Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (6): 147-156.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1174

Previous Articles     Next Articles

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

YANG Jia-bao1(), ZHOU Zhi-ming1, ZHANG Zhan2, FENG Li1, SUN Li1()   

  1. 1. College of Life Sciences,Shihezi University,Shihezi 832003
    2. Bingtuan Xingxin Vocational and Technical College,Bazhou 841007
  • Received:2021-09-13 Online:2022-06-26 Published:2022-07-11
  • Contact: SUN Li E-mail:2516040371@qq.com;lis@shzu.edu.cn

Abstract:

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. RT-PCR 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 1 980 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 stresses and oil accumulation in the process of seed development.

Key words: Helianthus annuus, HaLACS1, subcellular localization, expression patterns, yeast functional complementation