杉木木材形成过程扩展蛋白基因的克隆与表达分析

Cloning and Expression Analysis of Expansin Genes during Wood Formation in Chinese Fir

扩展蛋白是一类细胞壁结合蛋白,在一定的pH值下无需任何激活蛋白就可诱导细胞壁的伸展。以杉木木材形成的特异表达基因文库中获得的扩展蛋白ESTs序列为基础,通过RLM-RACE技术成功克隆到3条全长扩展蛋白基因。其中ClEXPA1 cDNA全长1033 bp,包括247个氨基酸的开放阅读框(ORF)区;ClEXPA2cDNA全长1374bp,包括268个氨基酸的开放阅读框;ClEXLA1cDNA全长1023bp,包括272个氨基酸的开放阅读框。进化树重建结果表明:ClEXPA1和ClEXPA2属于α-expansin(EXPA);ClEXLA1属于γ-expansin(EXLA)。实时定量PCR显示:ClEXPA1和ClEXPA2具有较为相同的表达模式,在形成层区域表达最高,在针叶及茎尖中度表达,成熟木质部表达较低,而根和花粉中几乎不表达;ClEXLA1在形成层及针叶中均有较高的表达,在木质部及茎尖中度表达,花粉中表达较低,而根中几乎检测不到。该组扩展蛋白的成功克隆,将为系统研究扩展蛋白在木材形成过程中的表达、功能及调控机制提供基因资源。

Expansins are a kind of cell wall binding proteins, and can induce cell wall extension without any activating protein at a certain pH condition. On the basis of ESTs isolated from a xylogenesis-specific cDNA library of Chinese Fir（Cunninghamia lanceolata）, we successfully cloned 3 full-length expansin genes using RLM-RACE. Sequence analysis showed that ClEXPA1 cDNA was 1 033 bp in length capable of encoding 247 amino acids, ClEXPA2 cDNA was 1 374 bp in length capable of encoding 268 amino acids, and ClEXLA1 cDNA was 1 023 bp in length capable of encoding 272 amino acids. Phylogenetic reconstruction indicated that ClEXPA1 and ClEXPA2 belonged to α-expansin （EXPA）; whereas ClEXLA1 belonged to γ-expansin （EXLA）. Expression patterns of ClEXPA1 and ClEXPA2 were similar, with high transcript levels detected in cambium region, and intermediate levels observed in needle and shoot tip; very low levels in mature xylem, and almost no transcript in root and pollen. In Contrast ClEXLA1 had different expression pattern： high transcript levels were observed in cambium region and needle; intermediate levels were detected in shoot tip and xylem; low expression levels in pollen and barely detectable in root. These isolated genes would provide an important pool in systematically studying the expression, function and regulation mechanism of expansins during wood formation.