甜樱桃GalDH、GalLDH的克隆及在果实发育过程的表达

doi:10.16036/j.issn.1000-2650.2019.01.008

摘要/Abstract

摘要： 【目的】探究GalDH和GalLDH在甜樱桃果实AsA生物合成中的作用。【方法】本研究以甜樱桃‘佐藤锦’（Satonishiki）果实为材料,采用改良CTAB法提取总RNA,运用RT-PCR法,克隆并分析了抗坏血酸L-半乳糖合成途径PacGalDH和PacGalLDH基因的cDNA全长序列。此外,采用实时荧光定量PCR分析了两基因在甜樱桃果实发育过程的表达。【结果】获得了1 253 bp长的PacGalDH cDNA序列,其中含有975 bp完整的开放阅读框（ORF）,编码324个氨基酸残基。获得了1 914 bp长的PacGalLDH cDNA序列,含有1 791 bp完整的ORF,编码596个氨基酸。通过氨基酸序列比对发现,PacGalDH和PacGalLDH氨基酸序列均与梅和桃对应的氨基酸序列具有较高的同源性,达98%。实时荧光定量PCR分析表明,PacGalDH 和PacGalLDH在甜樱桃果实不同发育阶段均有表达,均在花后10 d达到最大表达量,随后逐渐下降。并且,果实总抗坏血酸（T-AsA）含量在花后0 d最高,达44.7 ng/g,此后呈下降趋势。【结论】在果实生长发育过程中,PacGalDH的表达量变化与T-AsA水平变化趋势基本一致,初步推测PacGalDH可能是甜樱桃AsA生物合成的关键酶。

关键词: 甜樱桃, 抗坏血酸, GalDH, GalLDH, 克隆, 表达分析

Abstract: 【Objective】 The aim is to explore the role of enzymes GalDH and GalLDH in AsA biosynthetic L-galactose pathway in sweet cherry fruit. 【Method】 The fruits of Satonishiki were used as material to extracte the total RNA by modified CTAB method. The full-length cDNAs of PacGalDH and PacGalLDH were cloned by RT-PCR and sequences were analyzed using bioinformatics' methods. In addition, real-time quantitative PCR was used to analyze the expression levels of two genes during the fruit development. 【Result】 The 1 253 bp-long cDNA sequence of PacGalDH was obtained, consisting of 975 bp open reading frame (ORF) which encoded 324 amino acids. The 1 914 bp-long PacGalLDH cDNA sequence was obtained, containing 1 791 bp-long OFR, enconded 596 amino acids. The results of multiple alignment indicated that GalDH and GalLDH in Prunus avium had the highest identity to those in plum and peach to 98%, respectively. Furthermore, during the whole fruit development, the of PacGalDH and PacGalLDH reached the maximum at 10 d after anthesis, then gradually decreased. Correspondingly, T-AsA content in fruit was highest at 0 d after anthesis, reaching 44.7 ng/g, thereafter decreased persistently. 【Conclusion】 The expression patterns of PacGalDH were basically consistent with the change of AsA content during the fruit development, indicating GalDH may control AsA biothesythesis in sweet cherry with other enzymes.

Key words: sweet cherry, ascorbic acid, GalDH, GalLDH, clone, expression analysis

中图分类号:

S662.5

黄晓婧, 赵雪雯, 吕秀兰, 梁东. 甜樱桃GalDH、GalLDH的克隆及在果实发育过程的表达[J]. 四川农业大学学报, 2019, 37(01): 47-52.

HUANG Xiaojing, ZHAO Xuewen, LYU Xiulan, LIANG Dong. Cloning and Expression of GalDH and GalLDH during Fruit Development in Prunus avium[J]. Journal of Sichuan Agricultural University, 2019, 37(01): 47-52.

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