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Study on the Interactions Between the Truncated Fragments of SCR and eSRK from Brassica oleracea L. by a Yeast Two-Hybrid System

YANG Hong; ZHU Li-quan; ZHANG He-cui;YANG Yong-jun; XUE Li-yan; YANG Kun; YU Hao; PENG Yi-bo; LUO Bing; WU Zhi-gang; HUANG Dan; GAO Qi-guo; WANG Xiao-jia   

  1. 1、College of Agronomy and Biotechnology, Southwest University, Chongqing 400716;
    2、Chongqing Key Laboratory of Olericulture, Chongqing 400716
  • Received:2010-12-30 Online:2011-05-05 Published:2011-01-30

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Abstract: 【Objective】 Self-incompatibility of Brassica oleracea L. var capitata L. was determined by interaction between the female S-locus receptor kinase (SRK) and the male S-locus cysteine rich protein/S-locus protein 11 (SCR/SP11). In this study, a yeast two-hybrid system was adopted to investigate their potential interaction domains between SCR/SP11 and the extracellular fragment of SRK (eSRK).【Method】The full length SCRB3, SCRB3-1 and SCRB3-2 amplified from Brassica oleracea L. B3 were cloned into the pGBKT7 to construct the recombinant bait plasmids which were then transformed into yeast Y2HGold cells by PEG/LiAc method. The DNA fragments including eSRKB3, eSRKB3-1 and eSRKB3-2 were cloned into the pGADT7 to construct the recombinant activation domain (AD) plasmids which were transformed into yeast Y187 cells. Each transformant of Y2HGold [pGBKT7-SCRB3-s] was crossed with one of three transformants Y187 [pGADT7-eSRKB3-s]. Then the crossed yeast cells were screened consecutively on SD/-Trp-Leu/x-α-gal/AbA (DDO/x/A) and SD/-Trp-Leu-Ade-His/x-α-gal/AbA(QDO/x/A) nutritional media. 【Result】 The restriction enzyme analysis and DNA sequencing indicated that the above recombinant plasmids were correct. The yeast Y2HGold cells transformed with each of the three recombinant bait plasmids could grow on SD/-Trp nutritional media without autonomous activation effect on the reporter gene MEL1 and AUR1-C. The yeast Y187 cells transformed with each of the three recombinant AD plasmids could grow on SD/-Leu nutritional media without activation of the reporter gene MEL1. And these recombinant plasmids were no toxic to yeast cells. Four crossed yeast cell lines could grow on QDO/x/A nutritional media with transcription activation of the reporter genes AUR1-C, MEL1, HIS3, and ADE2.【Conclusion】The yeast two-hybrid system could be used to study the interaction domain between SCR and eSRK. The transmembrane domain of SRK is present or not which will have no effect on studying the interaction between SCR and SRK.

Key words: Brassica oleracea L. var capitata L. , self-incompatibility , S-locus receptor kinase (SRK) , S-locus cysteine-rich protein (SCR) , yeast two-hybrid

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