番茄脂肪氧化酶基因TomloxD的毕赤酵母表达及遗传转化研究
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摘要
植物脂肪氧化酶(LOX)是一类由单一的多肽链组成的含有非血红素铁、不含硫的过氧化物酶,它催化具有顺,顺-1, 4-戊二烯结构的多元不饱和脂肪酸的双加氧反应,如亚油酸(linoleic acid,LA)、亚麻酸(α-linolenic acid,LeA)、花生四烯酸(arachidonic acid,AA)。LOX参与了植物的伤害反应、病原攻击、种子萌芽、果实熟化、植物衰老以及脱落酸(Abscisic acid, ABA)和茉莉酸(Jasmonic acid, JA)合成等生理生化反应。
番茄中有五个LOX同工酶基因,即TomloxA、TomloxB、TomloxC、TomloxD、TomloxE。TomloxA、TomloxB和TomloxE相互之间的氨基酸序列的一致性是72%-77%,而TomloxC和TomloxD与TomloxA蛋白的一致性分别是42%和47%,TomloxC与TomloxD的蛋白的氨基酸一致性是46%。TomloxA、TomloxB和TomloxE在果实中表达;TomloxC在成熟果实的转色期和红熟期可以检测到,在叶片和花器中也有表达;而TomloxD主要在番茄叶中表达。乙烯能增强TomloxB和TomloxC的表达,而TomloxA的表达降低。其中TomloxC和TomloxD具有叶绿体导向信号,定位于叶绿体。这些结果表明不同的LOX异构体受到不同的调节,在番茄的生长发育过程中有不同的功能。到目前为止,番茄TomloxD基因的功能尚不清楚,为进一步阐明TomloxD基因在植物抗病性、抗逆性中的作用,本研究的主要内容与结果如下:
1.从质粒pBluescript TomloxD上PCR扩增TomloxD全长基因序列,利用pDH5.1质粒的35S CaMV启动子和终止子,构建了超表达载体pBINl9-TomloxD;通过序列比对扩增同源性较高的290 bpTomloxD基因片段序列,利用pDHG5.2中间载体构建TomloxD基因RNAi载体pBINl9-TomloxDi。
2.分别将双元载体转入根癌农杆菌LB4404,以AC++番茄子叶为外植体,通过农杆菌介导法,将目的基因转入番茄,经过50 mg/L Kan及500 mg/L的Sm筛选,获得一部分生根的再生植株,通过PCR检测表达载体上特有的NPTⅡ基因筛选到阳性植株。
3.以番茄Actin基因为内参,通过半定量RT-PCR,研究TomloxD基因在番茄不同组织的表达模式,结果表明该基因在根、成熟叶片,幼叶中表达,在果实中表达量很低。机械损伤处理后,TomloxD基因被诱导表达,在2 h时表达量最高。测定损伤处理2 h时番茄叶片脂肪氧化酶活性,未处理番茄叶片作为对照。
4.野生型番茄叶片损伤处理2 h后,提取脂肪氧化酶粗提液,对其酶活性进行了测定,结果表明,处理后LOX活性要高于未处理的对照组。
5.构建TomloxD-pPIC9K毕赤酵母表达载体,外源表达脂肪氧化酶蛋白并对其活性测定。
Lipoxygenases (linoleate: oxygen oxidoreductase, EC 1.13.11.12; LOXs) are monomeric non-heme, non-sulfur iron dioxygenases, which catalyze the incorporation of molecular oxygen into polyunsaturated fatty acids containing a cis, cis-1, 4-pentadiene moiety, such as linoleic acid(LA),α-linolenic acid(LeA), arachidonic acid,(AA). The function of various LOXs in plants has been implicated include wounding, pathogen attack, seed germination, fruit ripening, plant senescence, and synthesis of Abscisic acid (ABA) and Jasmonic acid (JA).
