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Acta Agron Sin ›› 2014, Vol. 40 ›› Issue (06): 1011-1019.doi: 10.3724/SP.J.1006.2014.01011

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Interaction between Transcription Factors SPT and HEC1 of Pistil of Brassica oleracea L.var. capitata L.

XU Jun-Qiang,SUN Zi-Jian,LIU Zhi-Yu,YANG Pu-Li,TANG Qing-Lin,WANG Zhi-Min,SONG Ming*,WANG Xiao-Jia*   

  1. Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education / Chongqing Key Laboratory of Olericulture / College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400715, China?
  • Received:2013-07-01 Revised:2014-03-04 Online:2014-06-12 Published:2014-04-08
  • Contact: 王小佳, E-mail: wxj@swu.edu.cn; 宋明,E-mail: swausongm@163.com, Tel: 023-68251093

Abstract:

To explore interaction of SPT and HECs in cabbage pistil development, we took stigma of self-incompatibility line E1 to obtain total RNA for first-strain cDNA synthesis, and cloned SPT gene fragment with the ORF of 1062 bp and HEC1 gene with 696bp by using primers according to SPT and HEC1 genes in Arabidopsis. Amino acid sequence analysis showed that SPT and HEC1we cloned encoded 353 and 231 amino acid residues, respectively; the predicted molecular weight of SPT protein was 37.67 kD, with pI of 6.83; the predicted molecular weight of HEC1 protein was 25.26 kD, with pI of 10.23. Relative expression of SPT was the highest in the fruit and pistil, that of HEC1 was the highest in the root and bud. Prokaryotic expression plasmids pCold I-SPT and pGEX-HEC1 were constructed and then transformed into E. coli Rosetta (DE3). The Pull-down assay showed that the two tagged expression products could interact with each other. To verify the interactions furtherly, we constructed yeast expression vectors pGBKT7-SPT, pGADT7-HEC1, and pGADT7-SPT, pGBKT7-HEC1 to transform into yeast Y2HGold and Y187 strains, respectively. Finally, diploid yeast showed positive results in SD/–Ade–Trp–Leu–His/X-α-gal/AbA synthetic dropout. It demonstrated that interaction between SPT and HEC1 can activate HIS3, AUR1-C,and ADE2 reporter genes downstream. Yeast two-hybrid experimental results as well as the pull-down assay showed that SPT and HEC1 can form heterodimer to regulate the development of pistil.

Key words: Brassica oleracea, Pistil, SPT, HEC1, Interaction, Yeast two-hybrid

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