日本落叶松纤维素合酶基因片段的克隆及单核苷酸多态性分析

易敏; 张守攻; 谢允慧; 孙晓梅

日本落叶松纤维素合酶基因片段的克隆及单核苷酸多态性分析

1.
林木遗传育种国家重点实验室, 中国林业科学研究院林业研究所, 北京 100091
基金项目:
国家"973"计划项目(2012CB114506)
中图分类号: S791.223

Isolation and Single Nucleotide Polymorphisms Analysis of Cellulose Synthase Gene Fragment in Larix kaempferi

1.
State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China
CLC number: S791.223

摘要: [目的] 纤维素合酶(cellulose synthase, CesA)在植物纤维素合成途径中发挥主要调节作用, 是控制木材纤维品质和产量的重要基因。从日本落叶松中分离克隆与纤维素合成相关的LkCesA基因, 并对其进行核苷酸多样性以及连锁不平衡分析, 为在日本落叶松中开展基于LkCesA基因的连锁不平衡作图及其辅助日本落叶松木材纤维性状的分子育种提供理论依据。[方法] 依据日本落叶松转录组数据库检测到的纤维素合酶(CesA)基因ESTs序列设计引物, 从日本落叶松中分离获得LkCesA基因片段。在此基础上, 利用DnaSP5.0软件对日本落叶松40株基因型个体的LkCesA序列进行核苷酸多样性和连锁不平衡分析。[结果] 从日本落叶松中成功克隆了CesA基因片段:该片段长1 209 bp, 包含部分开放阅读框, 长度为1 053 bp, 可编码350个氨基酸, 所推导的蛋白质氨基酸序列与火炬松PtCesA2的蛋白质氨基酸序列同源性为95.4%。在日本落叶松40株基因型个体的LkCesA序列中共检测到83个SNP位点, SNP发生频率为1/21 bp, 多样性指数πT为0.006 05。在这些SNPs中, 69个属于转换, 14个属于颠换, 其中19个为常见SNPs, 64个为罕见SNPs。在外显子区域, 共检测到54个SNP位点, 其中34个为错义突变, 20个为同义突变。进一步的连锁不平衡分析显示, 随着核苷酸序列长度的增加, SNP连锁不平衡程度逐渐减弱。[结论] 克隆到的LkCesA为植物CesA基因家族中的一员。LkCesA基因的连锁不平衡在基因内部就已衰退, 说明选择该基因作为候选基因, 在日本落叶松中开展连锁不平衡作图用于指导日本落叶松的定向培育及木材品质改良是可行的。此外, 在LkCesA基因中检测到多个常见SNP位点, 为进一步开展该基因的连锁不平衡作图提供了材料。
- 日本落叶松
- / 纤维素合酶基因
- / 基因克隆
- / 单核苷酸多态性
Abstract: Objective Cellulose synthase (CesA) plays a key role in the biosynthesis pathway of plant cellulose,which controls the quality and yield of wood fiber. This work aims at cloning the homologous genes cellulose-related from Larix kaempferi, and further analyzing the nucleotide diversity and linkage disequilibrium, which may provide important genetic foundation associated with LkCesA gene and gene-assisted breeding of new germplasms with desirable wood fiber traits in L.kaempferi.Method According to the expressed sequence tags (ESTs) of cellulose synthase from L.kaempferi transcriptome database, a cDNA fragment encoding CesA was isolated from L. kaempferi by gene-specific PCR amplification. The genomic sequences of LkCesA in 40 individuals were cloned and sequenced, then the single nucleotide polymorphisms (SNPs) diversity and linkage disequilibrium of LkCesA were analyzed using DnaSP5.0 software.Result The CesA fragment was 1 209 bp in length with a partial open reading frame (ORF, 1 053 bp) which would be capable to encode a predicted protein of 350 AA. The sequence indicated that the deduced amino acids shared 95.4% identity with PtCesA2 from Pinus taeda. A total of 83 SNPs was detected, and the frequency and diversity of SNPs (πT) were 1/21 bp and 0.006 05, respectively. There were 69 SNPs belonging to transition type and 24 belonging to transversion type, including 19 common SNPs and 64 rare SNPs. In total, 54 SNPs were detected in the coding regions of LkCesA, among which 20 were synonymous mutation and 34 were missense mutation. The results of linkage disequilibrium analysis showed that the LD declined rapidly with the nucleotide length.Conclusion The results of this study indicate that LkCesA is a member of CesA gene family. The linkage disequilibrium declined rapidly within the gene regions of LkCesA suggests that the gene could be used as the candidate gene for LD mapping, which contribute to the directional cultivation and wood quality improvement in Japanese larch. In addition, many common SNPs were detected in LkCesA which could be used in further LD mapping.
- Larix kaempferi
- / cellulose synthase
- / gene cloning
- / single nucleotide polymorphism

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日本落叶松纤维素合酶基因片段的克隆及单核苷酸多态性分析

Isolation and Single Nucleotide Polymorphisms Analysis of Cellulose Synthase Gene Fragment in Larix kaempferi

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日本落叶松纤维素合酶基因片段的克隆及单核苷酸多态性分析

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Isolation and Single Nucleotide Polymorphisms Analysis of Cellulose Synthase Gene Fragment in Larix kaempferi

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日本落叶松纤维素合酶基因片段的克隆及单核苷酸多态性分析

Isolation and Single Nucleotide Polymorphisms Analysis of Cellulose Synthase Gene Fragment in Larix kaempferi

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日本落叶松纤维素合酶基因片段的克隆及单核苷酸多态性分析

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Isolation and Single Nucleotide Polymorphisms Analysis of Cellulose Synthase Gene Fragment in Larix kaempferi

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