花生2-甲基-6-植基-1,4-苯醌甲基转移酶基因VTE3的克隆及多态性分析

doi:10.3864/j.issn.0578-1752.2012.09.003

摘要/Abstract

摘要： 【目的】分离花生2-甲基-6-植基-1,4-苯醌甲基转移酶（MPBQ MT）基因VTE3，揭示其分子生物学特征及遗传多态性。【方法】利用EST拼接、RT-PCR以及以DNA为模板的PCR扩增技术，从花生属栽培种中分离VTE3 全长cDNA；从不同类型栽培品种和花生属花生区组二倍体野生种（Arachis duranensis 和A. ipaensis ）中分离VTE3全长DNA，进行VTE3序列多态性分析，并构建VTE3的进化树。【结果】从3个栽培品种中分别克隆得到2条VTE3 cDNA序列（命名为rVTE3-1和rVTE3-2），rVTE3-1和rVTE3-2的编码区长均为1 059 bp，二者同源性97.8%，存在10个变异位点，其中8个为SNP变异；二者均编码351个氨基酸，氨基酸序列同源性98.6%，存在5个氨基酸差异。从13个栽培品种分别克隆得到2条VTE3 DNA序列（命名为gVTE3-1和gVTE3-2），13个品种间gVTE3-1的同源性为99.9%，gVTE3-2的同源性为100%。其中丰花2号gVTE3-1序列长2 710 bp，存在3个内含子，分别位于44—163、772—1 295和1 603—2 437 bp处；gVTE3-2序列长2 706 bp，也存在3个内含子，分别位于44—169、778—1 291和1 599—2 433 bp处。丰花2号gVTE3-1和gVTE3-2同源性96.6%，内含子区域存在36个SNP位点和3个限制性内切酶识别的多态性位点。从A. duranensis分离的VTE3 DNA序列命名为gVTE3-A，从A. ipaensis分离的VTE3 DNA序列命名为gVTE3-B。利用栽培种丰花2号gVTE3-1和gVTE3-2以及野生种gVTE3-A和gVTE3-B 4条DNA序列进行进化分析，推测序列gVTE3-1和gVTE3-2分别来自丰花2号的A、B染色体组。花生MPBQ MT氨基酸序列与其它物种的同源性较高，具有很强的保守性。【结论】本研究克隆了花生VTE3的全长cDNA和DNA；推断栽培品种的gVTE3-1和gVTE3-2分别来自A、B染色体组，不同染色体组的VTE3多态性位点丰富；不同栽培品种间等位基因核苷酸序列差异很小，所检测13个栽培品种的gVTE3-1以及野生种的gVTE3-A间存在等位变异，13个栽培品种的gVTE3-2以及野生种gVTE3-B间未发现等位变异。

关键词: 花生, 维生素E, 2-甲基-6-植基-1, 4-苯醌甲基转移酶, 基因克隆

Abstract: 【Objective】 The objective of this study is to isolate VTE3 (encoding MPBQ MT) in peanut, and to reveal the molecular characteristics and investigate polymorphism of the genes.【Method】A putative VTE3 of peanut was obtained via EST sequence splicing. cDNAs of VTE3 were cloned from cultivated varieties of A. hypogaea by RT-PCR, genomic DNAs of VTE3 were cloned from cultivated varieties and two wild species in Arachis by PCR. Polymorphism analysis of VTE3 from cultivated varieties and two wild species were performed, and phylogenetic tree was constructed by PHYLIP software. 【Result】 Two cDNA sequences of VTE3 (designated as rVTE3-1 andrVTE3-2) were isolated from each of the three cultivated varieties (Arachis hypogaea L.). rVTE3-1 and rVTE3-2 both had a DNA sequence of 1 059 bp in length, with the homology of 97.8% between the two sequences, and eight single-nucleotide polymorphisms (SNPs) exist within the sequences. The two cDNA sequences both encode 351 amino acids, and the homology of the two proteins was 98.6% with five amino acid differences. The two DNA sequences of VTE3 from each cultivar were designated as gVTE3-1 and gVTE3-2. The sequences of gVTE3-1 from the thirteen cultivars share a homology of 99.9% in nucleotide acid level and the thirteen sequences of gVTE3-2 are identical. gVTE3-1 of cultivated peanut samples from Fenghua 2 had a length of 2 710 bp, with three introns located at 44-163, 772-1 295 and 1 603-2 437 bp, and gVTE3-2 from Fenghua 2 had a length of 2 706 bp with three introns located at 44-169, 778-1 291 and 1 599-2 433 bp. Thirty-six SNPs and three variation sites of endonuclease recognition were identified between the intrones of the two sequences. Meanwhile, the VTE3 DNA (designated as gVTE3-A and gVTE3-B) was isolated from wild species A. duranensis (A-genome) and A. ipaensis (B-genome), respectively. The sequences of gVTE3-1 (Fenghua 2), gVTE3-2 (Fenghua 2), gVTE3-A and gVTE3-B were aligned and the phylogenetic tree was constructed. The tree demonstrated that FhgVTE3-1 and gVTE3-A clusters form a group and FhgVTE3-2 and gVTE3-B clusters form as an additional group. Sequence alignment analysis revealed that the homology of FhgVTE3-1 and gVTE3-A was 98.7% and the homology of FhgVTE3-2 and gVTE3-B was 100%. The amino acid homologies of MPBQ MT among different species shared higher similarities.【Conclusion】Full-length cDNA and DNA sequences in VTE3 of peanut were cloned. The present study demonstrated that abundant polymorphisms were observed in VTE3 from A-genome and B-genome of A. hypogaea, respectively. Additionally, polymorphisms were observed in the gVTE3-1 alleles of the thirteen cultivars and wild species of A. duranensis. However, no variation of gVTE3-2 was found in the gVTE3-2 alleles of the thirteen cultivars and wild species of A. ipaensis.

Key words: Arachis hypogaea L., vitamin E, 2-methyl-6-phytyl-1, 4-benzoquinone methyltransferase (MPBQ MT), gene cloning

郭安强, 万勇善, 刘风珍. 花生2-甲基-6-植基-1,4-苯醌甲基转移酶基因VTE3的克隆及多态性分析[J]. 中国农业科学, 2012, 45(9): 1685-1695.

GUO An-Qiang, WAN Yong-Shan, LIU Feng-Zhen. Cloning and Polymorphism of 2-Methyl-6-Phytyl-1, 4-Benzoquinol Methyltransferase Gene (VTE3) in Arachis[J]. Scientia Agricultura Sinica, 2012, 45(9): 1685-1695.

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