花生酰基载体蛋白基因的克隆与功能分析
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摘要
酰基载体蛋白(acyl carrier protein, ACP)是一类具有保守的丝氨酸残基的小分子量的酸性蛋白质。在脂肪酸合成过程中,ACP携带酰基链完成缩合、还原和脱氢等酶促反应。它还是不同长度酰基链的脂肪酸的酰基ACP去饱和反应和质体类酰基转移酶作用的辅助因子。植物贮藏脂肪酸中不饱和脂肪酸的含量、组成以及它们在总脂肪酸中所占的比例,与ACP异构体的种类及差异表达有密切关系。
已有研究表明,在拟南芥中表达由35S启动子驱动的带有其上游400bp调控区ACP1基因,引起转基因植株叶组织中ACP1基因的表达量增加了3-8倍,而在种子中没有明显的变化。同时,也使叶组织中脂肪酸组成发生了变化,16:3的含量明显减少,而相应的增加了亚麻酸18:3的含量,但总脂肪酸含量不变。
本研究在克隆获得花生AhACP1-1基因和AhACP1-2基因的基础上,对这两个基因在根、茎、叶、花和种子表达模式进行了分析;并在拟南芥、烟草和花生中利用过量表达和反义抑制表达策略验证这两个基因的功能。主要研究结果如下:
1.用半定量RT-PCR方法分析AhACP1-1和AhACP1-2在根、茎、叶、花和种子中表达模式,发现AhACP1-1在茎中几乎不表达,在种子的表达量最高,在根、叶和花中表达量相近;AhACP1-2各个部位均有表达,且表达量在种子和叶片中较高,在根和花中次之,茎中最弱。
2.以pROKⅡ为原始载体,将AhACP1-1和AhACP1-2这两个基因编码区及其上游约54 bp和下游约20 bp正向和反向插入载体的相应位点,由此获得4个植物表达载体。其中,上游约54 bp包括保守的七核苷酸基序“CTCCGTC”和“CT-rich”区。
3.拟南芥、烟草和花生的遗传转化
(1)在拟南芥中过量表达和抑制表达花生的AhACP1-1基因和AhACP1-2基因,获得部分卡那抗性拟南芥,经筛选和PCR检测分别获得转基因T1代各10株。
(2)在烟草SR1中过量表达和抑制表达花生的AhACP1-1基因和AhACP1-2基因,获得25株卡那抗性烟草植株,经分子检测均为转基因阳性植株,其中AhACP1-1基因过量表达和反义抑制各6棵,AhACP1-2基因过量表达7棵,反义抑制6棵。与野生烟草相比,在绝大多数独立的转基因株系中外源基因的表达量明显提高。
(3)以鲁花14中的胚小叶为外植体,建立了高效的遗传转化体系;并通过农杆菌介导的转化法过量表达和抑制表达花生的AhACP1-1基因和AhACP1-2基因,共获得15株卡那抗性花生植株,PCR检测结果均为阳性植株,其中过量表达获得5株,反义抑制表达获得10株。
4.转基因烟草植株中AhACP1-1基因和AhACP1-2基因功能验证
用气相色谱法检测部分转基因烟草植株叶片脂肪酸含量,结果显示有两株转基因植株叶片不饱和脂肪酸含量比野生烟草明显提高;用分光光度法对经过低温处理的转基因植株叶片中的丙二醛、游离脯氨酸和可溶性糖的含量进行测定,结果显示,在低温胁迫下,与对照烟草相比,转基因植株叶片中游离脯氨酸和可溶性糖的含量具有不同程度提高,而丙二醛含量则有所降低。
综上所述,本研究优化了花生再生和遗传转化体系;通过基因工程手段在不同物种中过量表达和抑制表达了花生的AhACP1-1基因和AhACP1-2基因;转基因功能分析表明,在烟草中组成型表达这两个基因可以改变转基因植株叶片脂肪酸组成,并且叶片中不饱和脂肪酸含量增加可能提高转基因植株的抗寒性;下一步我们将在转基因拟南芥和花生中进一步分析这两个基因的功能,并在花生种子中过量表达这两个基因,最终期望通过基因手段改良花生种子脂肪酸含量及组成。
Acyl carrier protein (ACP) is a small acidic protein with a conservative serine residue. In the de novo synthesis of fatty acid in plastid, ACP carrying acyl-chains with different lengths takes part in the cycles of condensation, reduction, and dehydration steps. ACP is also a cofactor for desaturation and acyl-transfer reaction of fatty acid with different chains catalyzed by stearoyl-ACP desarurase and acyl transferase .It has much to do with the species and differential expression of the isomer of ACP,the content and the composition of unsaturated fatty acid in the storage fatty acid in the plants and the proportion of unsaturated fatty acid in the total fatty acid .
