尾叶桉GLU4无性系F5H基因的克隆表达及序列分析

Cloning,expression and sequence analysis of F5H gene in Eucalyptus urophylla clone GLU4

[目的]克隆尾叶桉(Eucalyptus urophylla)GLU4中F5H基因全长,并对其进行序列和表达模式分析。[方法]根据NCBI公布的F5H基因保守序列设计引物,以尾叶桉GLU4茎部组织为试验材料克隆其g DNA和c DNA片段,利用荧光定量PCR分析其在植株不同组织中的表达模式。[结果]该基因g DNA长2 358 bp,c DNA长1 610 bp,含有2个外显子、1个内含子。开放阅读框编码529个氨基酸,Eu F5H编码的蛋白为一个带负电荷、存在跨膜结构、定位在内质网起始分泌途径中的蛋白质,二级结构包含典型的的α-螺旋和β-折叠,根据三级结构推测其可能具有羟化酶活性。Eu F5H蛋白与蓝桉亲缘关系较近,Eu F5H在不同组织中表达水平差异显著,其中半木质化茎中表达水平相对最高。[结论]成功获得了尾叶桉GLU4木质素合成酶基因F5H的全长序列。

[Objective] Cloning expression and sequence analysis of F5H gene in Eucalyptus urophylla clone GLU4. [Methods] Specific primers based on the highly conserved sequences of plant which publicized in NCBI were used. The g DNA and c DNA fragments were cloned from the young stem of Eucalyptus urophylla clone GLU4. The expression modes in different tissues of plants were analyzed by fluorescence quantitative PCR. [Results]Sequence analysis indicated that its g DNA and c DNA sequence were 2 358 bp and 1 610 bp respectively,which had two exons and one intron. Its open reading frame encoding 529 amino acid residues. Eu F5H encoding protein was a protein with negative charge,intermembrane structure and localized in the initial secretion pathway of endoplasmic reticulum and its secondary structure has typical α-helix and β-sheet. Eu F5H may have similar hydroxylase activity from the tertiary structure of protein. Phylogenetic analysis showed that it has close relative relation with Eucalyptus globules. The expression levels of Eu F5H of semi lignified stems were the highest. [Conclusion] F5H gene sequences were cloned from Eucalyptus urophylla GLU4 for the first time.