向日葵HaLACS1的克隆、表达及酵母功能互补鉴定  

Cloning,Expression of Helianthus annuus HaLACS1 Gene and Identification of Its Functional Complementation in Saccharomyces cerevisiae

作　　者：杨佳宝 周至铭 张展 冯丽[1] 孙黎[1] YANG Jia-bao;ZHOU Zhi-ming;ZHANG Zhan;FENG Li;SUN Li(College of Life Sciences,Shihezi University,Shihezi 832003;Bingtuan Xingxin Vocational and Technical College,Bazhou 841007)

机构地区：[1]石河子大学生命科学学院,石河子832003 [2]兵团兴新职业技术学院,巴州841007

出　　处：《生物技术通报》2022年第6期147-156,共10页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31760064,31360052)。

摘　　要：探究长链酰基辅酶A合成酶(long chain acyl-CoA synthetase,LACS)基因在向日葵(Helianthus annuus L.)油脂积累和逆境响应中的功能,为其在向日葵油脂合成和抗逆中的应用奠定基础。通过RT-PCR克隆得到向日葵HaLACS1的CDS序列,运用生物信息学方法分析HaLACS1的特点。利用实时荧光定量PCR(qRT-PCR)技术检测HaLACS1的组织表达特性及对NaCl、PEG和ABA的响应情况。通过构建GFP和HaLACS1的融合表达载体,转化拟南芥原生质体进行亚细胞定位分析。将HaLACS1转入酿酒酵母突变型菌株YB525中进行功能互补试验,并进行底物偏好性分析。结果显示,HaLACS1开放阅读框1 980 bp,编码659个氨基酸。蛋白进化树分析表明,HaLACS1与拟南芥AtLACS1和莴苣(Lactuca sativa)LsLACS1具有较高的相似性。拟南芥原生质体瞬时表达分析显示HaLACS1定位于内质网。实时荧光定量PCR结果显示,HaLACS1在所有组织中均有表达,但在种子发育的早期表达量较高,花次之。NaCl、PEG和ABA处理均能诱导HaLACS1在向日葵根、茎和叶中的表达。酵母功能互补试验证明HaLACS1蛋白具有LACS酶活性。底物偏好性分析表明HaLACS1偏好棕榈酸(C16:0)和油酸(C18:1)。表明HaLACS1与向日葵种子发育过程中的油脂积累和非生物胁迫响应相关。This work aims to investigate the functionality of long chain acyl-CoA synthetase(LACS)gene in Helianthus annuus L.in the oil accumulation and responses to abiotic stresses,for laying a foundation in the its oil synthesis and application in the tress responses.RTPCR was applied to clone the CDS sequence of the HaLACS1,and bioinformatics method was to analyze the characteristics of the HaLACS1.Real-time quantitative PCR was employed to detect the expression patterns of HaLACS1 in different tissues and in responses to NaCl,PEG and ABA treatments.The fusion protein of GFP and HaLACS1 was constructed and transformed into Arabidopsis thaliana protoplasts for subcellular localization analysis.The HaLACS1 gene was transformed into a LACS-deficient yeast strain YB525 to examine the complementation effect and analyze its substrate.The results showed that the length of HaLACS1’s open reading frame was 1980 bp,encoding 659 amino acids.The phylogenetic tree analysis demonstrated that HaLACS1 was highly similar with the AtLACS1 in A.thaliana and LsLACS1 in Lactuca sativa.The transient expression assays revealed that HaLACS1 was located in endoplasmic reticulum.qRT-PCR result showed that HaLACS1 was ubiquitously expressed in all tissues of H.annuus,but highly expressed in seeds at early developmental stage,followed in the flowers.The transcription levels of HaLACS1 were induced by NaCl,PEG and ABA in H.annuus roots,stems and leaves.Expression of HaLACS1 in the yeast strain YB525 demonstrated that HaLACS1 has LACS enzyme activity.Substrate preference analysis showed that HaLACS1 preferred palmitic acid(C16:0)and oleic acid(C18:1). All these results suggest that HaLACS1 is related to H. annuus in response to abiotic stressesand oil accumulation in the process of seed development.

关 键 词：向日葵 HaLACS1 亚细胞定位 表达模式 酵母功能互补 

分 类 号：S565.5[农业科学—作物学] Q943.2[生物学—植物学]