/L NAA, buds elongation media is MSB+ 2.5mg/L BA. These could be as accepter system for peanut genetic transformation.
4 Comparing on transformation efficiency of different genotypes mediated by Agrobacterium tumefaciens
The experiment conducted genetic transformation research on peanut using vector harboringγ-tmt and bar genes, and leaflet of 21 genotypes as explants. The results indicated that the ten genotypes which have higher regeneration rate are Fenghua2、02P181、Fenghua3、Bujieliuxi、Fenghua1、Baisha1016、Fenghua6、05D651、Haihua1、PengLaiyiwohou;Five genotypes with higher transformation efficiency are Fenghua2、Fenghua1、Fenghua3、Baisha1016、02P181;Fenghua2 is the highest one for 2.67%;PCR positive plants were obtained from the five genotypes.
1花生油体蛋白Oleosin基因启动子的克隆及其种子特异表达载体的构建
根据GeneBank上发表的花生Oleosin17.8基因的序列(EF695400)设计引物,用丰花3号基因组DNA为模板进行PCR扩增花生Oleosin启动子,琼脂糖凝胶电泳回收PCR扩增的目的片段,并与克隆载体pGM-T easy连接,获得重组质粒pT-OL1584,序列分析表明,获得的启动子序列长1584bp,与GenBank发表的Oleosin17.8启动子同源性为99.37%,除含有TATA、CAAT基本转录元件外,还有2个富含AT序列、2个RY重复序列元件、1个ACCCCA元件,2个TACACAT盒、5个CATG盒、5个E-Box、6个GATA-Box及14个DOF蛋白的AAAG基序等种子特异型启动子所具有的顺式作用元件。OL1584的GeneBank登录序列号为EU518466。重组子pT-OL1584用HindⅢ、BamHⅠ双酶切,并回收目的OL1584基因片断和切除35S启动子的pBI121载体DNA,经连接构建成种子特异表达载体pB-OL1584。并且在OL1584内部设计2个上游引物以重组质粒pT-OL1584为模板,进行PCR扩增,获得含Oleosin启动子5’端缺失片段的重组子pT-OL755和pT-OL375。
2.花生致敏蛋白基因Ara h1启动子的克隆及其种子特异表达载体的构建
参考王静(2005)和Olga M(2003)报道的花生Ara h1基因的序列设计引物,用丰花3号基因组DNA为模板进行PCR扩增花生Ara h1启动子,1.2%琼脂糖凝胶电泳回收PCR扩增的目的片段,并与克隆载体pGM-T easy连接,获得重组质粒pT-Ah1968,序列分析表明,Ah1968(GeneBank登录号为EU526898)启动子序列长1968bp,与王静发表的序列同源性为99.14%,与Olga M发表的序列同源性为98.27%.Ah1968除含有基本转录元件TATA、CAAT外,还含有3个AT-Rich、2个RY元件、3个CATG、1个ACCCCA、2个E-Box、2个TACACAT类似序列、2个TAACACA、10个GATA-Box和13个AAAG(DOF蛋白结合基序)等种子特异型启动子所具有的顺式作用元件。重组子pT-Ah1968和质粒pBI121分别用HindⅢ、BamHⅠ双酶切,并回收目的基因片断(约1716bp)和切除35S启动子的pBI121载体DNA,经连接构建成种子特异表达载体pB-Ah1716。在Ah1968内部设计3个上游引物以重组质粒pT-Ah1968为模板,进行PCR扩增,获得了Ara h1启动子5’端缺失片段的重组子pT-Ah1300、pT-Ah1000和pT-Ah500。
3花生胚小叶离体再生技术体系的优化
(1)不同品种适宜芽丛诱导培养基的筛选
小花生品种丰花2号适宜的芽丛诱导培养基为MSB + 3.0mg/L BA + 0.7mg/L NAA,芽丛诱导率为81.49%,再生率为94.39%;大花生品种丰花3号适宜的芽丛诱导培养基为MSB + 4.5mg/L BA + 0.2mg/L NAA,芽丛诱导率为72.33%,再生率为10.70%。
(2)不同品种适宜芽丛成苗培养基的筛选
丰花2号适宜的芽丛成苗培养基为MSB+ 2.5mg/L BA,芽丛出苗率、再生率分别为:172.00%和122.10%;丰花3号适宜的芽丛成苗培养基为MSB+ 5.0mg/L KIN,芽丛出苗率、再生率分别为:33.33%、4.03%。
不同基因型之间的成苗芽丛百分率、芽丛出苗率和植株再生率差异显著,基因型不同是造成花生再生率差异的主要因素。以胚小叶为外植体获取较高植株再生率的三因素最佳组合为:花生品种丰花2号、芽丛诱导培养基为MSB + 3.0mg/L BA + 0.7mg/L NAA,芽丛成苗培养基为MSB+ 2.5mg/L BA。可作为花生基因转化良好的受体系统。
4不同基因型花生基因转化效率的比较
以花生胚小叶为外植体,用携带有γ-维生素E甲基转移酶基因(γ-tmt)和筛选标记基因bar的植物表达载体(质粒为pGBVE ,根癌农杆菌为GV3101),对花生21个基因型进行了基因转化。结果表明:农杆菌侵染后再生率较高的10个基因型为:丰花2号、02P181、丰花3号、不结瘤系、丰花1号、白沙1016、丰花6号、05D651、海花1号、蓬莱一窝猴;基因转化率较高的5个基因型为:丰花2号、丰花1号、丰花3号、白沙1016、02P181,丰花2号的基因转化效率最高2.67%,获得的转基因植株经PCR检测呈阳性。
Peanut (Arachis hypogaea L.), the world’s fourth largest oilseed crop, is the oil and economic crop grown worldwide, also is the oil crop that having highest single yield、total yield and export income in our country. So improving peanut trait by genetic transformation is the important field of molecular breeding. Two seed-specific promoters were isolated from genome DNA of Fenghua3 by homology cloning, then seed-specific expression vectors were constructed; the effect of genotype、medium for the inducement of buds and medium for the elongation of buds to regeneration frequency were researched, regeneration system was Optimized in three factors; Genetic transformation frequencies of 21 genotypes were compared; Genetic transformation seeding were obtained, and identified by PCR. All these establish basics for molecular breeding of improving peanut quality. The main results were as follows:
1 Cloning of the Oleosin promotor from peanut and constructing of its seed- specific expression vector
