Construction and primary analysis of subtractive library induced by soybean mosaic virus (SMV).
- Author:
Chun-Yan LIU
1
;
Wei-Quan WANG
;
Qing-Shan CHEN
;
Cui-Ping YANG
;
Wen-Bin LI
;
Da-Wei XIN
;
Zhen-Guo JIN
;
Ying-Bo SONG
Author Information
1. Soybean Research Institute, Northeast Agricultural University, Harbin 150030, China.
- Publication Type:Journal Article
- MeSH:
Expressed Sequence Tags;
Gene Library;
Mosaic Viruses;
Nucleic Acid Hybridization;
methods;
Plant Diseases;
genetics;
virology;
Polymerase Chain Reaction;
Soybeans;
genetics;
virology
- From:
Chinese Journal of Biotechnology
2005;21(2):320-322
- CountryChina
- Language:Chinese
-
Abstract:
SMV is one of main diseases of soybean, which could affect yields and quality of soybean seriously. It was effective to soybean breeding by studying the expression of resistant gene to SMV with molecular technology. In this study, a soybean resistance line, DongNong 8143, was used to construct a subtractive cDNA library by SSH from soybean leaves inoculated by SMV No.1 at primary stage. cDNA dominantly or specifically expressed in infected leaves was purified using PCR Purification Kit and cloned into pGEM-T easy vector. Colonies were grown on LB-agar plates containing ampicillin, X-gal and IPTG. A subtractive plasmid library was constructed by SSH. Then the library was transformed to host bacteria E. coli DH5alpha, and the titer of the library was measured as 2 x 10(3) . 64 clones were picked up randomly and sequenced. Of them there is 50 clones which result of sequenced were good. The length of EST fragment varied from 136bp to 691bp, and the average length is 456bp. Among them, 41 sequences has poly(A). Through ESTs were compared with sequences in unigene database of GeneBank with BLASTn and BLASTx algorithm, 38 ESTs of them had comparatively clear results and the percent of them in acquired ESTs is 74%. The EST expression profile showed that the resistance-related genes include cell protection, signal transduction, restrict pathogen growth, system acquired resistance, and house-keeping gene. There are 12 ESTs, which have not comparatively clear results, that maybe new genes.
