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)

【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。

【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.