《植物生理学报》 2015, 51(11): 1982-1990

油桐LACS4和LACS8基因克隆及其表达分析

李捷宇, 龙洪旭, 张琳, 王哲, 李泽, 谭晓风^*

中南林业科技大学经济林培育与保护省部共建教育部重点实验室, 长沙410004

通信作者：谭晓风；E-mail: tanxiaofengcn@126.com；Tel: 0731-85623416

摘　要：

以油桐的近成熟种子为材料, 根据油桐转录组测序结果设计引物, 采用RT-PCR技术克隆了油桐长链脂酰辅酶A合成酶基因家族的两个成员的全长cDNA序列, 分别命名为VfLACS4 (GenBank登录号: KP996689)和VfLACS8 (GenBank登录号: KT362743), 通过实时定量PCR的方法检测了不同组织部位和种子不同发育时期该基因的表达水平, 并采用索式提取法测定了种子发育时期的油脂含量。结果表明: VfLACS4的CDS长度为1 989 bp, 编码662个氨基酸, 与其他物种的LACS4氨基酸序列具有较高的相似性, 其中与蓖麻的LACS4相似性最高; VfLACS8的CDS长度为2 199 bp, 编码732个氨基酸, 与麻疯树和蓖麻的LACS8具有较高的相似性; 实时定量RT-PCR结果表明VfLACS4基因在花、茎、叶和根中均有少量表达, 并在种子发育的10月10日表达量达到最大; VfLACS8基因在种子、花、茎、叶和根中均有一定量的表达, 在雌蕊中的表达量最高; 油桐种子发育过程中的油脂积累呈现出“S”型, VfLACS8基因的在发育种子中的表达模式与之相一致, 推测VfLACS8基因与种子发育过程中的油脂积累相关。

关键词：油桐; LACS4; LACS8; 基因克隆; 基因表达

收稿：2015-08-26   修定：2015-10-30

资助：国家林业公益性行业科研专项重大项目(201204403)、湖南省研究生科研创新项目(CX2015B292)和中南林业科技大学研究生科技创新基金(CX2015B11)。

Isolation and Expression Analysis of LACS4 and LACS8 from Vernicia fordii

LI Jie-Yu, LONG Hong-Xu, ZHANG Lin, WANG Zhe, LI Ze, TAN Xiao-Feng^*

The Key Laboratory of Cultivation and Protection for Non-Wood forest Trees, Ministry of Education, Central South University of forestry and Technology, Changsha 410004, China

Corresponding author: TAN Xiao-Feng; E-mail: tanxiaofengcn@126.com; Tel: 0731-85623416

Abstract:

Primers were designed according to the results of the transcriptome sequencing analysis of Vernicia fordii, two LACS family members named VfLACS4 and VfLACS8 were isolated from nearly ripe seed in Vernicia fordii with the method of RT-PCR. Their expression in different tissues and different developmental stages of seed was checked with qRT-PCR. The oil content of different developmental stages of seed was determined by soxhlet extraction. The CDS of VfLACS4 is 1 989 bp which codes a predicted protein of 662 amino acids. The amino acid identity compared with others is highly conserved and showed the highest similarity with the LACS4 of Ricinus communis. The CDS of VfLACS8 is 2 199 bp which codes a predicted protein of 732 amino acids. The amino acid identity compared with others is highly conserved and showed the highest similarity with the LACS8 of Jatropha curcas and Ricinus communis. The real-time RT-PCR result showed that VfLACS4 expressed not much in flower, stem, leaf and root, but reached the highest level in October 10th in developing seed; VfLACS8 expressed in seed, flower, stem, leaf and root, and reached the highest level in pistil. The expression mode of VfLACS8 in developmental seed is as same as the pattern of oil accumulation, VfLACS8 gene was speculated that it was related to oil accumulation in the process of seed development.

Key words: Vernicia fordii; LACS4; LACS8; gene cloning; gene expression