茶树橙花叔醇合成酶CsNES是催化(E)-橙花叔醇生物合成的关键酶。基于茶树全长转录本及基因组数据分析显示,橙花叔醇合成酶基因CsNES除全长转录本外,还存在两个较短的可变剪接基因亚型。利用5′RACE方法验证了CsNES基因分别在第二、三个外显子上存在可变的转录起始位点,并命名为CsNES-2和CsNES-3。体外蛋白重组显示,CsNES-2和CsNES-3表达产物均能够催化底物法尼基焦磷酸FPP生成(E)-橙花叔醇。基因表达分析显示,CsNES-2和CsNES-3在花中的表达量较高,在叶片中的表达水平较弱,而在嫩叶中的表达水平显著高于成熟叶片。在激素GA和MeJA处理下,CsNES-2和CsNES-3能够被MeJA诱导并上调表达。结果表明,CsNES基因存在5′端可变剪接事件,且其可变剪接产物具有催化活性,这为后续研究基因可变剪接机制对茶树萜类代谢的调控有重要指导意义。
Abstract
CsNES is a key enzyme for the formation of (E)-nerolidol in tea plants. According to the analysis of full-length transcriptome and genome data, it is shown that CsNES has two shorter alternative splicing transcripts besides the full-length transcript. The rapid amplification of cDNA ends (RACE) result indicates that the CsNES undergoes 5' alternative splicing in the second and third exons, respectively, which were named as CsNES-2 and CsNES-3. The recombinant proteins of CsNES-2 and CsNES-3 possess strong activities to catalyze the FPP into (E)-nerolidol and weak activities to hydrolyze FPP into (Z)-nerolidol. The gene expression analysis shows that CsNES-2 and CsNES-3 had higher expression levels in tea flower compared to those in tea leaves. Their expressions in young leaves were higher than those in mature leaves. Furthermore, the expression levels of CsNES-2 and CsNES-3 were induced by the MeJA treatment. This study identified two alternative splicing transcripts of the CsNES gene and gave insights into the regulation of gene alternative splicing in terpenoids metabolism in Camellia sinensis.
关键词
表达分析 /
茶树 /
橙花叔醇合成酶 /
功能分析 /
可变剪接
Key words
Camellia sinensis /
alternative splicing /
expression analysis /
function analysis /
nerolidol synthase
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基金
国家自然科学基金（32002096）、安徽省农业科学创新团队（2021YL036）
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