Identification of differentially-expressed genes potentially implicated in drought response in pitaya (Hylocereus undatus) by suppression subtractive hybridization and cDNA microarray analysis.
Drought is one of the most severe threats to the growth, development and yield of plant. In order to unravel the molecular basis underlying the high tolerance of pitaya (Hylocereus undatus) to drought stress, suppression subtractive hybridization (SSH) and cDNA microarray approaches were firstly combined to identify the potential important or novel genes involved in the plant responses to drought stress. The forward (drought over drought-free) and reverse (drought-free over drought) suppression subtractive cDNA libraries were constructed using in vitro shoots of cultivar 'Zihonglong' exposed to drought stress and drought-free (control). A total of 2112 clones, among which half were from either forward or reverse SSH library, were randomly picked up to construct a pitaya cDNA microarray. Microarray analysis was carried out to verify the expression fluctuations of this set of clones upon drought treatment compared with the controls. A total of 309 expressed sequence tags (ESTs), 153 from forward library and 156 from reverse library, were obtained, and 138 unique ESTs were identified after sequencing by clustering and blast analyses, which included genes that had been previously reported as responsive to water stress as well as some functionally unknown genes. Thirty six genes were mapped to 47 KEGG pathways, including carbohydrate metabolism, lipid metabolism, energy metabolism, nucleotide metabolism, and amino acid metabolism of pitaya. Expression analysis of the selected ESTs by reverse transcriptase polymerase chain reaction (RT-PCR) corroborated the results of differential screening. Moreover, time-course expression patterns of these selected ESTs further confirmed that they were closely responsive to drought treatment. Among the differentially expressed genes (DEGs), many are related to stress tolerances including drought tolerance. Thereby, the mechanism of drought tolerance of this pitaya genotype is a very complex physiological and biochemical process, in which multiple metabolism pathways and many genes were implicated. The data gained herein provide an insight into the mechanism underlying the drought stress tolerance of pitaya, as well as may facilitate the screening of candidate genes for drought tolerance.
Guizhou Key Laboratory of Agricultural Bioengineering, Guizhou University, Guiyang 550025, Guizhou Province, PR China.
Qing-Jie
QJ
Fan
Feng-Xia
FX
Yan
Guang
G
Qiao
Bing-Xue
BX
Zhang
Xiao-Peng
XP
Wen
D001483:Base Sequence; D029421:Cactaceae / Q000235:genetics*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D055864:Droughts; D020224:Expressed Sequence Tags; D017343:Genes, Plant; D020411:Oligonucleotide Array Sequence Analysis; D018749:RNA, Plant / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D013382:Subtraction Technique
D017931:DNA Primers; D018076:DNA, Complementary; D018749:RNA, Plant
AAP8; ABA; ABAR PYL8; ACTB; CAM; CAT; DEGs; Differentially-expressed genes; Drought stress; ESTs; FDR; FWC; GGAT; GGT; GO; GSH; GSR; ISSR; OSB–CoA ligase; PEG; PEPC; PLA(2); PLDα; Phospholipase Dα; Pitaya (Hylocereus undatus); RT-PCR; SHMT; SSH; Suppression subtractive hybridization; TAT; TPS; abscisic acid; cDNA microarray; catalase; crassulacean acid metabolism; differentially expressed genes; expressed sequence tags; false discovery rate; field water capacity; gene encoding O-succinylbenzoate–CoA ligase; gene encoding abscisic acid receptor PYL8; gene encoding amino acid permease 8; gene encoding gamma-glutamyltranspeptidase; gene encoding gamma-tocopherol methyltransferase; gene encoding glutamate–glyoxylate aminotransferase; gene encoding glutathione reductase; gene encoding glycine hydroxymethyltransferase; gene encoding phosphoenolpyruvate carboxylase; gene encoding phospholipase A(2); gene encoding trehalose-6-phosphate synthase; gene encoding tyrosine aminotransferase; gene encoding β-actin; gene ontology; glutathione; inter-simple sequence repeat; polyethylene glycol; qRT-PCR; quantitative real-time PCR; reverse transcriptase polymerase chain reaction; suppression subtractive hybridization; γ-TMT