樟树长链脂肪酰基CoA合成酶基因9克隆与表达分析

Identification and expression analysis of long chain fatty aycl-CoA synthetase gene 9 from Cinnamomum camphora

关键词:  樟树, 长链脂肪酰基CoA合成酶9, 籽油,基因表达分析, 酵母互补检测

Key words:  Cinnamomum camphora, long chain fatty aycl-coA synthetase 9, seed oil, gene expression analysis, yeast complementation

摘要:

该研究以樟树转录组数据为基础,筛选克隆了拟南芥AtLACS9同源候选基因CcLACS9,二者序列相似性为75%。相关软件预测CcLACS9享有植物LACS亚家族成员3个特征motifs,且N端含有定位质体信手肽。在Δlacs缺陷型酵母互补测试中,以油酸作为唯一外源脂肪酸、转化了CcLACS9的突变型酵母恢复正常生长,证明CcLACS9具有典型的脂肪酰基CoA合成酶的功能。为探究CcLACS9是否参与了樟树籽油生物合成,进一步研究了其组织表达模式和在种子发育过程中其表达量与籽油累积量之间的关系。实时荧光定量PCR分析显示CcLACS9基因在种仁与花中优势表达,种仁中相对表达量是根中的17.74倍。随机测定了30棵成年樟树成熟期种子千粒重、籽油含量和中链脂肪酸比例等指标。根据仁油含量将测试群体划为高、中、低三个不同品级,并在各品级中挑选3棵单株、逐月关联分析其仁油含量与CcLACS9相对表达量。结果表明:在种仁发育前期,仁油含量和CcLACS9表达量都持续上升且二者呈正相关性,8月份为CcLACS9表达量峰值期; 9月下旬后,仁油含量趋向稳定但CcLACS9表达量仍处于较高水平但呈现下降趋势,二者无明显相关性。LACS亚家族在植物进化中较为保守,同源基因在不同植物中具有相同或相似的功能。该研究结果暗示CcLACS9可能拥有AtLACS9相似的生物学功能,即在樟树种仁油酯合成和累积过程中起重要作用。

Abstract:

In this study, the cDNA encoding AtLACS9 homologous gene was identified and cloned based on transcriptomes data of Cinnamomum camphora, sharing 75% sequence similarity to AtLACS9 and being registered to CcLACS9. Multiple sequences alignment showed CcLACS9 possessed three plant LACS-specific motifs and plastidical targeting signal in N-terminal. Using oleic acid as a substrate, CcLACS9 could activate free fatty acids into acyl-CoA thioesters in a yeast mutant deficient in LACS complementation test. The tissue-specific expression profile further revealed that CcLACS9 was predominantly expressed in developing seeds and flowers, but fewer in leaf and stem by real-time quantitative PCR analysis. There were 17.74-folds relative quantitative expression of CcLACS9 in kernels relative to roots. In an effort to better understand whether CcLACS9 involved seed oil biosynthesis in camphor kernel, the correlation between expression of CcLACS9 and seed oil content was surveyed. The seeds of thirty adult camphor trees were randomly sampled in November and thousand seed weight, seed oil content and the percentage of decanoic acid and lauric acid were tested and counted. According to seed oil content, the 30 individuals were subdivided into three groups and three representative plants were selected from each group to be subjected to association analysis between seed oil yield and CcLACS9 expression level in developing kernel. The results of three groups all showed that both the contents of seed oil and expression level of CcLACS9 continued to rise from June to August, and there were significant positive correlations between them. The peak of expression level of CcLACS9 in kernel was found in August. After September, the contents of seed oil tended to be stable, however, CcLACS9 keeped high expression level and had no correlation with contents of seed oil. In plant, the homoeologous genes of LACSs subfamily tend to carry the common function. These resluts implies that CcLACS9 possibly play an important role in seed oil accumulation in kernel of camphor tree.