There are five lipoxygenases (TomloxA, TomloxB, TomloxC, TomloxD and TomloxE) present in tomato. TomloxA, TomloxB and TomloxE are 72% to 77% identical with each other at the amino acid level, while TomloxC and TomloxD show 42% and 47% identity, respectively, to the TomloxA protein, and 46% identity to each other. TomloxA, TomloxB and TomloxE are expressed at fruit. TomloxC is expressed during fruid ripening and also in leaf and flower, whereas TomloxD is mainly expressed at leaf. TomloxB and TomloxC expression is enhanced by the ripening hormone ethylene, whereas TomloxA expression declines. TomloxC and TomloxD contain chloroplast-targeting signals and are imported into chloroplasts in vitro. These results indicated that multiple isomers of LOX were regulated by different ways and have different functions in the growth and development of tomato. Nowadays, the fouction of TomloxD gene was not clear. To furtherly clarited the function of TomloxD gene in plant defense response, as follows:
1. The full length of TomloxD gene, which was obtained from the plasmid pBluescript TomloxD by PCR, was inserted into the plasmid pDH5.1. and was then inserted into the plasmid pBINl9 that digested by EcoR I to construct the over-expression vector pBINl9-TomloxD. The conserved fragment of TomloxD, which was length of 290 bp, was inserted into the middle plasmid pDH5.2. The aim fragment was excised from pDH52-TomloxD by EcoR I, and was then inserted into the plasmid pBINl9 as above.
2. The pBINl9-TomloxD and pBINl9-TomloxDi vector was transformed into Agrobactenum tumefacious strain LBA4404. Transformed tomato plants was obtained. Finally, we select the positive tomatos by PCR of NPTⅡgene.
3. The tomato-specific gene expression of Actin as control, Semi-quantitative RT-PCR analysis showed that the the TomloxD gene was mainly expressed in young leaf, mature leaf and root, and nearly has no expression in fruits. The expression of the TomloxD gene was induced by wounding, and reached the sumit at 2 h.
4. After AC++ tomato leaves were treated 2 h by wounding respose, crude enzymes were extracted and enzymes activity was determined. The result showed that activity of wounding treatment tomato leaf was higher than the control.
5. The TomloxD-pPIC9K expression vector was constructed, which expressed the protein of TomloxD in Pichia pastoris GS115, lipoxygenase activity analysis also demonstrated that the TomloxD expressed protein has lipoxygenase activity.
番茄中有五个LOX同工酶基因,即TomloxA、TomloxB、TomloxC、TomloxD、TomloxE。TomloxA、TomloxB和TomloxE相互之间的氨基酸序列的一致性是72%-77%,而TomloxC和TomloxD与TomloxA蛋白的一致性分别是42%和47%,TomloxC与TomloxD的蛋白的氨基酸一致性是46%。TomloxA、TomloxB和TomloxE在果实中表达;TomloxC在成熟果实的转色期和红熟期可以检测到,在叶片和花器中也有表达;而TomloxD主要在番茄叶中表达。乙烯能增强TomloxB和TomloxC的表达,而TomloxA的表达降低。其中TomloxC和TomloxD具有叶绿体导向信号,定位于叶绿体。这些结果表明不同的LOX异构体受到不同的调节,在番茄的生长发育过程中有不同的功能。到目前为止,番茄TomloxD基因的功能尚不清楚,为进一步阐明TomloxD基因在植物抗病性、抗逆性中的作用,本研究的主要内容与结果如下:
1.从质粒pBluescript TomloxD上PCR扩增TomloxD全长基因序列,利用pDH5.1质粒的35S CaMV启动子和终止子,构建了超表达载体pBINl9-TomloxD;通过序列比对扩增同源性较高的290 bpTomloxD基因片段序列,利用pDHG5.2中间载体构建TomloxD基因RNAi载体pBINl9-TomloxDi。
2.分别将双元载体转入根癌农杆菌LB4404,以AC++番茄子叶为外植体,通过农杆菌介导法,将目的基因转入番茄,经过50 mg/L Kan及500 mg/L的Sm筛选,获得一部分生根的再生植株,通过PCR检测表达载体上特有的NPTⅡ基因筛选到阳性植株。
