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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

Keywords: 日本落叶松 纤维素合酶基因 基因克隆 单核苷酸多态性

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Abstract:

[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

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