无载体无标记转植酸酶基因大豆的获得
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摘要
植物转基因技术的迅猛发展对传统的作物育种已产生了深刻的影响，然而，自从1998年有人提出转基因生物的安全性问题以后，转基因农作物的种植面积增长速度明显减慢。原因是由于以往的转基因操作中，转化的载体上不可避免的带有载体骨架序列、抗性标记基因、以及外源的启动子，这些因素可能造成转基因植物具有潜在的生物安全隐患。因此，转基因植物安全性问题迅速成为公众关心的焦点和制约转基因农作物产业化的瓶颈。随之产生的去除标记基因和载体骨架序列的新的植物分子育种方法成为植物转基因技术研究的热点。
本研究以半固体发酵方式培养泡盛曲霉(Aspergillus awamori)生产植酸酶，通过超滤、阴离子交换层析，凝胶层析步骤，分离纯化了植酸酶蛋白。SDS-PAGE结果显示该酶的分子量约为118kDa，糖基化程度为40.6％。酶学性质研究结果表明：泡盛曲霉植酸酶的最适反应温度为55℃，最适pH为2.5和5.5，37℃条件下以植酸钠为底物的K_m值为1.03nM，V_(max)为2.13μM／min。金属离子对酶活性影响的研究结果表明，EDTA基本不影响植酸酶活性，Ca~(2+)，Mg~(2+)，Mn~(2+)有轻微的抑制作用，Fe~(2+)，Zn~(2+)抑制作用显著。对该酶的耐热性研究表明，在较高温度条件处理后，仍有较高残余酶活性，与当今商品化的植酸酶相比，有较强的耐热性。
利用PCR技术克隆了泡盛曲霉(A.awamori 3.324)植酸酶基因及其编码区全序列，命名为phyA(GenBank accession no.DQ192035)。phyA结构基因全长1515bp，其中含有真菌内含子特征保守序列。核苷酸和氨基酸序列均与丝状真菌的同源性较高，其中与无花果曲霉(A.ficuum)NRRL3135 phyA的核苷酸同源性为92.1％(不计内含子，二者的内含子序列相差很大)，氨基酸同源性为95.9％；与A.niger963 phyA_2的核苷酸同源性为95％，氨基酸同源性为94％。该基因编码的植酸酶蛋白理论分子量为51042.8Da，共编码467个氨基酸，序列中存在组氨酸酸性磷酸酯酶的活性位点保守序列(RHGXRXP)和催化中心位点(HD)。根据信号肽序列的结构规则推断，N端的21个氨基酸为信号肽序列，由基因推导出的氨基酸序列上有10个潜在的糖基化位点。
构建了植物表达载体pBI121-phyA，通过农杆菌介导法将植酸酶基因导入模式植物烟草，获得卡那霉素抗性植株，通过PCR，Southern-blotting证明外源植酸酶基因已成功整合在烟草基因组上。对转基因烟草叶片的重组植酸酶活性分析表明植酸酶基因在烟草中获得高效表达。对烟草中的植酸酶酶学性质研究表明，重组植酸酶的最适反应温度与泡盛曲霉植酸酶的性质相同，耐热性略有下降，其中一个pH发生偏移。初步说明泡盛曲霉植酸酶基因能够在模式植物中得到正确表达。
利用PCR技术从植物表达载体pBI121-phyA上扩增获得无载体骨架序列、无抗性标记基因的含有植酸酶基因的最小线性表达元件，通过花粉管通道技术转化大豆。共对279朵花进行了转化，共获得99株T_1代植株。PCR检测结果表明，13株的扩增结果呈阳性，转化率为13％。473株T_2代个体的PCR结果显示，102株结果为阳性。对T_3代两个株系L14-11-2和L14-19-1部分植株的Southern-blotting结果分析表明，外源基因以低拷贝的方式整合在大豆基因组上。
RT-PCR分析表明，外源植酸酶基因已在大豆体内转录。测定了L14-11-2和L14-19-1两个转基因株系后代生长过程中植酸酶的积累情况。重组植酸酶的含量在大豆生长的第二周至第四周不断增加，随后的两周表达水平趋于平稳，在第七周开始有所下降。其中L14-19-1株系后代个体的植酸酶活性在第四生长周时达到最高，为150 U／mg蛋白，几乎是对照的3倍(56 U／mg蛋白)。在转基因大豆种子中植酸酶含量比对照平均提高100％。热稳定性分析表明，转基因大豆种子中植酸酶的残余酶活性在60℃，65℃和70℃时，分别保留70％，40％和10％，而对照样品在65℃以后几乎没有残余酶活性保留，明显高于未转基因大豆。Western-blotting分析结果表明，大豆体内表达的重组植酸酶的蛋白分子量大小为73 kD。
通过上述分析，证明外源植酸酶基因在大豆体内实现了遗传和表达，初步建立了一种新的植物分子育种转化体系——具有生物安全性的植物基因工程操作方法，已获得国家知识产权局的专利授权(专利号：ZL 02 1 328382)。目前，这转基因大豆的两个株系L14-11-2(命名为1411)和L14-19-1(命名为1412)已被国家农业部认定其生物安全等级为Ⅱ级，并批准进行中间试验。
Rapid development of plant transgenic techniques had impacted on traditional cropbreeding deeply. However, the field test of transgene crops has been slowered down in 1998with the issue of transgenics biosafty, particularly the concern for the unexpected biosaftyproblem from the vector such as its backbone sequence, resistant marker and promoter genesincorporated into it. The issue of transgenic plants biosafty attracts pulic concerns andrestricts the progress of transgenic crops industrialization. Therefore, developing vector- andmarker-free plant molecular breeding technics is one of the frontier areas in intrasngenicplants.
