茶树△12-脂肪酸去饱和酶基因FAD2和rFAD6的克隆与表达分析

摘要

本研究在茶树转录组测序的基础上,以铁观音茶树的芽叶为材料,采用RT-PCR技术,克隆了茶树不饱和脂肪酸合成途径中的关键限速酶—△12-FAD(△12-脂肪酸去饱和酶)基因的包含完整ORF的cDNA序列(CsFAD2和CsFAD6).生物信息学分析结果表明,CsFAD2的全长为1184 bp,其开放阅读框(ORF)长度1149 bp,编码382个氨基酸,定位于内质网上,其氨基酸序列与油茶FAD2的同源性最高达97%;CsFAD6的全长为1425 bp,其ORF长度为1311 bp,编码436个氨基酸,定位于叶绿体上,其氨基酸序列与葡萄FAD6同源性达81%.荧光定量PCR结果表明,铁观音茶树幼苗在4℃低温胁迫处理72 h过程中,这两个基因的表达均受低温的诱导,其表达量随着处理时间的延长而升高,在处理48 h时,表达量水平最高;在100 g·L-1的PEG胁迫处理12 h过程中,这两个基因的表达均受PEG胁迫处理的诱导;在ABA(100μmol·L-1)胁迫处理72 h过程中,在处理6~24 h期间,CsFAD2的表达量显著升高,而CsFAD6的表达不受ABA处理的影响,CsFAD6的表达量在处理72 h时显著降低;在NaCl(250 mmol·L-1)胁迫72 h过程中,CsFAD2的表达量全程降低,而CsFAD6在处理24~72 h期间表达量显著升高.%Based on the transcriptome database of tea plants, the cDNAs ( CsFAD2 and CsFAD6) including full ORFs of the key rate-limiting enzyme-FAD (△ 12-fatty acid desaturase) in the unsaturated fatty acid synthesis pathway were cloned from the buds and leaves of tea cultivar Tieguanyin by RT-PCR .The full length of cDNA of CsFAD2 was 1184 bp, which contained a 1149 bp ORF encoding 382 amino acids with 97% homologous to the CoFAD2. Subcellular localization prediction showed that CsFAD2 was localized to the endoplasmic reticulum. The full length of cDNA of CsFAD6 was 1425 bp and contained a 1311 bp ORF encoding 436 amino acids, which was 81%homologous to VvFAD6. It was predicted to be located on the chloroplast. Quantitative PCR analysis showed that the expression of CsFAD2 and CsFAD6 were induced by cold stress (4℃), which had the highest expression levels at 48 h.Similarly, CsFAD2 and CsFAD6 were induced by 100 g·L-1 PEG treatment for 12 h. The expression of CsFAD2 was significantly up-regulated under the treatment of ABA(100 μmol·L-1 ) for 6-24 h. While the expression of CsFAD6 was not affected by ABA during this period and dramatically down-regulated at 72 h. The expression of CsFAD2 was repressed under NaCl (250 mmol·L-1) treatment. Inversely, the expression of CsFAD6 was significantly induced by NaCl treatment from 24 to 72 h.