It has been shown that transgenic Arabidopsis plants, transformed with ACP-1 and its upstream 400 bp region of the transcription start site which is driven by the cauliflower mosaic virus 35S promoter, expressed high levels of ACP-1, and expression of this isoform increased 3 to 8 fold in leaf tissue, but no significant changes in seed. It also revealed that overexpression of ACP-1 in leaf tissue alters fatty acid composition. Levels of 16:3 significantly decreased while 18:3 increased, however, the levels of total fatty acid had no changes.
Based on the cloning of peanut AhACP1-1 and AhACP1-1 gene, the expression patterns of these two genes in peanut root, shoot, leaf, flower and seed were analyzed. and, to identify the functions of these genes, transformation of Arabidopsis, tobacco and peanut with construct pROKⅡ:AhACP1-1 and pROKⅡ:AhACP1-2 was carried out. The main results were shown as follows.
1. The expression patterns of AhACP1-1 and AhACP1-2 in root, shoot, leaf, flower and seed were analyzed by RT-PCR. It was found that the expression level of AhACP1-1 was highest in seed while nearly no expression in shoot, and similar in root, leaf and flower, and that AhACP1-2 expressed in every parts of transgenic plants, but, the highest expression level was in seed and leaf, and the second in root and flower, and the least in shoot.
2. Used pROKⅡas the initial vector, the code region, upstream 54bp and downstream 20bp of AhACP1-1 and AhACP1-2 was forward, or reversely, inserted in the same site of vector, and 4 plant expression vectors were constructed.
3. the genetic transformation of Arabidopsis, tobacco and peanut
(1) The constructs of pROKⅡ:AhACP1-1 and pROKⅡ:AhACP1-2 were introduced into Arabidopsis by Agrobacterium-mediated transformation. After selection on kanamycin-containing medium and PCR analysis, 10 lines of transformants for each construct were obtained. The subsequent generations of transgenic plants were still selected on kanamycin-containing medium.
(2) The constructs of pROKⅡ:AhACP1-1 and pROKⅡ:AhACP1-2 were introduced into tobacco by Agrobacterium-mediated transformation. After selection on kanamycin-containing medium and PCR analysis, 25 transformants were obtained. Among the transformants obtained, for AhACP1-1, 6 transformants were overexpression type and 6 were antisense expression type, and for AhACP1-2, 7 transformants were overexpression type and 6 were antisense expression type. In comparison with that of the control tobacco, the expression level of the foreign gene in most transgenic tobacco plants was increased.
(3) To improve the efficiency of transformation, the efficient system of genetic transformation as the explants of leaflet of LuHua14 was established; and the constructs of pROKⅡ:AhACP1-1 and pROKⅡ:AhACP1-2 were introduced into peanut by Agrobacterium-mediated transformation. After selection on kanamycin containing medium and PCR analysis, 15 transformants were obtained. Among of them, 5 transformants were overexpression type and 10 were antisense expression type.
4. Functional Identification of the AhACP1-1and AhACP1-2 in the transgenic tobacco
The content of fatty acid in leaf of some transgenic tobacco was detected by gas chromatograph. The results indicated that the content of unsaturated fatty acid was increased in comparison with the control. Then, the concentration of malondialdehyde, free proline and soluble sugar of the transgenic plants and the control plant, which were treated at 8℃in 0h,0.5h,1h,2h,4h,6h and 8h, was detected by the method of Spectrophotometer detection The results indicated that the concentration of free proline and soluble sugar of the transgenic plants were increased in comparison with the control, and that the concentration of malondialdehyde was reduced.
