Plant Molecular Biology Reporter

, Volume 20, Issue 1, pp 49–57

Application of suppression subtractive hybridization (SSH) to cloning differentially expressed cDNA inDunaliella salina (chlorophyta) under hyperosmotic shock

  • Xiao-Ning Zhang
  • Zhi-Cai Qu
  • You-Zhong Wan
  • Hong-Wei Zhang
  • Da-Leng Shen
Protocols

DOI: 10.1007/BF02801932

Cite this article as:
Zhang, XN., Qu, ZC., Wan, YZ. et al. Plant Mol Biol Rep (2002) 20: 49. doi:10.1007/BF02801932

Abstract

Two subtracted cDNA libraries ofDunaliella salina (Volvocales, Chlorophyceae) under different hyperosmotic shock were constructed using the suppression subtractive hybridization (SSH) method. The mRNA isolated from algae grown without stress was used as a “driver”, and the mRNAs isolated from algae 16 h (short-term treatment) or 7 d (long-term treatment) after salt stress were used as “testers”. The differentially expressed cDNA fragments inD. salina under salt stress were identified by screening these 2 libraries. Two cDNA fragments,D27 andD114, were identified from clones pL27 and pL114 after the long-term treatment. Three cDNA fragments,D21, D39, andD88, were identified from clones pSh21, pSh39, and pSh88 after the short-term treatment. The homology analysis revealed that D27 was highly similar (91%) to the subunit V of PS I reaction center inChlamydomonas reinhardtii. D21 was similar to fructose-1,6-diphosphate aldolase (78.4%). After searching GenBank with the sequences ofD39, D88, andD114, no similar sequences were found. Northern analysis revealed that the expression levels of all 5 cDNAs were increased significantly after salt stress. This means that SSH can be used in cloning differentially expressed cDNAs inD. salina under salt stress. The expression ofD27, D21, andD88 wasde novo induced by salt stress, and the expression ofD114 andD39 was increased from a relatively lower level; this indicates that all 5 cDNAs might exert an influence on the alga under hyperosmotic shock.

Key words

differential expression differential screening Dunaliella salina hyperosmotic shock suppression subtractive hybridization 

Abbreviations

CTCK

C-terminal cystine knot

FDP

fructose-1,6-diphosphate

SSH

suppression subtractive hybridization

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Copyright information

© International Society for Plant Molecular Biology 2002

Authors and Affiliations

  • Xiao-Ning Zhang
    • 1
  • Zhi-Cai Qu
    • 1
  • You-Zhong Wan
    • 1
  • Hong-Wei Zhang
    • 1
  • Da-Leng Shen
    • 1
  1. 1.Institute of GeneticsFudan UniversityShanghaiPR China

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