薄壳山核桃原花青素合成关键酶基因的克隆与表达分析

doi:10.12302/j.issn.1000-2006.202009065

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南京林业大学学报（自然科学版） ›› 2022, Vol. 46 ›› Issue (5): 49-57.doi: 10.12302/j.issn.1000-2006.202009065

薄壳山核桃原花青素合成关键酶基因的克隆与表达分析

贾展慧(), 贾晓东(), 许梦洋, 莫正海, 翟敏, 宣继萍, 张计育, 王刚, 王涛, 郭忠仁

江苏省中国科学院植物研究所(南京中山植物园),江苏省农业种质资源保护与利用平台,江苏南京 210014

收稿日期:2020-09-30 修回日期:2021-03-24 出版日期:2022-09-30 发布日期:2022-10-19
通讯作者: 贾晓东
基金资助:
国家自然科学基金项目(31901347);江苏省科技厅苏北科技专项项目(SZ-YC2019038);江苏省中国科学院植物研究所自主科研项目(JSPKLB202055);江苏省林业科技创新与推广项目(LYKJ-句容[2020]01);江苏省科技厅重点研发计划项目(BE2021349)

Cloning and expression analyses of the key enzyme gene of procyanidins biosynthesis in pecan (Carya illinoinensis)

JIA Zhanhui(), JIA Xiaodong(), XU Mengyang, MO Zhenghai, ZHAI Min, XUAN Jiping, ZHANG Jiyu, WANG Gang, WANG Tao, GUO Zhongren

Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, The Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing 210014, China

Received:2020-09-30 Revised:2021-03-24 Online:2022-09-30 Published:2022-10-19
Contact: JIA Xiaodong

摘要/Abstract

摘要：

【目的】原花青素是广泛存在于植物中一种重要的次级代谢产物,其强抗氧化性增强了植物自身的抗逆能力,同时赋予植物清除人体自由基的保健作用。研究薄壳山核桃种仁中原花青素生物合成途径,对改良薄壳山核桃的种质与品质均具有重要意义。【方法】以薄壳山核桃‘波尼’115 和135 d种仁混合样品为材料,通过RT-PCR扩增、克隆和测序后,获得了薄壳山核桃原花青素合成关键酶相关基因CiDFR、CiLAR和CiANR的基因序列,并进行了生物信息学分析和表达水平分析。【结果】CiDFR基因长1 148 bp,包含1 020 bp的开放阅读框(ORF),编码339个氨基酸;CiLAR基因长1 390 bp,包含1 050 bp的ORF,编码349个氨基酸;CiANR基因长度为1 104 bp,包含1 014 bp ORF,编码337个氨基酸。荧光定量PCR检测结果显示,CiDFR和CiANR基因在种仁发育中期(95~105 d)表达量较高,之后快速下降至较低值;CiLAR基因在95 d表达量较高,之后快速降低,在155 d样品中表达量又升至最高点。【结论】CiDFR和CiANR基因表达量与酚类代谢物含量变化相关性较强,而CiLAR基因与之相关性较弱。

关键词: 薄壳山核桃, 酚类代谢, 基因克隆, 生物信息学分析, 荧光定量PCR

Abstract:

【Objective】Procyanidins are a kind of important secondary metabolites widely existing in plants. They have higher antioxidant activity increasing plant stress resistance, and endow plants with the health care function of scavenging free radicals at the same time. The study of procyanidins biosynthesis pathway in the pecan kernel is of great significance for improving the germplasm and quality of pecan. 【Method】The sequences of CiDFR, CiLAR and CiANR gene, which are extracted from mixed samples of ‘Pawnee’ 115 and 135 d pecan kernel. These key genes related to the phenolic metabolic pathway, were obtained by RT-PCR amplification, cloning and sequencing.【Result】 CiDFR gene is 1 148 bp long and contains 1 020 bp open reading frame (ORF), encoding 339 amino acids. The CiLAR gene is 1 390 bp long and contains 1 050 bp of ORF, encoding 349 amino acids. CiANR gene is 1 104 bp long, contains 1 014 bp ORF, and encodes 337 amino acids. Fluorescence quantitative PCR showed that CiDFR and CiANR were expressed at high levels in the middle of seed kernel development (95-105 d) and then decreased rapidly to a lower level, while CiLAR was expressed at a higher level at 95 d and then decreased rapidly, and then increased to the highest level at 155 d again. 【Conclusion】CiDFR and CiANR gene expression had a strong correlation with phenolic metabolites, while CiLAR gene had a weak correlation.

Key words: pecan(Carya illinoinensis), metabolism of phenols, gene cloning, bioinformatics analysis, fluorescence quantitative PCR

中图分类号:

Q943.2
S722

贾展慧,贾晓东,许梦洋,等. 薄壳山核桃原花青素合成关键酶基因的克隆与表达分析[J]. 南京林业大学学报（自然科学版）, 2022, 46(5): 49-57.

JIA Zhanhui, JIA Xiaodong, XU Mengyang, MO Zhenghai, ZHAI Min, XUAN Jiping, ZHANG Jiyu, WANG Gang, WANG Tao, GUO Zhongren. Cloning and expression analyses of the key enzyme gene of procyanidins biosynthesis in pecan (Carya illinoinensis)[J].Journal of Nanjing Forestry University (Natural Science Edition), 2022, 46(5): 49-57.DOI: 10.12302/j.issn.1000-2006.202009065.

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基因名称 gene name	引物名称 primer name	核酸序列 nucleotide sequence (5'-3')
CiDFR	DFR-F	TGATGGAAATCACATCAGC
	DFR-R	CGTTCGGAAGGAATAAGC
CiLAR	LAR-F	CCCATTCACATCAGTAGCA
	LAR-R	ATCAATCACCCTTTCCCT
CiANR	ANR-F	AAGAGGTAAAGGTAGCACTG
	ANR-R	GAAACAACCAAGATAGGACA

基因名称 gene name	上游引物 forward primer (5'-3')	下游引物 reverse primer (5'-3')
Actin	GCTGAACGGGAAATTGTC	AGAGATGGCTGGAAGAGG
CiDFR	TGGATGGATGTATTTCGTGTC	CAAGAGGTGGGATGATAGTGA
CiLAR	CCCTCCTTTCTTCCTTGCT	CGGTCATATTGCTGGTTGC
CiANR	GAGACATAGCCAGACCAACAC	TTATCCTGCTTCCAAGACACT

薄壳山核桃原花青素合成关键酶基因的克隆与表达分析

Cloning and expression analyses of the key enzyme gene of procyanidins biosynthesis in pecan (Carya illinoinensis)

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