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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (9): 1703-1711.doi: 10.3724/SP.J.1006.2021.04247

• RESEARCH PAPERS • Previous Articles     Next Articles

Characterization of the promoter and 5'-UTR intron in AhFAD2-1 genes from peanut and their responses to cold stress

SHI Lei1,2(), MIAO Li-Juan1,2, HUANG Bing-Yan1,2, GAO Wei3, ZHANG Zong-Xin1,2, QI Fei-Yan1,2, LIU Juan3, DONG Wen-Zhao1,2, ZHANG Xin-You1,2,*()   

  1. 1Henan Academy of Crops Molecular Breeding, Henan Academy of Agricultural Sciences / Key Laboratory of Oil Crops in Huanghuaihai Plains, Ministry of Agriculture and Rural Affairs / Henan Provincial Key Laboratory for Oil Crops Improvement / National and Provincial Joint Engineering Laboratory for Peanut Genetic Improvement, Zhengzhou 450002, Henan, China
    2Henan Biological Breeding Center Co., Ltd., Zhengzhou 450002, Henan, China
    3Industrial Crops Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan, China
  • Received:2020-11-17 Accepted:2021-03-19 Online:2021-09-12 Published:2021-03-31
  • Contact: ZHANG Xin-You E-mail:leis100@163.com;haasz@126.com
  • Supported by:
    National Key Research and Development Program of China “Physiological Basis and Agronomic Management for High-quality and High-yield of Field Cash Crops”(2018YFD1000900);China Agriculture Research System(CARS-13);Henan Province Agriculture Research System(S2012-5)

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Abstract:

Delta-12 fatty acid desaturase 2 (FAD2) catalyzes the conversion of oleic acid to linoleic acid and is the determination of the level of oleic to linoleic acid ratio (O/L) in peanut oil. Peanuts with high oleic acid content are more susceptible to cold stress than those with normal oleic acid content, suggesting that FAD2 plays important roles in response to cold stress. To explore the role ofFAD2s during the process of cold stress acclimation in peanut, the genomic structures of AhFAD2-1A/Bwere determined; the function of promoters, intron of AhFAD2-1A/Band their response to cold stress were characterized by using β-glucuronidase (GUS) gene reporter system in transgenic Arabidopsis. The results were as follows: AhFAD2-1A/B genes consisted of two exons and one intron within their 5'-UTR; promoter ofAhFAD2-1A was too weak to be detected and the promoter of AhFAD2-1B poorly activated the expression level of GUS in cotyledon tip of seedlings; the promoter of AhFAD2-1pseudogene activated GUS expression limited to cotyledon, hypocotyl, and seed. The intron of AhFAD2-1B demonstrated promoter-like activity which was restricted in cotyledon and hypocotyl, and not only enhanced the gene expression efficiency but also expanded gene expression range. Intron-mediated enhancement was an essential aspect of AhFAD2-1expression. Activities of 5'-flanking region of AhFAD2-1A/B were repressed by the cold stress.

Key words: peanut (Arachis hypogaea L.), Δ12-fatty acid desaturase gene (FAD2), promoter, intron-mediated enhancement, cold stress, GUS reporter gene

Table 1

Sequences of primers used in this study"

目的片段
Target fragment 		上游引物
Forward primer (5°-3°) 		下游引物
Reverse primer (5°-3°)
PAhFAD2-1A+intron ACGCGTCGA (Sal I) CTTCCGCCAGAATGAGGAACTAAA TCACAG GACATCTAGA (Xba I) GTTGTGTTGTTAAAGCT CCTGTTACCAATG
PAhFAD2-1B+intron CGGGGTACC (Kpn I) ATGTTTCATAGAATTTAAGCTCAGA CACG
PAhFAD2-1B(pseudo) ACGCGTCGA (Sal I) CACCAAGTAGCTTCTCAATGGCTCA GATTCG
AhFAD2-1B-intron ACGCGTCGA (Sal I) CATAGGAGAAGCACTCACTTCTCTT CTCTC
AhFAD2-1B-intron681 ACGCGTCGA (Sal I) CATAATGGCTTCTGGGCCCTCAC
AhFAD2-1B-intron363 ACGCGTCGA (Sal I) CATAATGGCTTCTGGGCCCTCAC GACATCTAGA (Xba I) TATCATGCCAAGTGACT AGTATGA
PAhFAD2-1A ACGCGTCGA (Sal I) CTTCCGCCAGAATGAGGAACTAAA TCACAG GACATCTAGA (Xba I) GATGAATCTCGCAGCCA CGTTT
PAhFAD2-1B CGGGGTACC (Kpn I) ATGTTTCATAGAATTTAAGCTCAGA CACG

Fig. 1

Comparison of FAD2-1A/B/pseudo promoters and genomic structures in peanut TSS: the transcription start site."

Fig. 2

Histochemical analyses of AhFAD2A/B/pseudo promoters and introns in transgenic Arabidopsis plants "

Fig. 3

GUS histochemical staining of transgenic Arabidopsis with PAhFAD2-1A+intron under cold stress A: PAhFAD2-1A+intron transgenic Arabidopsis plants; B: CaMV35S transgenic Arabidopsis plants. 1-day: one day after treating; 2-day: two days after treating; 4-day: four days after treating. "

Fig. 4

GUS histochemical staining of transgenic Arabidopsis with PAhFAD2-1B +intron under cold stress 1-day: one day after treating; 2-day: two days after treating; 4-day: four days after treating."

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