Identification of differentially expressed genes related with Jujube fruitshrink disease by suppression subtractive hybridization

Abstract：【Objective】Jujube fruit shrink disease caused by Alternaria alternate is one of main fruit dis⁃eases of Ziziphus jujuba. It could reduce the yields and quality of jujube severely. Although the diseasehas been investigated at the tissue and physiological level, the molecular response of jujube fruits to A. alternate infection is still unclear. Investigation on the differentially expressed genes can help us better un⁃derstand the molecular processes involved in the interactions between pathogen and jujube fruits. In thisstudy, a suppression subtractive hybridization (SSH) technique was used to identify the differentially expressed genes in jujube fruits infected by A. alternate in order to illustrated the molecular response of ju⁃jube fruits to the disease.【Methods】The physiological race (ZS091) of A. alternata was provided byHenan Academy of forestry, China. The isolate was cultured on PDA medium for one week and the proliferated spores were dissolved in Tween-80, and adjusted to 108 conidia per mL in distilled water as the in⁃oculum. The inoculum was artificially inoculated on the fruits of Z. jujuba‘Fengmiguan’in the white mature stage in the Jujube Experimental Station of Northwest A & F University, Qingjian county, Shaanxiprovince, China. The fruits inoculated with distilled water were set as the reference. We picked threefruits 0.5, 1, 2, 3 and 4 d after inoculation, respectively. The fruit samples were immediately frozen in liq⁃uid nitrogen, and transported to laboratory in dry ice and stored at -80 ℃ before RNA extraction. TotalRNA was extracted with the‘MiniBEST Plant RNA Extraction Kit’reagent (Takara) according to manu⁃facturer’s instruction. cDNA subtractive library on differential expression induced by the infection of A.alternate by SSH was constructed with PCR-SelectTM cDNA Subtraction Kit (Clontech). Double-strandedcDNAs of tester (the pooled samples of A. alternata-inoculated fruits) and driver (non-inoculated fruits)were synthesized from 1 μg of mRNA, respectively. The secondary PCR amplicons were purified with Ad⁃vantage 2 PCR Kits. The subtracted products were cloned in pMD19-T vectors and transformed to DH5αcompetent cells (TaKaRa). Blue/white selection of transformants was carried out using X-gal and IPTG.Selected positive clones were subjected to sequence on ABI3730xl. The obtained sequences were editedmanually using DNA Star Software for removing vector sequences and then aligned in the CAP3 to gener⁃ate consensus sequences. The obtained consensus sequences were subjected to homology analysis on NC⁃BI using BLASTn and BLASTx to search for homologous sequences in the non-redundant nucleotide andprotein databases, respectively.【Results】SSH cDNA library was successfully constructed from the A. alternate-inoculated‘Fengmiguan’fruits and the non-inoculated fruits in the white mature stage. In total,1 000 cDNA library clones were obtained, among which 200 positive clones were selected randomly for se⁃quencing, and finally 182 high quality ESTs were obtained. And 118 Unigenes, including 7 Contigs and111 Singlet, were obtained after cluster analyses. Homologous analysis revealed that 84 (86.44%) of theESTs matched known proteins while 34 (13.56%) of the ESTs had no significant similarity with depositedsequences. Those best matched proteins mainly involves in protein synthesis, protein degradation, diseasedefense response, protein modification and transportation, signal transduction, cell structure, transcription⁃al regulation, metabolism and energy, etc. For example, serine/threonine protein kinase, C2 domain-con⁃taining protein, scarecrow-like protein and Zinc finger protein genes were supposed to function as signaltransduction and transcription regulating genes in response of plant to infection of pathogens. Superoxidedismutase, thaumatin-like protein, metallothionein protein might be involved in the defense response ofjujube fruits to the disease. Some abiotic stress induced genes, including the genes for wound-responsivefamily proteins and heat shock proteins, also occurred several folds, which indicated that there was crosstalk between biotic and abiotic stresses. Genes involved in photosynthesis and energy metabolism were also included in acquired ESTs, indicating primary metabolites might function in jujube resistance to A. alternate.【Conclusion】In this study, 118 differentially expressed genes were identified from the jujubefruits artificially inoculated by A. alternate, among which several genes were involved in the plant diseaseresistanceresponse. The expression profiles would provide a good starting point for understanding the mo⁃lecular processes involved in the plant-pathogen interactions. This study may provide insights into the molecular pathogenesis mechanism of jujube fruit shrink disease.