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Acta Agron Sin ›› 2012, Vol. 38 ›› Issue (04): 578-588.doi: 10.3724/SP.J.1006.2012.00578


Cloning and Expression Characteristics of EXO70A1 from Brassica oleracea, Brassica campestris and Brassica napus

YANG Kun1,2,*,**,ZHOU Yong-Xiang3,**,ZHANG He-Cui1,ZHAO Yong-Bin4,YANG Yong-Jun1,LU Jun-Xing1,ZHU Li-Quan1,2,*,XUE Li-Yan1,LÜ Jun1, 2,GAO Qi-Guo5   

  1. 1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400716, China; 2 Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400716, China; 3 College of Resources and Environments, Chongqing 400716, China; 4 College of Life Science, Jilin Normal University, Siping 136000, China; 5 Key Laboratory in Olericulture of Chongqing, Southwest University, Chongqing 400716, China?
  • Received:2011-08-16 Revised:2011-12-19 Online:2012-04-12 Published:2012-02-14
  • Contact: 朱利泉, E-mail: zhuliquan@swu.edu.cn, Tel:02368250794; 杨昆, E-mail: ykun0827@163.com

Abstract: EXO70A1 is an important signal element of self-incompatibility in Brassica. In this study, coding sequences (CDS) of EXO70A1 and the corresponding gDNA were cloned by PCR. Sequence analysis was carried out by means of bioinformatics. RT-PCR was used to analyze expression characteristics of EXO70A1 in Brassica oleracea. And BoEXO70A1 was transformed into yeast Y187 to explore its expression. The results showed: That the gene lengths of BnEXO70A1, BrEXO70A1 and BoEXO70A1 were 3 797, 3 752, and 3 770 bp, respectively, and all consisted of 12 exons and 11 introns; their identity positions reached 91%. Sequence conservation was higher in exons than in introns excluding 4th, 5th, 6th, and 8th intron. CDS of three EXO70A1 genes consisted of
1 917 base pairs and sequence similarity was 97.1%. Three proteins of EXO70A1 conduced all consisted of 638 amino acids; the similarity and identity of EXO70A1 in the three species were 99.8% and 98.1%, respectively. BnEXO70A1, BrEXO70A1, and BoEXO70A1 shared similar secondary structures and three-dimensional structures. All introns started from the sequence GU and ended with the sequence AG (in the 5' to 3' direction). They are referred to as the splice donor and splice acceptor site, respectively. Another important sequence “CU(A/G)A(C/U)” in all introns of EXO70A1 genes was located at 20–50 bases upstream of the acceptor site. The corresponding sequence lengths of all 12 exons of EXO70A1 were identical among three Brassica species and Arabidopsis thaliana; and similarity of four coding sequences was 90.1%. The similarity and identity of EXO70A1 proteins in the four species reached 99.8% and 93.7%, respectively. EXO70A1 was a subunit of EXO70 family which showed high conservative in Magnoliophyta. The results showed that BoEXO70A1 presented weak expression in Y187, while expressed in stamens, young stems, petals, pistils, young roots and leaves. However, its expression quantity was different in different tissues, with strong expression in pistils and weak in stamens. Thus conclusions can be drawn that: EXO70A1, in Brassica, is high conservative; EXO70A1 perhaps plays very important roles in plant cells because of its different expression characteristics in different plant tissues.

Key words: EXO70A1, Cloning, Sequence analysis, Expression characteristics

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