摘要
目的克隆表达蚬壳花椒种子萌发过程中油脂代谢关键调控基因,研究该基因在蚬壳花椒种子外源激素赤霉素(GA)促萌发下调控蚬壳花椒种子人工萌发分子机制。方法根据蚬壳花椒转录组测序结果,筛选克隆油脂代谢关键基因,并利用原核表达体系表达纯化目的蛋白,对其编码产物进行生物信息学分析和预测。种子萌发实验分为清水对照组与GA促萌发实验组,设定0、6、12、24、48 h为萌发取样点,相对荧光定量分析该基因表达量变化。结果通过克隆该基因片段及序列分析,确定该基因编码异柠檬酸裂解酶(ICL),将其命名为ZdICL。该基因开放阅读框全长为1 728 bp,编码产物包含575个氨基酸。构建原核表达载体pET-28a-ZdICL,并在大肠杆菌BL21(DE3)中获得高效表达,对该基因编码产物的氨基酸序列进行聚类分析表明,其与甜橙同源性最高,而与模式生物拟南芥同源性最低。在种子萌发过程中其表达量在GA促萌发过程中第48小时前逐步降低,到第48小时表达量升高,与外源激素GA对种子萌发过程中脂肪动员代谢调控相吻合。结论通过对ZdICL基因进行克隆表达以及表达量分析,初步认为蚬壳花椒种子自然繁育率低下的原因之一可能是自身内源激素GA含量不足,导致ICL活性不足,无法有效保护种子抵御氧化损伤,致死种现象较多,同时为探索该基因在蚬壳花椒种子萌发过程中的调控功能奠定基础。
Objective To clone and express the key genes regulating oil metabolism during seed germination of Zanthoxylum dissitum, and investigate the molecular mechanism of the gene regulating artificial seed germination of Z. dissitum under the stimulation of exogenous hormone gibberellin（GA） of its seeds. Methods According to the transcriptome sequencing results of Z. dissitum, the key genes related to lipid metabolism were screened and cloned, the target protein was expressed and purified by prokaryotic expression system, and the encoded products were analyzed and predicted by bioinformatics analysis. Meanwhile, seed germination experiment was designed with the classification of two groups, including clear water control group and GA germination promoting experiment group. Germination sampling points were set at 0, 6, 12, 24, and 48 h. Furthermore, the relative fluorescence quantitative analysis was carried out to evaluate the changes in the expression of target genes. Results By gene cloning and sequences analysis, this gene was identified as isocitrate lyase（ICL） gene, which named as ZdICL. The total length of such open reading frame of the gene was 1 728 bp, and the coding product included 575 amino acids. Moreover, clustering analysis of the amino acid sequences of ZdICL gene coding product indicated that it had the highest homology with Citrus sinensis, but it has the lowest homology with model organism Arabidopsis thaliana. Then the prokaryotic expression vector pET-28 a-ZdICL was constructed with high-efficiency expression in Escherichia coli BL21（DE3）. In the seed germination process, the expression quantity gradually declined gradually in the GA aided germination process before 48 h, and increased at 48 h. In the seed germination process under the effect of exogenous hormones GA, it was identical to the lipid mobilization metabolism regulation. Condusion In our study, clone expression of ZdICL gene and analysis of expression quantity suggested that one of the reasons for the low natural breeding rate
作者
周韬
荣健
孙吉康
王平
李强
ZHOU Taol;RONG Jian;SUN Ji-kang;WANG Ping;LI Qiang(College of Life Science and Technology,Central South University of Forestry and Technology,Changsha 410004,China;College of Environmental Science and Engineering,Central South University of Forestry and Technology,Changsha 410004,China;Runan County Forestry Technology Promotion Station,Luoyang 471200,China)
出处
《中草药》
CAS
CSCD
北大核心
2018年第19期4628-4635,共8页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金资助项目(31370612)
湖南省高校科技成果产业化繁育项目(15CY011)
关键词
蚬壳花椒
ZdICL
克隆
原核表达
大肠杆菌
Zanthoxylum dissitum Hemsl
ZdlCL gene
cloning
prokaryotic expression
bioinforrnatics
Escherichia coli
