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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (2): 426-437.doi: 10.3724/SP.J.1006.2023.14251

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Cloning and function analysis of a HaLACS9 gene in Helianthus annuus L.

YANG Jia-Bao1(), ZHANG Zhan2, ZHOU Zhi-Ming1, LYU Xin-Hua1, SUN Li1,*()   

  1. 1College of Life Sciences, Shihezi University, Shihezi 832003, Xinjiang, China
    2Bingtuan Xingxin Vocational and Technical College, Tiemenguan 841007, Xinjiang, China
  • Received:2021-12-29 Accepted:2022-06-07 Online:2022-07-07 Published:2022-07-07
  • Contact: SUN Li E-mail:2516040371@qq.com;sunlishz@126.com
  • Supported by:
    National Natural Science Foundation of China(31760064);National Natural Science Foundation of China(31360052)

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Abstract:

Long-chain acyl-coenzyme A (CoA) synthetase (LACS) catalyzes the formation of acyl-CoAs from free fatty acids, which plays important roles in lipid metabolism in plant. Here, a LACS family gene named HaLACS9 was screened and cloned from sunflower (Helianthus annuus L.). Homologous protein comparison showed that HaLACS9 had conserved LACS domains and had higher homology with Arabidopsis thaliana, Lactuca sativa, and Cynara cardunculus LACS9 proteins. Subcellular localization prediction indicated that HaLACS9 was localized in the chloroplast. The promoter region of HaLACS9 gene contained a large number of hormone and stress responses related elements. The qRT-PCR revealed that HaLACS9 gene was ubiquitously expressed in sunflower organs, and the highest expressed in seeds at 10 days after flowering. The transcription levels of HaLACS9 were induced by drought, salt, ABA, and GA3 treatments in sunflower roots, stems, and leaves. The relative expression pattern of HaLACS9 at seed developmental stages in sunflower was higher at the early seed developmental stages, related to sunflower seed oil rapid accumulation periods. The relative expression of HaLACS9 was gradually decreased at late stages of seed development and the slow accumulation rate of oil. HaLACS9 was proved to have the synthetase activity of acyl-CoA in complementation test of deficient yeast mutant and prefers to activate oleic acid substrate. It was speculated that HaLACS9 participated in the regulation of sunflower seed lipid biosynthesis and abiotic stress responses.

Key words: sunflower, HaLACS9, expression patterns, yeast complementation

Table 1

Primers used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 用途
Function
HaLACS9-F ATGAGTGCATATATTGTTGGAGTTT 全长cDNA克隆
Full-length cDNA cloning
HaLACS9-R TCAAGACTGGTAGAACTTTTCTAGC
18S rRNA-F CTACCACATCCAAGGAAGGCAG 实时荧光定量PCR
qRT-PCR
18S rRNA-R CGACAGAAGGGACGAGTAAACC
HaLACS9-qRT-F ACTCTCGCCCTTCGGAAATC 实时荧光定量PCR
qRT-PCR
HaLACS9- qRT-R TTTCAAATGCCCTCCCGTCT
pHaLACS9-F GGGGTACCATGAGTGCATATATTGTTGG (Kpn I) pYES2-HaLACS9载体构建
Construction of pYES2-HaLACS9 vector
pHaLACS9-R TGCTCTAGATTAAGACTGGTAGAACTTTT (Xba I)

Fig. 1

Cloning (A) and chromosome location (B) of HaLACS9 gene in Helianthus annuus L."

Fig. 2

Sequence analysis of HaLACS9 and its homologous proteins At: Arabidopsis thaliana; Ha: Helianthus annuus L.; Cc: Cynara cardunculus; Ps: Prunus sibirica; Os: Oryza sativa; Ta: Triticum aestivum; Ls: Lactuca sativa."

Fig. 3

Conserved motif distribution (A) and conservative motif (B) of LACS9 in different plant species At: Arabidopsis thaliana; Ha: Helianthus annuus L.; Cc: Cynara cardunculus; Ps: Prunus sibirica; Os: Oryza sativa; Ta: Triticum aestivum; Ls: Lactuca sativa."

Fig. 4

Structural analysis of HaLACS9 A: the tertiary structure prediction of HaLACS9 and the AMP binding domain are shown in red box. B: the signal peptide prediction of HaLACS9."

Fig. 5

Phylogenetic tree of HaLACS9 and its homologous proteins At: Arabidopsis thaliana; Ha: Helianthus annuus L.; Ls: Lactuca sativa; Cc: Cynara cardunculus; Si: Sesamum indicum; Rc: Ricinus communis; Ps: Prunus sibirica; Gm: Glycine max; Ah: Arachis hypogaea; Eg: Elaeis guineensis; Bn: Brassica napus; Zm: Zea mays; Os: Oryza sativa; Ta: Triticum aestivum. Red triangle indicates the target protein."

Fig. 6

Cis-acting elements of HaLACS9 promoter"

Fig. 7

Relative expression patterns of HaLACS9 genes Different lowercase letters indicate there are significantly different at P < 0.05."

Fig. 8

Effects of PEG (A) and NaCl (B) stress on the relative expression of HaLACS9 genes Different lowercase letters indicate there are significantly different at P < 0.05."

Fig. 9

Effects of ABA (A) and GA3 (B) stress on the relative expression of HaLACS9 genes Different lowercase letters indicate there are significantly different at P < 0.05."

Fig. 10

Relative expression level of HaLACS9 and oil contents in developing kernel of sunflower Different lowercase letters indicate there are significantly different at P < 0.05."

Fig. 11

Yeast growth status Different lowercase letters indicate there are significantly different at P < 0.05."

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