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Acta Agron Sin ›› 2017, Vol. 43 ›› Issue (04): 620-628.doi: 10.3724/SP.J.1006.2017.00620

• RESEARCH NOTES • Previous Articles    

Selection and Cloning of Thaumatin-like Protein (TLP) Gene from Winter Brassica rapa and Its Expression under Low Temperature Stress

MA Li1,**,YUAN Jin-Hai1,**,SUN Wan-Cang1,LIU Zi-Gang1,ZENG Xiu-Cun2,WU Jun-Yan1,FANG Yan1,LI Xue-Cai1,CHEN Qi1,XU Yao-Zhao2,PU Yuan-Yuan1,LIU Hai-Qing1,YANG Gang1,LIU Lin-Bo1   

  1. 1 Gansu Key Laboratory of Crop Improvement and Germplasm Enhancement / Rapeseed Engineering Research Center of Gansu Province / College of Agronomy, Gansu Agricultural University, Lanzhou 730070, China; 2 College of Agronomy and Biotechnology, Hexi University, Zhangye 734000, China
  • Received:2016-01-19 Revised:2016-11-03 Online:2017-04-12 Published:2016-12-14
  • Contact: Sun Wanchang, E-mail:18293121851@163.com, Tel: 18293121851 E-mail:348369277@qq.com
  • Supported by:

    This study was supported by the China Agriculture Research System(CARS-13), the National Natural Science Foundation of China (31460356, 31560397), The National Basic Research Program of China (973 Program) (2015CB150206), the Natural Science Foundation of Gansu Province (145RJZG050), the part of National Key Research and Development Program(2016YFD0101300), and the research and demonstration project of high efficient planting pattern in the core area of the Silk Road Economic Zone in Xinjiang.

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Abstract:

Thaumatin-like protein (TLP) is an essential protein induced by low temperature stress and enhances plant resistance to adverse circumstances. In our study, we selected differential protein spots from Longyou 7 leaves under low temperature stress and separated TLP closely related to cold resistance by using two-dimensional electrophoresis and mass spectrometry. Using the primers of TLP cDNA sequences and reverse transcription PCR (RT-PCR), we cloned the complete open reading frame of TLP gene of winter Brassica rapa cultivar Longyou7. The sequence length of TLP from Brassica rapa was 732 bp, encoding a protein of 243 amino acid residues with a predicted molecular weight of 26.02 kD and a theoretical pI of 9.02. Bioinformatics analysis showed a similarity of 99.18% in TLP amino acid sequence between B. rapa and Brassica napus, the predicted TLP contained a conserved amino acid sequence belonging to the plant GH69-TLP-SF superfamily. The TLP had one signal peptide which belongs to hydrophilic protein. Moreover, the detection of subcellular localization suggested the synthetic process of this protein is in the endoplasmic reticulum. The prediction of the second structures indicated that TLP is a labile protein consisting of random coils and extended strands. The expression analysis of TLP gene by utilizing real time and Semi-quantitative RT-PCR suggested that the TLP expression could be up-regulated in response to lower temperature. In conclusion, TLP gene of the winter Brassica rapa cultivar Longyou 7 might play a significant role in response to cold stress.

Key words: Winter Brassica rapa, Two-dimensional gel electrophoresis, Lower temperature, TLP

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