Extracellular phytase produced by Aspergillus awamori 3.324 through semi-solidcultivation was purified by ultrafiltration followed by chromatography using ion exchange,gel filtration and chromatofocusing columns, sequencely. The purified enzyme is a 118kDaprotein including 40.6% glycosylation. It possesses optimum temperature and pH values of55℃, 2.5 and 5.5. The K_m and V_(max) of the enzyme for dodecasodium phytate at 37℃are 1.03nM and 2.13μM/min, respectively. Phytase activity was observed not affected by EDTA, andinhibited moderately by Ca~(2+), Mg~(2+), Mn~(2+), and significantly by Fe~(2+) and Zn~(2+). The enzymeexhibits better thermostability at high temperature than the commercial phytase product.
The phytase gene was obtained by PCR amplification, and named as phyA (GenBankaccession no.DQ192035), with a size of 1515 bp and fungi characteristic intron sequence.phyA exhibits a high homology with filamentous fungi on both nucleotide and amino acidsequences. Compared with A.ficuum NRRL3135 and A.niger 963 phytase genes, itsnucleotide sequence shows 92.1% and 95.0% homologies (except its intron sequence, whichshows a big difference), while the amino acid sequence deduced from phyA shows 95.9% and94.0% homologies. The molecular weight of the phytase is 51042.8Da, which encodes apolypeptide of 467 amino acids. The amino acid sequences deduced from phyA contains theactive site septa-petide RHGXRXP, catalytically active dipeptide HD and 10 glycosylationsites, and 21 amino acids at N-ternimal are expected to be a signal peptides sequence.
The ORF of the phytase gene was cloned into the plant expression vector pBI121. Therecombinat plasmid pBI121-phyA was then transferred into tobacco via the Agrobacteriummediation. The results of PCR and Southern Blotting showed that the phytase gene wasintegrated into the tobacco genome. The phytase activity assay of tobacco leavesdemonstrated that the recombinant phytase gene was highly expressed. The characterization of the recombinant enzyme expressed indicated the same temperature profile with the phytaseproduced by Aspergillus awamori, although slightly lower thermostability and one-unit shiftof the optimum pH were observed.
A minimal linear transgenic DNA containing phyA gene without vector and marker wasobtained from the plant express vectorpBI121-phyA by PCR, which was introduced intosoybean by the pollentube pathway. A total of 279 flowers were treated with the lineartransgenic DNA. The PCR amplifications indicated that 13 in 99 T_1 plants contained the genecassette, with a transformation frequency of 13%, and 102 plants in 473 T_2 plants showedPCR-positive amplifications. T_3 individuals derived from the lines L14-11-2 and L14-19-1were investigated by Southern blot analysis, indicating their low copy number insertions.
RT-PCR results showed the presence of phyA transcripts. The T_3 progenies from the linesL14-11-2 and L14-19-1 were collected to analyze phytase accumulation in their leaves. Thephytase level increased from week 2 to week 4, remained at stable for the following 2 weeks,but slightly decreased during week 7. Phytase activity in the plants from the line L14-19-1was higher than that from the line L14-11-2 from weeks 2 to 6. The highest expression wasobserved in the line L14-19-1 during week 4 (150 U/mg protein), about 3 times higher thanthat of the untransformed control (56 U/mg protein). In seeds, the transgenic seed displayed a100% increase in phytase activity compared to that of the wild-type. The temperature stabilityof the plant-synthesized recombinant phytase in the T_3 transgenic soybeans was analysised.Upon incubation at 60, 65 and 70℃for 10 min, 70%, 40% and 10% of the activity remained,respectively, for the crude extracts of the transgenic seeds, whereas no enzyme activity wasdetected for the untransformed control. Western-blotting showed that the recombinant phytasemigrated with an apparent molecular mass of approximately 73 kDa.
