Suppressive subtractive hybridization method analysis and its application to salt stress in grapevine (Vitis vinifera L.)

Abstract

Two subtracted cDNA libraries of grapevines (Vitis vinifera. L) under short term salt stress incubation were constructed using the suppression subtractive hybridization (SSH) method combined with the differential screening approach. The mRNA isolated from leaves of the salt-tolerant grapevine cultivar Razegui grown without stress was used as a "driver," and the corresponding mRNAs isolated after a short-term treatment 6 or 24h of salt stress were used as "testers." The differentially expressed cDNA fragments were identified by differential screening of these 2 libraries. During SSH procedure, each step was operated exactly according to the manual of the kit and the results were verified correct before following step. The libraries consisted of about 7000 recombinant clones, with the average size being of 500 bp, ranging from 100 bp to 900 bp. Using a PCR-select differential screening kit, 1000 recombinant clones were randomly chosen from the subtracted cDNA libraries and hybridized with forward, reverse subtracted and unsubtracted probes for two rounds. As a result, 848 positive clones were obtained. Sequencing of randomly selected clones from the differential screening revealed that most of transcripts over-expressed by salt stress have been reported as responsive to abiotic stress response.