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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2009, Vol. 3 Issue (2) : 146-151     DOI: 10.1007/s11703-009-0045-7
RESEARCH ARTICLE |
Early stage SSH library construction of wheat near isogenic line TcLr19 under the stress of Puccinia recondita f. sp. tritici
Aihua YAN, Lifeng ZHANG, Yunwei ZHANG, Dongmei WANG()
College of Life Science, Agricultural University of Hebei, Baoding 071001, China
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Abstract  

cDNA library of wheat near isogenic line TcLr19 was constructed with suppression subtractive hybridization (SSH) 16 h after inoculation with race 366 of Puccinia recondita f. sp. tritici. This SSH library included 1337 positive clones and the insert sizes ranged from 200 bp to 600 bp, 237 clones were selected according to the result of reverse northern blotting, and then 35 ESTs were sequenced. EST similarity analysis was finished by comparing sequences with BLAST software in the non-redundant database of GenBank. The results showed that they were related to many biological processes including signal transduction, transcription regulation and hypersensitive response.

Keywords TcLr19      leaf rust      suppression subtractive hybridization (SSH)      early stage      ESTs     
Corresponding Authors: WANG Dongmei,Email:dongmeiwang63@hotmail.com   
Issue Date: 05 June 2009
URL:  
http://academic.hep.com.cn/fag/EN/10.1007/s11703-009-0045-7     OR     http://academic.hep.com.cn/fag/EN/Y2009/V3/I2/146
Fig.1  Integrity detection of total RNA of wheat leaves
Note: T and D represent tester and driver, respectively.
Fig.2  dscDNA synthesis
Note: T means tester, D means driver, and M means marker
Fig.3  Reduction of actin abundance by PCR-select subtraction
Note: 1-4 mean the actin product of the subtracted samples after 20, 25, 30 and 35 cycles, respectively; 5-8 represent the actin product of the nonsubtracted samples after 20, 25, 30 and 35 cycles, respectively.
Fig.4  Insert fragment detection
Fig.5  Reverse northern blotting
Note:——represents the point color on tester membrane that was significantly darker than that on driver’s;
———represents the points on tester membranes but not on driver membrane;
----represents the points in the same color on both membranes.
accessionlength/bpputative identificationE value
SL1288ADP-ribosylation factor (Triticum aestivum)2.00E-176
SL2504chloroplast psbA gene for herbicide-binding protein D1 (Secale cereale)1.00E-178
SL3374vacuolar ATP synthase subunit E (VATE) (Triticum aestivum)1.00E-81
SL10681partial CA4 gene for P-type ATPase (Hordeum vulgare)1.00E-90
SL15457NADP-ME2 for NADP dependent malic enzyme (Oryza sativa)3.00E-189
SL16425Cytochrome P450 (Triticum aestivum)1.00E-140
SL18787glycine max plamsma membrane-associated AAA-ATPase5.00E-174
SL21319multifunctional protein (Oryza sativa)1.00E-82
SL25404MBD2 (Triticum aestivum)7.00E-175
SL27179calcium-dependent protein kinase 3-like (CDPK3) (Triticum aestivum)3.00E-122
SL30130metallothionein-like protein (Wali1) (Triticum aestivum)3.00E-49
SL37607S-adenosylmethionine decarboxylase (Triticum monococcum)0
SL38419gstA2 (Triticum aestivum)0
SL39158glutamine-dependent asparagine synthetase (ASN1) (Triticum aestivum)2.00E-66
SL42268putative adenine phosphoribosyl transferase (Triticum aestivum)3.00E-117
SL483234-hydroxyphenylpyruvate dioxygenase (Triticum aestivum)8.00E-109
SL50331arabinoxylan arabinofuranohydrolase isoenzyme AXAH-II (Hordeum vulgare)4.00E-157
SL67322putative E2 SUMO conjugating enzyme (Triticum turgidum)6.00E-110
SL60257glutathione S-transferase (Triticum aestivum)1.00E-90
SL69363vacuolar proton-inorganic pyrophosphatase (Triticum aestivum)3.00E-173
Tab.1  Similarity analysis (BLASTx) of ESTs with the function identified genes in GenBank
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