关键词: 大豆, 花叶病毒病, 抑制消减杂交, 表达序列标签

Abstract:

Soybean is a major source of edible oil and high-quality protein in the world, but the disease make the yield of soybean lower, even have no harvests. To study the resistance mechanism to soybean mosaic virus(SMV), a soybean line, Dongnong 8143, with resistance to SMV, was used, and a subtractive cDNA library was constructed from soybean leaves infected by SMV No.1 at primary stage with SSH. DongNong 8143 was planted in culture pans. When the plants were at V1 stage, the fully-developed unifoliate leaves were inoculated with SMV No.1, then the sampling from inoculated plants and non-inoculated plants was made once every 24 hours and 8 times totally. Total RNA was extracted with TRIZOL Reagent according to the kit manual. To perform SSH, total RNA was pooled for both uninfected and infected samples. Poly(A)+RNA was purified with the PolyATract® mRNA Isolation Kit. SSH was performed using the PCR-select cDNA Subtraction Kit. SMV infected leaves RNA as the tester and uninfected leaves RNA was used as the driver. The cDNA was then digested with RsaⅠ. The tester cDNA then was subdivided into 2 portions, and each was ligated with a different cDNA adaptor. Two rounds of hybridization were performed. After filling in the ends by DNA polymerase, 2 rounds of PCR amplification were performed using the primers that matched the different adaptors to the 5′ and 3′ ends. 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 and IPTG. As a result, A subtractive plasmid library was constructed by SSH. Two thousands positive clones were acquired. 64 clones were picked up randomly and sequenced, and the results showed that the shortest length of ESTs was 136 bp, the longest length of ESTs was 691 bp, and the average length was 456 bp. 41 ESTs of them had poly(A)⁺ sequence. All ESTs were compared with sequences in unigene database of GenBank with BLASTn and BLASTx algorithm, and 49 ESTs of them had comparatively clear results. ESTs with known function involved in cellular protect of soybean, signal transduction, restrict pathogen growth, system acquired resistance, and housekeeping genes related with photosynthesis, respiration, and protein synthesis, etc. SAR genes were the richest not only in varieties but also in quantity. In the function unknown ESTs, many homologous ESTs are found from other biotic and abiotic-stresses selected cDNA libraries after compared with dbEST in GenBank.

Key words: Soybean, Soybean mosaic virus, SSH, Expressed sequence tags