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

  • Dong-Mei Li
  • Christian Staehelin
  • Wen-Tian Wang
  • Shao-Lin Peng
Article

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.

Keywords

Chitinase Cuscuta campestris Gene expression Glycoside hydrolase family 19 Mikania micrantha 

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

Notes

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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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Dong-Mei Li
    • 1
    • 2
  • Christian Staehelin
    • 1
  • Wen-Tian Wang
    • 3
  • Shao-Lin Peng
    • 1
  1. 1.State Key Laboratory of Biocontrol, School of Life SciencesSun Yat-Sen UniversityGuangzhouChina
  2. 2.Floricultural Research InstituteGuangdong Academy of Agricultural ScienceGuangzhouChina
  3. 3.South China Botanical GardenChinese Academy of SciencesGuangzhouChina

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