To sum up, this study modified the peanut regeneration and transformation system. The constructs of pROKⅡ:AhACP1-1 and pROKⅡ:AhACP1-2 were transformed into Arabidopsis, tobacco and peanut by Agrobacterium-mediated transformation; the functional analysis of the transgenic plants indicated that the constitutive expression of AhACP1-1and AhACP1-2 in tobacco could change the composition of fatty acid in leaf of the transgenic plants, and the increasing of content of unsaturated fatty acid may improve the cold resistance of the transgenic plants. Next,we would analyze the function of AhACP1-1and AhACP1-1 in transgenic Arabidopsis and peanut, and overexpress them in seed of peanut to change the content and the composition of fatty acid in seed of peanut by gene engineering.
已有研究表明,在拟南芥中表达由35S启动子驱动的带有其上游400bp调控区ACP1基因,引起转基因植株叶组织中ACP1基因的表达量增加了3-8倍,而在种子中没有明显的变化。同时,也使叶组织中脂肪酸组成发生了变化,16:3的含量明显减少,而相应的增加了亚麻酸18:3的含量,但总脂肪酸含量不变。
本研究在克隆获得花生AhACP1-1基因和AhACP1-2基因的基础上,对这两个基因在根、茎、叶、花和种子表达模式进行了分析;并在拟南芥、烟草和花生中利用过量表达和反义抑制表达策略验证这两个基因的功能。主要研究结果如下:
1.用半定量RT-PCR方法分析AhACP1-1和AhACP1-2在根、茎、叶、花和种子中表达模式,发现AhACP1-1在茎中几乎不表达,在种子的表达量最高,在根、叶和花中表达量相近;AhACP1-2各个部位均有表达,且表达量在种子和叶片中较高,在根和花中次之,茎中最弱。
2.以pROKⅡ为原始载体,将AhACP1-1和AhACP1-2这两个基因编码区及其上游约54 bp和下游约20 bp正向和反向插入载体的相应位点,由此获得4个植物表达载体。其中,上游约54 bp包括保守的七核苷酸基序“CTCCGTC”和“CT-rich”区。
3.拟南芥、烟草和花生的遗传转化
(1)在拟南芥中过量表达和抑制表达花生的AhACP1-1基因和AhACP1-2基因,获得部分卡那抗性拟南芥,经筛选和PCR检测分别获得转基因T1代各10株。
(2)在烟草SR1中过量表达和抑制表达花生的AhACP1-1基因和AhACP1-2基因,获得25株卡那抗性烟草植株,经分子检测均为转基因阳性植株,其中AhACP1-1基因过量表达和反义抑制各6棵,AhACP1-2基因过量表达7棵,反义抑制6棵。与野生烟草相比,在绝大多数独立的转基因株系中外源基因的表达量明显提高。
(3)以鲁花14中的胚小叶为外植体,建立了高效的遗传转化体系;并通过农杆菌介导的转化法过量表达和抑制表达花生的AhACP1-1基因和AhACP1-2基因,共获得15株卡那抗性花生植株,PCR检测结果均为阳性植株,其中过量表达获得5株,反义抑制表达获得10株。
4.转基因烟草植株中AhACP1-1基因和AhACP1-2基因功能验证
用气相色谱法检测部分转基因烟草植株叶片脂肪酸含量,结果显示有两株转基因植株叶片不饱和脂肪酸含量比野生烟草明显提高;用分光光度法对经过低温处理的转基因植株叶片中的丙二醛、游离脯氨酸和可溶性糖的含量进行测定,结果显示,在低温胁迫下,与对照烟草相比,转基因植株叶片中游离脯氨酸和可溶性糖的含量具有不同程度提高,而丙二醛含量则有所降低。
综上所述,本研究优化了花生再生和遗传转化体系;通过基因工程手段在不同物种中过量表达和抑制表达了花生的AhACP1-1基因和AhACP1-2基因;转基因功能分析表明,在烟草中组成型表达这两个基因可以改变转基因植株叶片脂肪酸组成,并且叶片中不饱和脂肪酸含量增加可能提高转基因植株的抗寒性;下一步我们将在转基因拟南芥和花生中进一步分析这两个基因的功能,并在花生种子中过量表达这两个基因,最终期望通过基因手段改良花生种子脂肪酸含量及组成。
Acyl carrier protein (ACP) is a small acidic protein with a conservative serine residue. In the de novo synthesis of fatty acid in plastid, ACP carrying acyl-chains with different lengths takes part in the cycles of condensation, reduction, and dehydration steps. ACP is also a cofactor for desaturation and acyl-transfer reaction of fatty acid with different chains catalyzed by stearoyl-ACP desarurase and acyl transferase .It has much to do with the species and differential expression of the isomer of ACP,the content and the composition of unsaturated fatty acid in the storage fatty acid in the plants and the proportion of unsaturated fatty acid in the total fatty acid .
