Sequence analysis of mitogen-activated protein kinases of common buckwheat, Fagopyrum esculentum
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摘要:目的
比较普通荞麦Fagopyrum esculentum种内丝裂原活化蛋白激酶基因(MAPK)序列的差异，研究MAPK基因序列在普通荞麦栽培进化过程中的变化。
方法以普通荞麦的9个栽培品种和3个落花落果野生种质为材料，PCR特异性扩增获得MAPK基因的保守片段，对基因片段序列进行差异分析和蛋白结构预测。
结果荞麦MAPK基因cDNA全长为2 835 bp，开放阅读框1 827 bp, 编码609个氨基酸，含有TDY的三肽模块，为植物D组MAPK蛋白。PCR扩增获得12个供试材料的MAPK序列，其单型不变位点为723个，多态位点为70个。9个栽培品种间开放阅读框(ORF)区域无序列差异，3个野生种质间ORF区域也无序列差异。栽培品种与野生品种的ORF区域序列含有8个差异位点，编码3个差异氨基酸。其中，ORF区域第13位点组氨酸(H)→酪氨酸(Y)发生置换，导致1个α-螺旋构象发生变化。
结论普通荞麦MAPK基因序列高度保守，栽培驯化对ORF区域第13位点差异氨基酸的选择具有高度一致性。
Abstract:ObjectiveTo identify the difference of mitogen-activated protein kinase gene (MAPK) sequences among intraspecific common buckwheat, Fagopyrum esculentum, and study the sequence change during the chronical domestication process.
MethodThe common buckwheat varieties, containing nine cultivars and three wild types with shattering habit, were selected for PCR amplification of the conservative fragments of MAPK gene. Sequences were analyzed and protein conformations were predicted.
ResultThe full length of buckwheat MAPK cDNA was 2 835 bp, the length of the open readling frame (ORF) was 1 827 bp, and 609 amino acids containing the TDY tripeptide module were encoded. The buckwheat MARK belonged to the group D of plant MARK proteins. A total of 723 invariable sites and 70 polymorphic sites in MAPK sequence of common buckwheat were identified in 12 tested materials. We did not find difference among the ORF sequences of nine cultivars, neither among three wild types. There were eight differential nucleotides in the ORF of MAPK gene which encoded three amino acid polymorphisms between the cultivars and wild types. We found a change of α-helix conformation which was induced by transforming histidine (H) to tyrosine (Y) at the 13th site in the ORF of MARK.
ConclusionThe MAPK sequence is highly conserved and the 13th amino acid site has been highly consistently selected during the chronical process of domestication.
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Keywords:
- Fagopyrum esculentum /
- MAPK /
- sequence analysis /
- amino acid site /
- protein conformation
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致谢: 感谢贵州师范大学黎瑞源老师给予的支持和帮助！
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图 1 荞麦MAPK基因的开放阅读框序列及其编码的氨基酸序列
*标记MAPK典型的TDY结构。
Figure 1. The open reading frame and its coding amino acid sequence of buckwheat MAPK gene
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图 2 普通荞麦特异引物PCR扩增结果
M：Marker条带；P:阳性对照。
Figure 2. Amplification products of common buckwheat DNA using specified primers
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图 4 不同植物MARK蛋白序列的聚类分析
Figure 4. Phylogenetic tree of MAPK protein sequences from different plant species
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图 5 普通荞麦MARK蛋白结构域分析
A：栽培品种；B:野生种质; C、D、E分别为栽培品种MAPK蛋白第3位点Y→C(C)、第13位点H→Y(D)和第23位点M→L(E)发生转换后的结构域；箭头指向结构发生改变的区域。
Figure 5. Domain analysis of MAPK protein of common buckwheat
表 2 普通荞麦栽培品种与野生种质间MARK ORF片段的碱基和氨基酸差异位点
Table 2 The differential sites of the nucleotides and amino acids in MARK ORF fragments between cultivars and wild types of common buckwheat
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