In conclusion, the phytase gene was inherited and expressed in the recombvinant soybeansdeveloped with the vecter- and marker-free plant molecular breeding approach, through whichthe biosafty dispute could be overcome. The patent for this new technique was approved byState Intellectual Property Office of P.R.China (ZL 02 1 328382). Now the biosafty of gradeⅡwas authorized to the two lines L14-11-2(1411) and L14-19-1(1412) by the Ministry ofAgriculture of P.R. China.
本研究以半固体发酵方式培养泡盛曲霉(Aspergillus awamori)生产植酸酶，通过超滤、阴离子交换层析，凝胶层析步骤，分离纯化了植酸酶蛋白。SDS-PAGE结果显示该酶的分子量约为118kDa，糖基化程度为40.6％。酶学性质研究结果表明：泡盛曲霉植酸酶的最适反应温度为55℃，最适pH为2.5和5.5，37℃条件下以植酸钠为底物的K_m值为1.03nM，V_(max)为2.13μM／min。金属离子对酶活性影响的研究结果表明，EDTA基本不影响植酸酶活性，Ca~(2+)，Mg~(2+)，Mn~(2+)有轻微的抑制作用，Fe~(2+)，Zn~(2+)抑制作用显著。对该酶的耐热性研究表明，在较高温度条件处理后，仍有较高残余酶活性，与当今商品化的植酸酶相比，有较强的耐热性。
利用PCR技术克隆了泡盛曲霉(A.awamori 3.324)植酸酶基因及其编码区全序列，命名为phyA(GenBank accession no.DQ192035)。phyA结构基因全长1515bp，其中含有真菌内含子特征保守序列。核苷酸和氨基酸序列均与丝状真菌的同源性较高，其中与无花果曲霉(A.ficuum)NRRL3135 phyA的核苷酸同源性为92.1％(不计内含子，二者的内含子序列相差很大)，氨基酸同源性为95.9％；与A.niger963 phyA_2的核苷酸同源性为95％，氨基酸同源性为94％。该基因编码的植酸酶蛋白理论分子量为51042.8Da，共编码467个氨基酸，序列中存在组氨酸酸性磷酸酯酶的活性位点保守序列(RHGXRXP)和催化中心位点(HD)。根据信号肽序列的结构规则推断，N端的21个氨基酸为信号肽序列，由基因推导出的氨基酸序列上有10个潜在的糖基化位点。
构建了植物表达载体pBI121-phyA，通过农杆菌介导法将植酸酶基因导入模式植物烟草，获得卡那霉素抗性植株，通过PCR，Southern-blotting证明外源植酸酶基因已成功整合在烟草基因组上。对转基因烟草叶片的重组植酸酶活性分析表明植酸酶基因在烟草中获得高效表达。对烟草中的植酸酶酶学性质研究表明，重组植酸酶的最适反应温度与泡盛曲霉植酸酶的性质相同，耐热性略有下降，其中一个pH发生偏移。初步说明泡盛曲霉植酸酶基因能够在模式植物中得到正确表达。
利用PCR技术从植物表达载体pBI121-phyA上扩增获得无载体骨架序列、无抗性标记基因的含有植酸酶基因的最小线性表达元件，通过花粉管通道技术转化大豆。共对279朵花进行了转化，共获得99株T_1代植株。PCR检测结果表明，13株的扩增结果呈阳性，转化率为13％。473株T_2代个体的PCR结果显示，102株结果为阳性。对T_3代两个株系L14-11-2和L14-19-1部分植株的Southern-blotting结果分析表明，外源基因以低拷贝的方式整合在大豆基因组上。
RT-PCR分析表明，外源植酸酶基因已在大豆体内转录。测定了L14-11-2和L14-19-1两个转基因株系后代生长过程中植酸酶的积累情况。重组植酸酶的含量在大豆生长的第二周至第四周不断增加，随后的两周表达水平趋于平稳，在第七周开始有所下降。其中L14-19-1株系后代个体的植酸酶活性在第四生长周时达到最高，为150 U／mg蛋白，几乎是对照的3倍(56 U／mg蛋白)。在转基因大豆种子中植酸酶含量比对照平均提高100％。热稳定性分析表明，转基因大豆种子中植酸酶的残余酶活性在60℃，65℃和70℃时，分别保留70％，40％和10％，而对照样品在65℃以后几乎没有残余酶活性保留，明显高于未转基因大豆。Western-blotting分析结果表明，大豆体内表达的重组植酸酶的蛋白分子量大小为73 kD。
通过上述分析，证明外源植酸酶基因在大豆体内实现了遗传和表达，初步建立了一种新的植物分子育种转化体系——具有生物安全性的植物基因工程操作方法，已获得国家知识产权局的专利授权(专利号：ZL 02 1 328382)。目前，这转基因大豆的两个株系L14-11-2(命名为1411)和L14-19-1(命名为1412)已被国家农业部认定其生物安全等级为Ⅱ级，并批准进行中间试验。
Rapid development of plant transgenic techniques had impacted on traditional cropbreeding deeply. However, the field test of transgene crops has been slowered down in 1998with the issue of transgenics biosafty, particularly the concern for the unexpected biosaftyproblem from the vector such as its backbone sequence, resistant marker and promoter genesincorporated into it. The issue of transgenic plants biosafty attracts pulic concerns andrestricts the progress of transgenic crops industrialization. Therefore, developing vector- andmarker-free plant molecular breeding technics is one of the frontier areas in intrasngenicplants.
