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Molecular cloning and characterization of the promoter for the multiple stress-inducible gene BjCHI1 from Brassica juncea

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Abstract

We have previously isolated a Brassica juncea cDNA encoding a novel chitinase BjCHI1 with two chitin-binding domains (Zhao and Chye in Plant Mol Biol 40:1009–1018, 1999). The expression of BjCHI1 was highly inducible by methyl jasmonate (MeJA) treatment, wounding, caterpillar feeding, and pathogenic fungal infection. These observations suggest that the promoter of BjCHI1 gene might contain specific cis-acting elements for stress responses. Here, we report the cloning and characterization of the BjCHI1 promoter. A 1,098 bp BjCHI1 genomic DNA fragment upstream of the ATG start codon was isolated by PCR walking and various constructs were made by fusing the BjCHI1 promoter or its derivatives to β-glucuronidase reporter gene. The transgenic Arabidopsis plants showed that the BjCHI1 promoter responded to wounding and MeJA treatment, and to treatments with either NaCl or polyethyleneglycol (PEG 6000), indicating that the BjCHI1 promoter responses to both biotic and abiotic stresses. A transient gene expression system of Nicotiana benthamiana leaves was adopted for promoter deletion analysis, and the results showed that a 76 bp region from −695 to −620 in the BjCHI1 promoter was necessary for MeJA-responsive expression. Furthermore, removal of a conserved T/G-box (AACGTG) at −353 to −348 of the promoter greatly reduced the induction by MeJA. This is the first T/G-box element identified in a chitinase gene promoter. Gain-of-function analysis demonstrated that the cis-acting element present in the 76 bp region requires coupling with the T/G-box to confer full magnitude of BjCHI1 induction by MeJA.

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Abbreviations

ABA:

Abscisic acid

CaMV 35S:

Cauliflower mosaic virus 35S promoter

ET:

Ethylene

GUS:

β-glucuronidase

JA:

Jasmonate

LUC:

Luciferase

MeJA:

Methyl jasmonate

PEG:

Polyethyleneglycol

PR:

Pathogenesis-related

SA:

Salicylic acid

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Acknowledgments

Special thanks are given to Prof Mee-Len Chye (The University of Hong Kong, Hong Kong, China) for her encouragement and supply of the B. juncea seeds. We are grateful to Xi-Ping Shi and Xue-Hui Sun (Institute of Biotechnology, Chinese Academy of Agricultural Sciences, China), Xiu-Ping Guo (Institute of Crop Science, Chinese Academy of Agricultural Sciences, China) for help in taking micrograph and fluorometric quantification, and Dr Shi-Lai Bao (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China) for help in the LUC assays. Great thanks are given to Dr Zhi-Liang Chu (Arena Pharmaceuticals, Inc. San Diego, USA) for editing English writing of the manuscript. This work was supported by the National Natural Science Foundation of China (30571199).

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Author notes

  1. En-Bei Xie

    Present address: Agricultural Vocation-Technical College, 530007, Nanning, Guangxi Province, People’s Republic of China

  2. Ying-Lun Fan

    Present address: School of Agriculture, Liaocheng University, 252059, Liaocheng, Shandong Province, People’s Republic of China

Authors and Affiliations

  1. National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture, Institute of Crop Science, Chinese Academy of Agricultural Sciences (CAAS), 100081, Beijing, People’s Republic of China

    Xue-Feng Wu, Chun-Lian Wang, En-Bei Xie, Ying Gao, Ying-Lun Fan & Kai-Jun Zhao

  2. Graduate School of the Chinese Academy of Agricultural Sciences, 100081, Beijing, People’s Republic of China

    Xue-Feng Wu

  3. Agricultural College, Guangxi University, 530005, Nanning, Guangxi Province, People’s Republic of China

    Pi-Qing Liu

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  1. Xue-Feng Wu
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Wu, XF., Wang, CL., Xie, EB. et al. Molecular cloning and characterization of the promoter for the multiple stress-inducible gene BjCHI1 from Brassica juncea . Planta 229, 1231–1242 (2009). https://doi.org/10.1007/s00425-009-0911-9

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