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谷胱甘肽过氧化物酶(glutathione peroxidase,GPXs)是一种重要的过氧化物分解酶,能清除活性氧,阻止脂膜氧化,起到保护细胞免受氧化胁迫的作用,具有重要的生理功能[1]。自CRIQUI等[2]从烟草Nicotiana tabacum中获得GPX基因后,先后从柑橘Citrus[3],番茄Lycopersicon esculentum[4],水稻Oryza sativa[5],香蕉Musa nana[6],荷花Nelumbo nucifera[7],枣Ziziphus jujube[8]等植物中获得GPX基因。近年来,对于植物GPXs的研究主要集中在生物胁迫和非生物胁迫(如高温、低温、干旱、重金属毒害、耐盐等)方面,尤其是GPXs在非生物胁迫中的功能作用,证实了GPX基因与植物抗逆性紧密相关。在非生物胁迫下,GPX基因在转录水平发生变化,参与相关氧化还原途径[9]。如苹果Malus MdGSTU1提高苹果的耐盐能力[10],过量表达NtGPX基因提高烟草的抗冻性[11],番茄LePHGPX基因的过量表达提高植株耐高温能力[12]。研究人员对拟南芥Arabidopsis thaliana GPX基因的功能研究取得较大进展,新的GPX被不断发掘(拟南芥目前已知8个),对其中7个GPX基因的研究证实,全部对非生物胁迫有应答反应,转基因可提高植物抵御胁迫的能力[13],如AtGPX3能显著增强植株的抗旱性[14],AtGPX1具有抗高温下的氧化胁迫能力[15]。目前虽有多种植物GPX基因被发现,并开展相关功能研究,但主要集中于单子叶和双子叶植物,对裸子植物的研究较少。马尾松Pinus massoniana属裸子植物松科Pinaceae松属Pinus植物,是中国南方重要的用材林树种[16],但在马尾松生长过程中常受到病虫害的危害,给林业经济带来严重损失,制约了马尾松人工林的健康发展。本研究在高通量测序分析基础上结合cDNA末端快速扩增(RACE)技术,获得谷胱甘肽过氧化物酶基因,对其进行生物学分析,利用实时定量聚合酶链式反应(qRT-PCR)技术研究其不同组织、不同抗性材料、不同时间表达模式,为探求马尾松虫害防御机制提供依据。
Cloning and expression analysis of the PmGPX gene encoding glutathione peroxidase in Pinus massoniana
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摘要: 克隆获得马尾松Pinus massoniana PmGPX基因,进行生物信息学分析以及在不同抗虫材料表达模式分析。在前期马尾松转录组测序获得该基因片段基础上,用cDNA末端快速扩增(RACE)技术获得基因全长,实时荧光定量聚合酶链式反应(qRT-PCR)技术分析该基因在不同抗虫材料中的表达模式。克隆获得基因cDNA全长序列,命名为PmGPX。该基因包含741 bp开放阅读框,编码246个氨基酸。序列分析表明:该基因具有GPX家族典型的保守结构域,等电点PI 8.29,蛋白分子量27.08 kDa,与针叶植物北美云杉Picea sitchensis的同源性较高,为91%。与单子叶和双子叶植物同源性较差,大部分在64%~80%,而在GPX家族典型保守结构域的同源性较高,为82%~100%。qRT-PCR技术分析表明:PmGPX在所有组织中均有表达,在针叶中表达量最高,嫩叶的表达量是根的33.45倍,在根、花和球果中的表达量较低,不同材料日表达模式相似,总体上随时间出现“升—降—升”模式。该基因在抗虫材料中启动较早,且表达量高于对照。推测该基因参与了马尾松抗虫防御体系。Abstract: The study PmGPX genes was played a role in Pinus massoniana insect resistance, that the great significance for in-depth insect-resistant resistance mechanism in Pinus massoniana. PmGPX, a cDNA region encoding glutathione peroxidase, was cloned in the transcriptome sequencing analysis of one insect-resistant material in Pinus massoniana. Analyses included an amino acid sequence and an expression (qRT-PCR) analysis. Results showed that the total length of open reading frame (ORF) was 741 bp encoding 246 amino acids. The amino acid sequence analysis showed three high conservative character fields of the GPX gene family for this gene. The isoelectric point of PmGPX was PI 8.29, and the protein molecular weight was 27.08 kDa. In genetic clustering, compared with some other plants, PmGPX showed a higher homology with Picea sitchensis (91%) but a low homology with other monocots and dicotyledonous plants (64%-80%). However, homologies of the three typical conservative GPX family domains (G1, G2, G3) reached a higher level with other plants (82%-100%). The expression analysis indicated that PmGPX was expressed in all kinds of tissues with higher expression levels in leaves (P < 0.01), especially in young leaves (33.45 times more than the expression level in roots). Daily expression patterns were same in different insect-resistant materials and showed up-down-up-regulated trend the day. In insect-resistant material, expression of PmGPX reached a higher point earlier than that of control (P < 0.05 in old leaves and tender stem). The expression analysis also indicated that PmGPX was involved in the insect-resistant defense system of P. massoniana. This study laid a foundation for further analysis of the PmGPX biological function and its role in insect-resistant defense with Pinus massoniana.
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https://zlxb.zafu.edu.cn/article/doi/10.11833/j.issn.2095-0756.2017.05.012