3.以番茄Actin基因为内参,通过半定量RT-PCR,研究TomloxD基因在番茄不同组织的表达模式,结果表明该基因在根、成熟叶片,幼叶中表达,在果实中表达量很低。机械损伤处理后,TomloxD基因被诱导表达,在2 h时表达量最高。测定损伤处理2 h时番茄叶片脂肪氧化酶活性,未处理番茄叶片作为对照。
4.野生型番茄叶片损伤处理2 h后,提取脂肪氧化酶粗提液,对其酶活性进行了测定,结果表明,处理后LOX活性要高于未处理的对照组。
5.构建TomloxD-pPIC9K毕赤酵母表达载体,外源表达脂肪氧化酶蛋白并对其活性测定。
Lipoxygenases (linoleate: oxygen oxidoreductase, EC 1.13.11.12; LOXs) are monomeric non-heme, non-sulfur iron dioxygenases, which catalyze the incorporation of molecular oxygen into polyunsaturated fatty acids containing a cis, cis-1, 4-pentadiene moiety, such as linoleic acid(LA),α-linolenic acid(LeA), arachidonic acid,(AA). The function of various LOXs in plants has been implicated include wounding, pathogen attack, seed germination, fruit ripening, plant senescence, and synthesis of Abscisic acid (ABA) and Jasmonic acid (JA).
There are five lipoxygenases (TomloxA, TomloxB, TomloxC, TomloxD and TomloxE) present in tomato. TomloxA, TomloxB and TomloxE are 72% to 77% identical with each other at the amino acid level, while TomloxC and TomloxD show 42% and 47% identity, respectively, to the TomloxA protein, and 46% identity to each other. TomloxA, TomloxB and TomloxE are expressed at fruit. TomloxC is expressed during fruid ripening and also in leaf and flower, whereas TomloxD is mainly expressed at leaf. TomloxB and TomloxC expression is enhanced by the ripening hormone ethylene, whereas TomloxA expression declines. TomloxC and TomloxD contain chloroplast-targeting signals and are imported into chloroplasts in vitro. These results indicated that multiple isomers of LOX were regulated by different ways and have different functions in the growth and development of tomato. Nowadays, the fouction of TomloxD gene was not clear. To furtherly clarited the function of TomloxD gene in plant defense response, as follows:
1. The full length of TomloxD gene, which was obtained from the plasmid pBluescript TomloxD by PCR, was inserted into the plasmid pDH5.1. and was then inserted into the plasmid pBINl9 that digested by EcoR I to construct the over-expression vector pBINl9-TomloxD. The conserved fragment of TomloxD, which was length of 290 bp, was inserted into the middle plasmid pDH5.2. The aim fragment was excised from pDH52-TomloxD by EcoR I, and was then inserted into the plasmid pBINl9 as above.
2. The pBINl9-TomloxD and pBINl9-TomloxDi vector was transformed into Agrobactenum tumefacious strain LBA4404. Transformed tomato plants was obtained. Finally, we select the positive tomatos by PCR of NPTⅡgene.
3. The tomato-specific gene expression of Actin as control, Semi-quantitative RT-PCR analysis showed that the the TomloxD gene was mainly expressed in young leaf, mature leaf and root, and nearly has no expression in fruits. The expression of the TomloxD gene was induced by wounding, and reached the sumit at 2 h.
4. After AC++ tomato leaves were treated 2 h by wounding respose, crude enzymes were extracted and enzymes activity was determined. The result showed that activity of wounding treatment tomato leaf was higher than the control.
5. The TomloxD-pPIC9K expression vector was constructed, which expressed the protein of TomloxD in Pichia pastoris GS115, lipoxygenase activity analysis also demonstrated that the TomloxD expressed protein has lipoxygenase activity.
引文
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