It has been shown that transgenic Arabidopsis plants, transformed with ACP-1 and its upstream 400 bp region of the transcription start site which is driven by the cauliflower mosaic virus 35S promoter, expressed high levels of ACP-1, and expression of this isoform increased 3 to 8 fold in leaf tissue, but no significant changes in seed. It also revealed that overexpression of ACP-1 in leaf tissue alters fatty acid composition. Levels of 16:3 significantly decreased while 18:3 increased, however, the levels of total fatty acid had no changes.
Based on the cloning of peanut AhACP1-1 and AhACP1-1 gene, the expression patterns of these two genes in peanut root, shoot, leaf, flower and seed were analyzed. and, to identify the functions of these genes, transformation of Arabidopsis, tobacco and peanut with construct pROKⅡ:AhACP1-1 and pROKⅡ:AhACP1-2 was carried out. The main results were shown as follows.
1. The expression patterns of AhACP1-1 and AhACP1-2 in root, shoot, leaf, flower and seed were analyzed by RT-PCR. It was found that the expression level of AhACP1-1 was highest in seed while nearly no expression in shoot, and similar in root, leaf and flower, and that AhACP1-2 expressed in every parts of transgenic plants, but, the highest expression level was in seed and leaf, and the second in root and flower, and the least in shoot.
2. Used pROKⅡas the initial vector, the code region, upstream 54bp and downstream 20bp of AhACP1-1 and AhACP1-2 was forward, or reversely, inserted in the same site of vector, and 4 plant expression vectors were constructed.
3. the genetic transformation of Arabidopsis, tobacco and peanut
(1) The constructs of pROKⅡ:AhACP1-1 and pROKⅡ:AhACP1-2 were introduced into Arabidopsis by Agrobacterium-mediated transformation. After selection on kanamycin-containing medium and PCR analysis, 10 lines of transformants for each construct were obtained. The subsequent generations of transgenic plants were still selected on kanamycin-containing medium.
(2) The constructs of pROKⅡ:AhACP1-1 and pROKⅡ:AhACP1-2 were introduced into tobacco by Agrobacterium-mediated transformation. After selection on kanamycin-containing medium and PCR analysis, 25 transformants were obtained. Among the transformants obtained, for AhACP1-1, 6 transformants were overexpression type and 6 were antisense expression type, and for AhACP1-2, 7 transformants were overexpression type and 6 were antisense expression type. In comparison with that of the control tobacco, the expression level of the foreign gene in most transgenic tobacco plants was increased.
(3) To improve the efficiency of transformation, the efficient system of genetic transformation as the explants of leaflet of LuHua14 was established; and the constructs of pROKⅡ:AhACP1-1 and pROKⅡ:AhACP1-2 were introduced into peanut by Agrobacterium-mediated transformation. After selection on kanamycin containing medium and PCR analysis, 15 transformants were obtained. Among of them, 5 transformants were overexpression type and 10 were antisense expression type.
