Cloning and function analysis of promoter of DcCDPK8 from Dendrobium catenatum

WANG Yuan1 GAO Yan-hui1 ZHU Yu-qiu1 SI Jin-ping

(1.Dendrobium State Forestry Engineering Research Center, State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Lin’an, Zhejiang Province, China 311300)

【Abstract】 DcCDPK8 is involved in abiotic stress such as low temperature stress and signal transduction of hormones ABA and MeJA, but the transcriptional regulation is still unclear. In order to study the core promoter region of DcCDPK8 gene in Dendrobium catenatum and explore its transcriptional regulation mechanism, the DcCDPK8 gene promoter sequence was cloned by PCR from D.catenatum. Promoter sequence function was studied by fusion of 5' terminal deletion and GUS gene. The results showed that the promoter sequence of DcCDPK8 gene has a low-temperature responsive element (LTR) between −1 749 bp and −614 bp, two MeJA responsive elements between −1 749 bp and −230 bp, and one ABA responsive element between −614 bp and −230 bp. Three 5'-end different deletion fragments were constructed to fuse the eukaryotic expression vectors p BI121 with GUS, which were transformed into tobacco leaves. The GUS activities under cold stress treatment were DcCDPK8-p1 > DcCDPK8-p2 > DcCDPK8-p3. GUS activities under exogenous ABA induction were DcCDPK8-p1 > DcCDPK8-p2 > DcCDPK8-p3, and GUS activities under exogenous MeJA induction were DcCDPK8-p1 > DcCDPK8-p2 > DcCDPK8-p3. It is speculated that the ABA response element (ARE) in the promoter sequences of DcCDPK8 plays a positive regulatory role in response to exogenous ABA, while the MeJA cis-acting element plays a negative role in response to exogenous MeJA.

【Keywords】 Dendrobium catenatum; DcCDPK8 promoter; transient transfection; histochemical staining;

【DOI】

【Funds】 National Key Research and Development Program (2017YFC1702201) National Natural Science Foundation of China (81603228)

Download this article

    References

    [1] Si JP, Zhang Y, Luo YB, et al. Herbal textual research on relationship between Chinese medicine “Shihu” (Dendrobium spp.) and “Tiepi Shihu” (D. catenatum) [J]. China Journal of Chinese Materia Medica, 2017, 42 (10): 2001 (in Chinese).

    [2] Si JP, Yu QX, Song XS, et al. Artificial cultivation modes for Dendrobium officinale [J]. China Journal of Chinese Materia Medica, 2013, 38 (4): 481 (in Chinese).

    [3] Harmon A C, Gribskov M, Gubrium E, et al. The CDPK superfamily of protein kinases [J]. New Phytol, 2010, 151 (1): 175.

    [4] Liu GS, Chen J. Roles of Calcium-dependent Protein Kinases (CDPKs) in Plant Calcium Signal Transduction [J]. Chinese Bulletin of Botany, 2003, 20 (2): 160 (in Chinese).

    [5] Schenk P W, Snaarjagalska B E. Signal perception and transduction:the role of protein kinases [J]. Biochim Biophys Acta, 1999, 1449 (1): 1.

    [6] Nishiyama R, Mizuno H, Okada S, et al. Two mRNA species encoding calcium-dependent protein kinases are differentially expressed in sexual organs of Marchantia polymorpha through alternative splicing [J]. Plant Cell Physiol, 1999, 40 (2): 205.

    [7] Cordingley M G, Riegel A T, Hager G L. Steroid-dependent interaction of transcriptionfactors with the inducible promoter of mouse mammary tumor virus in vivo[J]. Cell, 1987, 48 (2): 261.

    [8] Valdivia R H, Falkow S. Bacterial genetics by flow cytometry:rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction[J]. Mol Microbio, 1996, 22 (2): 367.

    [9] Sheng K, Gao YH, Si JP, et al. Cloning and Expression Analysis of DoCDPK6 and Promoter in Dendrobium officinale [J]. Journal of Agricultural Biotechnology, 2017, 25 (4): 588 (in Chinese).

    [10] Sheng K, Gao YH, Si JP, et al. Cloning and Expression Analysis of DoCDPK Genes in Dendrobium officinale [J]. Acta Horticulturae Sinica, 2016, 43 (12): 2412 (in Chinese).

    [11] Bendahmane A, Querci M, Kanyuka K, et al. Agrobacterium transient expression system as a tool fo the isolation of disease resistance genes:application to the Rx2 locus in potato [J]. Plant J, 2010, 21 (1): 73.

    [12] Jefferson R A, Kavanagh T A, Bevan M W. GUS fusions: betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants [J]. Embo J, 1987, 6 (13): 3901.

    [13] Sheng K. 铁皮石斛DcCDPKs基因的克隆及表达分析 [D]. Linan: Zhejiang A & F University, 2017 (in Chinese).

    [14] Liu YR, Lv LS, Cheng J, et al. Mechanism of CDPKs in Plant Adaptation to Abiotic Stress [J]. Plant Physiology Journal, 2015 (9): 1387 (in Chinese).

    [15] Wu ZG, Wu SJ, Wang YC, et al. 植物中钙依赖蛋白激酶 (CDPK) 的研究进展 [J]. Acta Prataculturae Sinica, 2018 (1): 204 (in Chinese).

This Article

ISSN:1001-5302

CN: 11-2272/R

Vol 44, No. 02, Pages 293-297

January 2019

Downloads:0

Share
Article Outline

Knowledge

Abstract

  • 1 Materials and methods
  • 2 Results and analysis
  • 3 Conclusion and discussion
  • References