核桃Y_2SK_2型脱水素基因JrDHN的克隆、表达和单核苷酸多态性分析

Molecular Cloning, Expression and Single Nucleotide Polymorphisms Analysis of Typical Y_2SK_2 Dehydrin in Juglans

采用RT-PCR和RACE技术从‘香玲’核桃(Juglans regia L.‘Xiangling’)叶片中克隆了1个脱水素基因JrDHN(GenBank登录号KC503061.1),其全长1 056 bp,具有528 bp的开放阅读框,编码175个氨基酸组成的多肽,具有LEA家族成员的特征多肽序列,属于典型的Y2SK2型脱水素。以核桃18S基因为内参,对‘香玲’核桃JrDHN在4℃胁迫下的表达模式进行了初步的研究,结果表明低温诱导下叶片中JrDHN的表达增加,4℃处理4 h后达到最大值;自然越冬条件下,花芽中JrDHN表达量呈先上升后下降的趋势,在12月份表达量最高,推测JrDHN在核桃对低温胁迫响应中发挥重要功能。‘香玲’JrDHN在雌花中表达量高,其次是叶片和树皮,在花芽中最低。单核苷酸多态性分析JrDHN序列中有30个SNPs位点和11个Indels;单倍型分析显示12份核桃材料可分为10个单倍型,单倍型多样性为0.9697。

Reverse transcription PCR(RT-PCR)and rapid amplification of cDNA ends(RACE)technologies were used to clone JrDHN(GenBank accession No. KC503061.1)from leaves of walnut(Juglans regia L.‘Xiangling'). Cloned JrDHN gene was 1 056 bp in length,containing 528 bp ORF encoded 175 amino acids. Amino acid sequence analysis indicated that JrDHN possessed typical characters of LEA family. qRT-PCR analysis showed that the expression of JrDHN in Xiangling walnut was induced by low temperature and peaked at 4 hours after exposed to 4 ℃ low temperature. Under the condition of natural overwintering,JrDHN gene expression increased firstly,reached to a peak in December,followed by decrease. It was deduced that JrDHN may play important roles in tolerating to cold stress. The transcripts of JrDHN could be detected in female flower,bark,flower bud and leaf of Xiangling walnut,especially high expression in female flower. Additionally,after analyzing single nucleotide polymorphism of JrDHN,30 SNPs and 11 Insertions/Deletions(Indels)were detected among 12 walnut accessions. Haplotype analysis showed that 12 walnut accessions were divided into ten haplotypes,and the haplotype diversity was 0.9697.