丝瓜苯丙氨酸解氨酶基因PAL克隆及表达分析
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福建省属公益类科研院所基本科研专项(2017R1026-6、2015R1026-9);福建省自然科学(2017J01062);福建省农业科学院创新团队PI项目(2016PI-40);福建省农业科学院创新项目(2015QC-6、PC2017-7)第一作者主要从事园艺植物生物技术与育种研究。E-mail:zhs0246@163.com


Cloning and Expression Analysis of Phenylalanine Ammonia-Lyase PAL Gene From Luffa cylindrical
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    摘要:

    采用RACE和RT-PCR技术从丝瓜果肉中分离到1条长达2406 bp的cDNA,具有完整的开放阅读框(ORF), 共2145个碱基;预测编码715个AA,理论分子量(Mw)为77.69 kD,等电点(pI)为6.11,编码的蛋白与黄瓜和香瓜的同源蛋白相似性均在93%以上,显示其高度的保守性,将基因命名为LcPAL,GeneBank登录号为:KP341758。系统进化树分析显示,丝瓜与同为葫芦科的黄瓜、香瓜的PAL蛋白的亲缘关系较近,聚为同一类。Motif Scan分析显示,LcPAL编码的蛋白包含有PAL-HAL(60 -525 位)、PLN02457(8 -715位)及phe_aml_yase(24-705位)三个保守结构域和1个酶活性中心序列(197-212位),属于典型的Lyase_I_Like超家族。Wolf Psort预测其亚细胞定位于叶绿体或内质网中。实时荧光定量PCR 分析显示,LcPAL基因在普通丝瓜果实、花、茎、叶和根中均有表达。LcPAL在所选的8个丝瓜品种中表达存在一定差异,在普通丝瓜中的表达量均高于有棱丝瓜;在普通丝瓜品种‘闽丝3 号’鲜切及采后储藏褐变过程中, LcPALV初期表达上调,后期表达量受到抑制,且与其酶活性的变化趋势基本一致。普通丝瓜LcPAL表达与果肉褐变的发生过程高度相关,LcPAL可能在普通丝瓜果肉褐变中发挥着作用。

    Abstract:

    A length of 2 406 bp cDNA was isolated from Luffa cylindrical by using Race and RT-PCR techniques,which contained a 2 145 bp open reading frame(ORF)that encoded 715 amino acids,with a predicted molecular weight of 77.69 kD and a hypothetical isoelectric point of 6.11. It shared over 93% identity with the homologous proteins from Cucumis sativus and Cucumis melo,proving that it was highly conservative. This gene was named LcPAL and the GenBank accession was KP341758. Phylogenetic analysis indicated that LcPAL and PAL of Cucumis sativus and Cucumis melo was gather to a same group. Wolf Psort protection indicated that LcPAL protein was located in chloroplast or endoplasmic reticulum,and Motif Scan analysis showed that LcPAL protein had three conserved domain databases (i.e., PAL-HAL,PLN02457 and phe_aml_yase)and an enzyme active center sequences in the position of 60 ~ 525,8 ~ 715,24 ~ 715 and 197 ~ 212 Sites,respectively,which suggested that LcPAL protein was one of typical Lyase_I_Like superfamily. Real-time PCR analysis revealed that LcPAL could be expressed in different tissues of Luffa cylindrical, including roots, stems, leaves, flowers and fruits. The levels of LcPAL were different among eight luffa varieties,and the expression in Luffa cylindrical was higher than that in Luffa acutangula Roxb. What is more, the expression level of LcPAL in‘Minsi 3’was up-regulated in the early, and then decreased during fresh-cut and post-harvest storage browning processes,which was consistent with the changes observed in peroxidase activity,suggesting that LcPAL gene may play a regulation role in luffa browning process.

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朱海生,温文旭,刘建汀,等.丝瓜苯丙氨酸解氨酶基因PAL克隆及表达分析[J].植物遗传资源学报,2018,19(2):305-313.

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  • 收稿日期:2017-05-31
  • 最后修改日期:2017-06-28
  • 录用日期:2017-07-04
  • 在线发布日期: 2018-02-23
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