Identification and expression analysis of early cold-induced genes from cold-hardy Citrus relative Poncirus trifoliata (L.) Raf.

Identification and Expression Analysis of Genes Induced in Response to Tomato chlorosis virus Infection in Tomato

“…A SSH was performed by PCR-Select cDNA Subtraction Kit (Ta-KaRa, Kusatsu, Japan) using Poly (A + ) RNA from ToCV or mock-inoculated control plants as previously outlined ( Şahin-Çevik, 2013 ; Şahin-Çevik and Moore, 2006 ; Şahin-Çevik et al, 2017 ). For construction of the subtractive cDNA library containing genes induced by ToCV, a forward subtraction was applied using the tester cDNA from ToCV-inoculated leaf samples and the driver cDNA from the leaf samples of mock-inoculated control.…”

“…Selection, sequencing and sequence analysis of clones from the subtractive library were conducted as previously described ( Şahin-Çevik, 2013 ; Şahin-Çevik and Moore, 2006 ; Şahin-Çevik et al, 2017 ) and briefly outlined here. Bacterial colonies confirmed to contain pGEM-T easy plasmids with a tomato cDNA inserts were randomly selected from the subtracted cDNA library.…”

“…To prepare nylon macroarrays, constitutively expressed housekeeping genes including, β-actin, tubulin, glyceraldehyde phosphate dehydrogenase, ubiquitin, genes were selected as internal controls for normalization. Two human genes Glycogen 2 (GenBank accession number: NM_001184703) and troponin T type 1 (GenBank accession number: NM_003283) were selected as negative controls ( Şahin-Çevik, 2013 ). All controls were amplified by two-step RT-PCR using PrimeScript RT-PCR kit (TaKaRa, Kusatsu, Japan) according to the manufacturer’s instructions using gene specific primers and appropriate annealing temperature for each primer set.…”

“…To prepare nylon macroarrays, PCR amplified cDNAs of selected from the library and control genes were first denatured with 0.4 N NaOH. Then, equal amount of denatured PCR products of cDNAs, control genes and water sample were spotted in duplicates on Hybond N+ nylon membranes (Roche, Basel, Switzerland) using a Bio-Dot Microfiltration Apparatus (Bio-Rad, Hercules, CA, U.S.A.) by a vacuum application ( Şahin-Çevik, 2013 ; Şahin-Çevik et al, 2017 ). Nylon macroarrays were prepared in duplicates for hybridization with probes from ToCV-inoculated and mock-inoculated samples.…”

“…The expression of cDNAs from ToCV-induced cDNA library was analyzed by macroarray hybridization with DIG labeled cDNA probes synthesized from RNA isolated from ToCV or mock-inoculated control plants as previously reported ( Şahin-Çevik, 2013 ). First, DIG labeled cDNA probes were synthesized from Poly (A + ) RNAs isolated from ToCV-inoculated and mock-inoculated control tomato seedlings by the Transcriptor reverse transcriptase (Roche, Basel, Switzerland) according to the manufacturer’s instructions.…”