4. Functional Identification of the AhACP1-1and AhACP1-2 in the transgenic tobacco
The content of fatty acid in leaf of some transgenic tobacco was detected by gas chromatograph. The results indicated that the content of unsaturated fatty acid was increased in comparison with the control. Then, the concentration of malondialdehyde, free proline and soluble sugar of the transgenic plants and the control plant, which were treated at 8℃in 0h,0.5h,1h,2h,4h,6h and 8h, was detected by the method of Spectrophotometer detection The results indicated that the concentration of free proline and soluble sugar of the transgenic plants were increased in comparison with the control, and that the concentration of malondialdehyde was reduced.
To sum up, this study modified the peanut regeneration and transformation system. The constructs of pROKⅡ:AhACP1-1 and pROKⅡ:AhACP1-2 were transformed into Arabidopsis, tobacco and peanut by Agrobacterium-mediated transformation; the functional analysis of the transgenic plants indicated that the constitutive expression of AhACP1-1and AhACP1-2 in tobacco could change the composition of fatty acid in leaf of the transgenic plants, and the increasing of content of unsaturated fatty acid may improve the cold resistance of the transgenic plants. Next,we would analyze the function of AhACP1-1and AhACP1-1 in transgenic Arabidopsis and peanut, and overexpress them in seed of peanut to change the content and the composition of fatty acid in seed of peanut by gene engineering.
引文
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[53] Hitz WD, Carlson TJ, Booth JR, et,al Cloning of a higher-plant plastidω-6 fatty acid desaturase cDNA and its expression in a cyanobacterium 1994
[54] Kinney AJ. Development of genetically engineered soybean oils for food applications. Journal of Food Lipids, 1996, 3: 273-292
[55] Reddy AS, Thomas TL. Expression of a cyanobacternal△6-desaturase gene result inγ-linolenic acid production in transgenic plants. Nat Biotechnol, 1996,14:639-642
[56] Cahoon EB, Shanklin J, Ohlrogge JB. Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco.Proc Natl Acad Sci USA,1992,89:11184～11188
[57]张宁洁,高国庆,张树林.回交育种分析花生脂肪酸含量的变化.中国种业,2008(2):46-47
[58]张小茜,单雷,唐桂英等.农杆菌介导的花生△12脂肪酸脱氢酶基因AhFAD2RNAi抑制表达遗传转化研究.中国油料作物学报,2007,29(4):409-415
[59]黄冰艳张新友苗利娟等.花生FAD2基因RNAi载体转化及转基因籽粒脂肪酸分析.中国油料作物学报,2008,30(3)
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[68] Ozias-Akins P,Anderson WE, Holbrok CC.Somatic embryogenesis in~Irachis hypogaea I:genotype comparison.PlantScience,1992,83(1):103-111.
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[70]庄振宏等.花生再生和转化体系的初步研究.福建师范大学学报(自然科学版),2001,17(4):88-92Tripepi, R. R. Adventitious shoot regeneration. In: Geneve, R. L.; Preece, J. E.;
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[74]谢玉英.膜脂组成与植物抗寒性的关系.安徽农学通报,2007,13(11):38-39
[75]刘叶菊,刘飞虎.干旱胁迫对烟草样品丙二醛含量和细胞膜透性的影响.亚热带植物科学,2004,33(4)
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[40] Song P, Allen RD. Identification of a cotton fiberspecific acyl carrier protein cDNA by differential display. Biochim Biophys Acta, 1997,1351:305-312
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[48] Loof B, Appleqvist LA. Plant breeding for improved gield and quality. In:Applequvist LA, Ohlson R, eds. Rapeseed: cultivation, composition,processing and utilization. Amsterdam:Elsevier, 1972:101-122
[49] Johnson RW, Fritz E. Fatty acids in Industry:Properties, Derivatives, Applications. New York:Marcel Dekker,1989
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[51] Kabbaj A, Vervoort V, Abbott AG, et,al. Polymorphism in Helianthus and expression of stearate, oleate and linoleate desaturase genes in sunflower with normal and high-oleic contents.Helia,1996,19:1–18.
