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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2010, Vol. 4 Issue (4) : 449-455     DOI: 10.1007/s11703-010-1034-6
RESEARCH ARTICLE |
Establishment of cDNA-AFLP technology system and stoneless gene difference expression in Ziziphus jujuba Mill.
Bin HAN, Ruixia BAI, Li LI, Lisha ZHANG, Chuan MA, Jiwei ZHAO, Jinxin WANG, Jianying PENG()
College of Horticulture, Agricultural University of Hebei, Baoding 071001, China
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Abstract  

An efficient and stable cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis system for Chinese jujube was established and successfully used for the studies of related stoneless gene difference expression in Ziziphus jujuba Mill. ‘Wuhejinsixiaozao’ fruit. Several main factors influencing cDNA-AFLP analysis were studied, including the preparation and purification of cDNA, restriction and ligation of cDNA, the preamplification reaction, selective amplification reaction, electrophoresis on denaturing polyacrylamide gels, and sliver staining. The results indicated that the total RNA extracted by modified SDS method was pure, complete, and suitable for reverse transcription to cDNA. Restriction digestion of cDNA was performed by using two restriction enzymes, around 150 ng DNA digested with three units of EcoRI and MseI enzymes, respectively, and incubated at 37°C for 5 h. The digested cDNA fragment was diluted 5 times and used as templates for preamplification, and the preamplification products were diluted 10 times and used as templates for selective amplification. The selective amplification fragments were subjected to PAGE electrophoresis and silver staining. By cDNA-AFLP analysis, it acquired three transcript-derived fragments (TDFs), DC1, DC5, and DC9, related with stoneless gene of Z. jujuba Mill. ‘Wuhejinsixiaozao’ fruit.

Keywords Chinese jujube      cDNA-AFLP      stoneless gene      difference expression     
Corresponding Authors: PENG Jianying,Email:pengzhuwen@sina.com   
Issue Date: 05 December 2010
URL:  
http://academic.hep.com.cn/fag/EN/10.1007/s11703-010-1034-6     OR     http://academic.hep.com.cn/fag/EN/Y2010/V4/I4/449
codeprimer combinations
E1M3E-AA, M-AC
E2M2E-AT, M-AT
E3M1E-AC, M-AA
E3M4E-AC, M-AG
E4M2E-CA, M- AT
E4M9E-CA, M-CG
E4M10E-CA, M-CC
E5M2E-CT, M-AT
E5M3E-CT, M-AC
E6M2E-CC, M-AT
E7M4E-CG, M-AG
E8M6E-TA, M-TA
E8M7E-TA, M-TT
E9M9E-TT, M- CG
E10M4E-TC, M-AG
E11M3E-TG, M-AC
E12M4E-AG, M- AG
Tab.1  cDNA-AFLP fragment detected for each primer combination
Fig.1  Agarose gel electrophoresis of total RNA from Chinese jujube fruit by improved SDS method
Fig.2  Agarose gel electrophoresis of the double stand cDNA
?Note: M is DL2000 Marker, 1–4 are the double strand cDNA of the ?Chinese jujube fruit.
elementsgradients
concentration of cDNA150 ng
time of restriction digestion5 h
dilution multiple of adaptor-ligated production5 ×
dilution multiple of pre-amplification production10 ×
Tab.2  The effects of different elements on cDNA-AFLP products
Fig.3  Amplified results using primer combination E4/M9
Fig.4  Specific fragment amplified by using primer combination E4/M9
Note: 1, 3, 5, 7, 9 and 11 are ‘Wuhejinsixiaozao’ fruit on 8 d, 15 d, 20 d, 30 d, 40 d, and 60 d after blooming, respectively; 2, 4, 6, 8, 10, and 12 are ‘Jinsixiaozao’ fruit on 8 d, 15 d, 20 d, 30 d, 40 d, and 60 d after blooming, respectively.
Fig.5  Specific fragment amplified by using primer combination E4/M10
Note: 1, 3, 5, 7, 9, and 11are ‘Wuhejinsixiaozao’ fruit on 8 d, 15 d, 20 d, 30 d, 40 d, and 60 d after blooming, respectively; 2, 4, 6, 8, 10, and 12 are ‘Jinsixiaozao’ fruit on 8 d, 15 d, 20 d, 30 d, 40 d, and 60 d after blooming, respectively.
Fig.6  Specific fragment amplified by using primer combination E10/M4
Note: 1, 3, 5, 7, 9, and 11are ‘Wuhejinsixiaozao’ fruit on 8 d, 15 d, 20 d, 30 d, 40 d, and 60 d after blooming, respectively; 2, 4, 6, 8, 10, and 12 are ‘Jinsixiaozao’ fruit on 8 d, 15 d, 20 d, 30 d, 40 d, and 60 d after blooming, respectively.
Fig.7  The result of the second selective amplification
Note: 1–2 are DC1; 3–4 are DC5; 5–6 are DC9; M is the DL2000 DNA Marker.
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