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

doi:10.13560/j.cnki.biotech.bull.1985.2021-1174

Abstract

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

YANG Jia-bao, ZHOU Zhi-ming, ZHANG Zhan, FENG Li, SUN Li. Cloning,Expression of Helianthus annuus HaLACS1 Gene and Identification of Its Functional Complementation in Saccharomyces cerevisiae[J]. Biotechnology Bulletin, 2022, 38(6): 147-156.

Figures/Tables 11

Table 1 Information of primers used in this study

引物名称 Primer	引物序列 Primer sequence（5'-3'）	功能 Function
L1F	CCCAAGCTTATGGATAAGCTG- CAGTTTGC	HaLACS1克隆及酵母表达载体构建
L1R	CCGCTCGAGTTAAGACTTGTT- CCCGCCTA	HaLACS1克隆及酵母表达载体构建
qF	GAGCTCGAAAACGGAACGTG	qRT-PCR
qR	ATTGCAAGCCTCCATCGCTA	qRT-PCR
PF	CGGACTAGTATGGATAAGCT- GCAGTTT	pAN580-HaLACS1-eGFP载体构建
PR	CATGCCATGGAGACTTGTTC- CCGCCTA	pAN580-HaLACS1-eGFP载体构建
18S-F	CTACCACATCCAAGGAAGG- CAG	qRT-PCR
18S-R	CGACAGAAGGGACGAGTA- AACC	qRT-PCR

Fig. 1 Cloning（A）and chromosome location（B）of the HaLACS1 gene in H. annuus

Fig. 2 Multiple alignment results of LACS1 proteins from different plants At：Arabidopsis thaliana. Ha：H. annuus L. Ls：Lactuca sativa. Bn：Brassica napus. Zm：Zea mays. Os：Oryza sativa

Fig. 3 Cis-acting elements of the H. annuus HaLACS1 gene promoter

Fig. 4 Phylogenetic relationships of the HaLACS1 protein in H. annuus with other plant LACS1

Fig. 5 Subcellular localization of the HaLACS1 protein in the protoplasts of Arabidopsis A：The green fluorescent signals of the HaLACS1 gene. B：Endoplasmic reticulum marker. C：Bright field. D：Merged

Fig. 6 Tissue expression analysis of the HaLACS1 gene Different letters indicate values are significantly different（P<0.05）. The same below

Fig. 7 Relative expressions of the HaLACS1 gene in H. annuus tissues under drought（A）and salt stresses（B）

Fig. 8 Expression pattern of HaLACS1 in H. annuus tissues under ABA treatment

Fig. 9 Growth status of recombinant yeast cells

Fig. 10 Sudan black B staining of recombinant yeast cells **P<0.01

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