The primers were designed according to the published sequence of Oleosin17.8 gene on GenBank (EF695400). Extracting genome DNA from peanut Fenghua3, the fragment of promotor OL1584 was obtained by PCR. The OL1584 was cloned into pGM-T easy vector for sequencing and named pT-OL1584. Sequencing analysis indicated that the inserted fragment was showing 99.37% homology to the reported sequence Oleosin17.8 .And the accession number of sequence OL1584 in GeneBank is EU518466. TATA、CAAT、two AT-Rich elemengts、two RY elements、five CATG boxes、five E-Boxes、six GATA-Boxes two TACACAT elements and fourteen AAAG boxes are existed in OL1584. The recombinant plasmid pT-OL1584 and pBI121 were digested by HindⅢand BamHⅠ, the aimed 1584bp fragment and the longer of pBI121 resected 35S gene were recycled, and ligated by T4 DNA Ligase.The result proved that OL1584 was ligated into the normal vector of pBI121. And the seed-specific expression vector pB-OL1584 was constructed. Then the OL1584 5’deletion plasmids pT-OL755 and pT-OL375 were constructed by PCR with pT-OL1584 for template and different up-primers.
2 Cloning of the Ara h1 promotor from peanut and constructing of its seed-specific expression vector
The primers were designed according to the published sequence of Ara h1 gene (WANG jing, 2005; Olga M, 2003). Extracting genome DNA from peanut Fenghua3, the fragment of promotor Ah1968 was obtained by PCR. Then Ah1968 was cloned into pGM-T easy vector for sequencing and named pT-Ah1968. Sequencing analysis indicated that the inserted fragment was showing 99.14% homology to the reported sequence by WANG Jing(2005), 98.27% homology to the reported sequence by Olga M(2003).And the accession number of sequence Ah1968 in GeneBank is EU526898.TATA、CAAT、three AT enhancers、two RY elements、three CATG boxes、two E-Boxes、ten GATA-Boxes two TACACAT elements and thirteen AAAG boxes are existed in Ah1968. The recombinant plasmid pT-Ah1968 and pBI121 were digested by HindⅢand BamHⅠ, the aimed 1716bp fragment and the longer of pBI121 resected 35S gene were recycled and ligated by T4 DNA Ligase.The result proved that Ah1716 was inserted into the normal vector of pBI121. Then the seed-specific expression vector pB-Ah1716 was constructed. The Ah1968 5’deletion plasmids pT-Ah1300、pT-Ah1000 and pT-Ah500 were constructed by PCR with pT-Ah1968 for template and different up-primers .
3 Optimization of the Regeneration Technique from independent Leaflet of Peanut
(1)Study on the suitable medium for the inducement of buds
The experimental results indicated that the suitable medium for the inducement of buds were MSB + 3.0mg/L BA + 0.7mg/LNAA for Fenghua2, frequencies of buds inducement and regeneration are 81.49% and 94.39% respectively. MSB + 4.5mg/L BA + 0.2mg/L NAA is suitable for Fenghua3, frequencies of buds inducement and regeneration are 72.33% and 10.70% respectively.
(2)Study on the suitable medium for the elongation of buds
The experimental results indicated that the best media for the elongation of buds were MSB+ 2.5mg/L BA for fenghua2, the rate of seeding in buds and regeneration were 172.00% and 122.10% respectively. And MSB+ 5.0mg/L KIN is suitable for Fenghua3, the rate of seeding in buds and regeneration were 33.33% and 4.03% respectively.
Conclusion:There are significant differences on bud differentiation and plant regeneration between the two genotypes. Genotype is the main element of regeneration difference. The lesser change of Plant hormone deepness is not enough to offset the regeneration difference of genotypes. The best combination of three factors for higher regeneration frequency: Fenghua2, buds inducement media is MSB + 3.0mg/L BA + 0.7mg/L NAA, buds elongation media is MSB+ 2.5mg/L BA. These could be as accepter system for peanut genetic transformation.
4 Comparing on transformation efficiency of different genotypes mediated by Agrobacterium tumefaciens
The experiment conducted genetic transformation research on peanut using vector harboringγ-tmt and bar genes, and leaflet of 21 genotypes as explants. The results indicated that the ten genotypes which have higher regeneration rate are Fenghua2、02P181、Fenghua3、Bujieliuxi、Fenghua1、Baisha1016、Fenghua6、05D651、Haihua1、PengLaiyiwohou;Five genotypes with higher transformation efficiency are Fenghua2、Fenghua1、Fenghua3、Baisha1016、02P181;Fenghua2 is the highest one for 2.67%;PCR positive plants were obtained from the five genotypes.
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