Early stage SSH library construction of wheat near isogenic line <italic>TcLr19</italic> under the stress of <italic>Puccinia recondita</italic> f. sp. <italic>tritici

doi:10.1007/s11703-009-0045-7

Front Agric Chin

2009, Vol. 3

Issue (2) : 146-151 https://doi.org/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 Author(s): WANG Dongmei,Email:dongmeiwang63@hotmail.com

Issue Date: 05 June 2009

Cite this article:

Aihua YAN,Lifeng ZHANG,Yunwei ZHANG, et al. Early stage SSH library construction of wheat near isogenic line TcLr19 under the stress of Puccinia recondita f. sp. tritici[J]. Front Agric Chin, 2009, 3(2): 146-151.

URL:

https://academic.hep.com.cn/fag/EN/10.1007/s11703-009-0045-7
https://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.

Tab.1 Similarity analysis (BLASTx) of ESTs with the function identified genes in GenBank

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