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[53] Hitz WD, Carlson TJ, Booth JR, et,al Cloning of a higher-plant plastidω-6 fatty acid desaturase cDNA and its expression in a cyanobacterium 1994
[54] Kinney AJ. Development of genetically engineered soybean oils for food applications. Journal of Food Lipids, 1996, 3: 273-292
[55] Reddy AS, Thomas TL. Expression of a cyanobacternal△6-desaturase gene result inγ-linolenic acid production in transgenic plants. Nat Biotechnol, 1996,14:639-642
[56] Cahoon EB, Shanklin J, Ohlrogge JB. Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco.Proc Natl Acad Sci USA,1992,89:11184～11188
[57]张宁洁,高国庆,张树林.回交育种分析花生脂肪酸含量的变化.中国种业,2008(2):46-47
[58]张小茜,单雷,唐桂英等.农杆菌介导的花生△12脂肪酸脱氢酶基因AhFAD2RNAi抑制表达遗传转化研究.中国油料作物学报,2007,29(4):409-415
[59]黄冰艳张新友苗利娟等.花生FAD2基因RNAi载体转化及转基因籽粒脂肪酸分析.中国油料作物学报,2008,30(3)
[60] Fridovich I. Superoxide dismutase .Joural of Bio2 logical Chemistry,1969,244:6049-6055
[61] Elster EF. Oxygen activation and oxugen toxicity.Ann Plant Physiol,1982,33:69-73
[62]王宝山.生物自由基与植物膜伤害.植物生理通讯,1988,33:73-96
[63] Dhindsa RS, Matowe W. Drought tolerance in two mosser:correlated with enzymatic defence against lipidper oxidation.J Exp Bot,1981,32:79-91
[64]刘友良等.植物抗冷性测定技术的原理和比较.植物生理学通讯,1985,21(1):40-43
[65] Lyons JM, Raison JK. Oxidative activity of mitochoncria isolated from plant tissues sensitive and resistant to chilling injury.Plant Physiol,1970,45:386-389
[66] Murata N. Molecular species composition of phosphatidyl glycerols from chilling-sensitive and chilling-resistant plants.Plant Cell Physiol,1983,24:81-86
[67] Sukhija PS, Palmquist DL. Rapid method for determination of total fatty acid content and composition of feedstuffs and feces. J Agric Food Chem. 1988,36:1202-1206
[68] Ozias-Akins P,Anderson WE, Holbrok CC.Somatic embryogenesis in~Irachis hypogaea I:genotype comparison.PlantScience,1992,83(1):103-111.
[69] Sugiyama T, Sadzuka Y, Nagasawa K, et,al. Membrane transport and antitum or activity of pirarubicin and comparison with those of doxorubicin. Jpn J Cancer Res, 1999,90(7):775-780
[70]庄振宏等.花生再生和转化体系的初步研究.福建师范大学学报(自然科学版),2001,17(4):88-92Tripepi, R. R. Adventitious shoot regeneration. In: Geneve, R. L.; Preece, J. E.;
[71] George, EF. Plant propagation by tissue culture. Part 1: The technology. Edington: Exegetics Ltd., 1993: 3-36.
[72] Merkle, SA., eds. Biotechnology of ornamental plants–biotechnology in agriculture series, No 16. Wallingford: CAB International, 1997: 45-71.
[73] Nasiruddin, KM., Begum, R., Yasmin, S. Protocorm like bodies and plantlet regenerationfrom Dedorbium formosum leaf callus. Asian J. Plant Sci., 2003, 2: 955-957.
[74]谢玉英.膜脂组成与植物抗寒性的关系.安徽农学通报,2007,13(11):38-39
[75]刘叶菊,刘飞虎.干旱胁迫对烟草样品丙二醛含量和细胞膜透性的影响.亚热带植物科学,2004,33(4)
[76]沈丽晓,陈力耕.植物抗寒基因工程的研究进展.生物学杂志, 1998,15(6):1-3
[77] Levitt J. Responses of Plants to Environmental Stresses. New York: Academic Press,1980
[78] Nanijo T, Kobaysshi M, Yoshiba Y. Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. FEBS Letter, 1999(461):205-221