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Molecular Cloning and Characterization of a Chitinase-Homologous Gene from Mikania micrantha Infected by Cuscuta campestris

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Abstract

Mmchi1, a putative chitinase gene from Mikania micrantha infected by the parasitic plant Cuscuta campestris, was cloned and characterized. Mmchi1 is predicted to encode a 35.42-kDa polypeptide with a isoelectric point of 5.69. The corresponding genomic sequence contains two introns (1,171 and 621 bp). Phylogenetic analysis showed that the predicted Mmchi1 protein is related to class I and class II chitinases (glycoside hydrolase family 19). Mmchi1 is likely a class II chitinase, as the protein sequence lacks a cysteine-rich hevein domain at its N terminus. Southern blot analysis suggests that sequences related to Mmchi1 exist in the genome of M. micrantha. Expression of Mmchi1 in different tissues was analyzed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Mmchi1 was constitutively expressed in shoots, whereas only low transcript levels were detected in other tissues. Transcript levels of Mmchi1 in shoots of M. micrantha infected by C. campestris, as analyzed by RT-PCR and real-time PCR, significantly increased after 2 days post infection but markedly decreased during the following days. Transcript levels of Mmchi1 determined by semiquantitative RT-PCR varied in noninfected shoots exposed to various stress factors. Elevated levels of Mmchi1 transcripts were accumulated in response to mechanical wounding and application of abscisic acid, salicylic acid, or ZnSO4, respectively. Under salt stress conditions, Mmchi1 transcripts accumulated at significant lower levels, however. Taken together, these data suggest that Mmchi1 is a stress-related gene of M. micrantha, which is stimulated in response to C. campestris infection at early post-penetration stage but significantly suppressed during later infection stages.

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Abbreviations

dpi:

Days post infection

MW:

Molecular weight

pI:

Isoelectric point

ORF:

Open reading frame

PCR:

Polymerase chain reaction

PR:

Pathogenesis-related

RACE:

Rapid amplification of cDNA ends

RT-PCR:

Reverse transcriptase-polymerase chain reaction

SSH:

Suppression subtractive hybridization

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Acknowledgements

The authors thank Dr. Ling-Wen Ding for providing helpful information on RNA extraction. This research was financially supported by the Key Project Foundation of the Chinese Education Ministry (project no. 704037).

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  1. Dong-Mei Li

    Present address: Floricultural Research Institute, Guangdong Academy of Agricultural Science, Guangzhou, 510640, China

Authors and Affiliations

  1. State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, China

    Dong-Mei Li, Christian Staehelin & Shao-Lin Peng

  2. South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, 510650, China

    Wen-Tian Wang

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  1. Dong-Mei Li
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Correspondence to Shao-Lin Peng.

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Li, DM., Staehelin, C., Wang, WT. et al. Molecular Cloning and Characterization of a Chitinase-Homologous Gene from Mikania micrantha Infected by Cuscuta campestris . Plant Mol Biol Rep 28, 90–101 (2010). https://doi.org/10.1007/s11105-009-0125-0

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