香椿TsCCR基因的克隆及表达分析

Cloning and Expression Analysis of Cinnamoyl-CoA Reductase Gene(TsCCR) from Toona sinensis

肉桂酰辅酶A还原酶(CCR)是木质素合成的关键酶,本研究根据香椿转录组数据从太和‘红油椿’中克隆得到1个肉桂酰辅酶A还原酶基因,命名为TsCCR,对其进行了生物信息学分析,并通过荧光定量PCR技术分析了TsCCR基因在香椿幼苗根、茎、叶及低温、高温、干旱、盐4种非生物胁迫条件下的表达特性。结果表明:香椿TsCCR基因含有975 bp的开放阅读框,共编码324个氨基酸,含有FR-SDR-e保守结构域,与柑橘的亲缘关系最近;TsCCR基因在茎中的表达量最高;4℃低温胁迫期间,TsCCR表达量短暂升高后逐渐降低;38℃高温胁迫期间,短暂降低后逐渐升高;200 mmol/L NaCl盐胁迫处理期间,TsCCR表达量在4 h时急剧降低,随后又急剧增加;200 g/L PEG6000干旱胁迫处理期间,TsCCR表达量呈现先下降后升高又下降的趋势。以上结果表明香椿TsCCR基因在香椿抵抗非生物胁迫方面可能发挥调控作用,本试验结果为通过基因工程改良香椿的栽培抗性提供了参考。

Cinnamoyl-CoA reductase plays an important role in lignin biosynthesis.In this research,one cinnamoyl-CoA reductase gene named TsCCR was cloned from Toona sinensis cultivar Taihe ＇Hongyou Toona sinensis＇ according to transcriptome sequencing data.The bioinformatics of TsCCR gene was analyzed,and TsCCR gene expression levels were also tested in different tissues（root,stem,leaf） of Toona sinensis seedlings,under four abiotic stress conditions（heat-temperature stress,cold-temperature stress,drought stress and salt stress） by fluorescent quantitative Real-time PCR.Sequence analysis revealed the Toona sinensis TsCCR gene contained an open reading frame of 975 bp encoding 324 amino acids,of which the TsCCR protein contained a FR-SDR-e conserved domain.Moreover,the relationship between Toona sinensis and Citrus sinensis CCR was closest.The TsCCR gene expression in stems was significantly higher than that in leaves and roots.The TsCCR gene expression increased temporarily first and then was down-regulated during 4℃ cold treatment.The expression trend was down-regulated temporarily and then was up-regulated during 38℃ heat treatment.The TsCCR gene expression decreased significantly in 4 h,and then increased rapidly again during 200 mmol/L Na Cl salt stress treatment.The TsCCR gene expression decreased,then increased,and then decreased again during 200 g/L PEG6000 drought stress treatment.All these results revealed that TsCCR gene might play an important role in resistance to abiotic stresses.These results might provide a reference for improving the cultivation of Toona sinensis by genetic engineering.