Extracellular phytase produced by Aspergillus awamori 3.324 through semi-solidcultivation was purified by ultrafiltration followed by chromatography using ion exchange,gel filtration and chromatofocusing columns, sequencely. The purified enzyme is a 118kDaprotein including 40.6% glycosylation. It possesses optimum temperature and pH values of55℃, 2.5 and 5.5. The K_m and V_(max) of the enzyme for dodecasodium phytate at 37℃are 1.03nM and 2.13μM/min, respectively. Phytase activity was observed not affected by EDTA, andinhibited moderately by Ca~(2+), Mg~(2+), Mn~(2+), and significantly by Fe~(2+) and Zn~(2+). The enzymeexhibits better thermostability at high temperature than the commercial phytase product.
The phytase gene was obtained by PCR amplification, and named as phyA (GenBankaccession no.DQ192035), with a size of 1515 bp and fungi characteristic intron sequence.phyA exhibits a high homology with filamentous fungi on both nucleotide and amino acidsequences. Compared with A.ficuum NRRL3135 and A.niger 963 phytase genes, itsnucleotide sequence shows 92.1% and 95.0% homologies (except its intron sequence, whichshows a big difference), while the amino acid sequence deduced from phyA shows 95.9% and94.0% homologies. The molecular weight of the phytase is 51042.8Da, which encodes apolypeptide of 467 amino acids. The amino acid sequences deduced from phyA contains theactive site septa-petide RHGXRXP, catalytically active dipeptide HD and 10 glycosylationsites, and 21 amino acids at N-ternimal are expected to be a signal peptides sequence.
The ORF of the phytase gene was cloned into the plant expression vector pBI121. Therecombinat plasmid pBI121-phyA was then transferred into tobacco via the Agrobacteriummediation. The results of PCR and Southern Blotting showed that the phytase gene wasintegrated into the tobacco genome. The phytase activity assay of tobacco leavesdemonstrated that the recombinant phytase gene was highly expressed. The characterization of the recombinant enzyme expressed indicated the same temperature profile with the phytaseproduced by Aspergillus awamori, although slightly lower thermostability and one-unit shiftof the optimum pH were observed.
A minimal linear transgenic DNA containing phyA gene without vector and marker wasobtained from the plant express vectorpBI121-phyA by PCR, which was introduced intosoybean by the pollentube pathway. A total of 279 flowers were treated with the lineartransgenic DNA. The PCR amplifications indicated that 13 in 99 T_1 plants contained the genecassette, with a transformation frequency of 13%, and 102 plants in 473 T_2 plants showedPCR-positive amplifications. T_3 individuals derived from the lines L14-11-2 and L14-19-1were investigated by Southern blot analysis, indicating their low copy number insertions.
RT-PCR results showed the presence of phyA transcripts. The T_3 progenies from the linesL14-11-2 and L14-19-1 were collected to analyze phytase accumulation in their leaves. Thephytase level increased from week 2 to week 4, remained at stable for the following 2 weeks,but slightly decreased during week 7. Phytase activity in the plants from the line L14-19-1was higher than that from the line L14-11-2 from weeks 2 to 6. The highest expression wasobserved in the line L14-19-1 during week 4 (150 U/mg protein), about 3 times higher thanthat of the untransformed control (56 U/mg protein). In seeds, the transgenic seed displayed a100% increase in phytase activity compared to that of the wild-type. The temperature stabilityof the plant-synthesized recombinant phytase in the T_3 transgenic soybeans was analysised.Upon incubation at 60, 65 and 70℃for 10 min, 70%, 40% and 10% of the activity remained,respectively, for the crude extracts of the transgenic seeds, whereas no enzyme activity wasdetected for the untransformed control. Western-blotting showed that the recombinant phytasemigrated with an apparent molecular mass of approximately 73 kDa.
In conclusion, the phytase gene was inherited and expressed in the recombvinant soybeansdeveloped with the vecter- and marker-free plant molecular breeding approach, through whichthe biosafty dispute could be overcome. The patent for this new technique was approved byState Intellectual Property Office of P.R.China (ZL 02 1 328382). Now the biosafty of gradeⅡwas authorized to the two lines L14-11-2(1411) and L14-19-1(1412) by the Ministry ofAgriculture of P.R. China.
引文
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