山葡萄苯丙氨酸解氨酶基因(PAL)的克隆与表达分析

doi:10.7668/hbnxb.2018.06.009

摘要/Abstract

摘要： 为了探究山葡萄果皮转色阶段影响花色苷组成的关键酶的表达，以山葡萄幼叶为试验材料，提取山葡萄基因组DNA，应用同源克隆方法获得了PAL基因（GenBank登录号：MH045991）的全长序列，并进行了生物学信息分析。采用Q-PCR方法对山葡萄果皮8个不同着色时期的表达量进行分析。结果显示，PAL基因的DNA序列全长是1 763 bp，具有一个1 671 bp的开放阅读框（ORF），编码556个氨基酸。分子量为61.07 ku，等电点为5.76，为稳定蛋白质。整个多肽链具有跨膜螺旋结构，无信号肽，分子进化树分析表明，与欧亚种葡萄同源性较高；PAL基因在山葡萄果皮8个时期的表达规律，分析PAL基因在后4个时期表达量的不规则变化，PAL基因在转色50%和转色100%这2个时期几乎不表达。PAL基因的表达调控受很多因素调节，在山葡萄生长发育前期呈一定规律变化，在后4个时期表达量呈不规则变化，PAL基因在转色50%和转色100% 2个时期几乎不表达，这可能受到某种抑制PAL酶活性因子的影响。

关键词: 山葡萄, 苯丙氨酸解氨酶, 基因克隆, 序列分析

Abstract: To investigate the expression of key enzymes affecting the composition of anthocyanins in the transformation stage of grape peel of Vitis amurensis. With the young leaves of Vitis amurensis as the test material, the genomic DNA of Vitis vinifera was extracted and the full length sequence of PAL (GenBank accession number:MH045991) was obtained by homologous cloning, and conducted a biological information analysis. The Q-PCR method was used to analyze the expression levels of 8 grapevine skins at different pigmentation stages. The results showed that the DNA sequence of the PAL gene was 1 763 bp in length and had a 1 671 bp open reading frame (ORF) encoding 556 amino acids. The molecular weight was 61.07 ku and the isoelectric point was 5.76, which was a stable protein. The whole polypeptide chain had a transmembrane helix structure with no signal peptide. Molecular phylogenetic tree analysis showed that the homology was higher with European and Asian grape species; The expression pattern of PAL gene in the 8 stages of the grape rind of P. villosa. The PAL gene was analyzed in the last 4 periods. With irregular expression levels, the PAL gene was hardly expressed during both the 50% color shift and 100% color shift. The regulation of PAL gene expression was regulated by many factors. It changed regularly in the early stage of growth and development of Vitis vinifera. In the last 4 periods, the expression level changes irregularly. PAL gene was in 50% color rotation and 100% color rotation. Almost no expression, which may be affected by some kind of inhibition of PAL enzyme activity factors.

Key words: Vitis amurensis, Phenylalaninammo-nialyase, Gene cloning, Sequence analysis

中图分类号:

Q78
S663.03

陈蒙, 张雪, 张宇, 杨铭慧, 刘海峰. 山葡萄苯丙氨酸解氨酶基因(PAL)的克隆与表达分析[J]. 华北农学报, 2018, 33(6): 64-71. doi: 10.7668/hbnxb.2018.06.009.

CHEN Meng, ZHANG Xue, ZHANG Yu, YANG Minghui, LIU Haifeng. Cloning and Expression Analysis of Phenylalanine (PAL) Gene in Vitis amurensis[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(6): 64-71. doi: 10.7668/hbnxb.2018.06.009.

http://www.hbnxb.net/CN/Y2018/V33/I6/64

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