Screening Of Differential Expression Genes Under Cold Stress In Apple And Function Identification Of A Novel Anti-stress Gene MdTLP7

Plants are sessile organisms and hence they cannot escape unfavourable environmental conditions within their life cycle. Low temperature is a major environmental constraint on the plant growth and distribution worldwide. To improve the plant anti-stress ability via gene engineering is an effectively way to improve the crop produtivity, quality and security, moreover, various of anti-stress related genes’isolation and identifion provided many candidates for the genetic improvement. In this study, we selected a cold resistant variety of apple tree M.26. We presented genome-wide analysis of gene expression changes under cold stress using tag sequencing on a Solexa Illumina platform. A large quantity of novel and differentially expressed genes were identified to be involved in cold stress. This study was intended to increase significantly our understanding of the molecular mechanisms and the genetic basis of cold stress tolerance in apple. The essential results were showed as the following:(1) Based on determining the time of cold treatment, we performed the transcriptome sequencing of the stressed samples. To determine the time of cold treatment and compare the ability of stress resistance between the apple rootstocks Mailing26(M.26) and cultivation variety Gala, we tested the physiological indices of electrolyte conductivity and malondialdehyde (MDA) content. These results suggested that1h and6h were two important cold exposure time points. Plants were treated at4℃and RNA was extracted for transcriptome sequencing.318576,300676and640732distinct tags were achieved in the0h,1h and6h libraries, respectively. Then, tags recorded only once were wiped off and matched to the reference genes, and14219,11176and16116valid genes were obtained.(2) Screening of differentially expressed genes (DEGs) and analyzation of function. We analyzed the DEGs with differential fold bigger than4(|log2ratio|=2). In0h vs.1h,139 genes were upregulated and1499were downregulated; While in1h vs.6h,1085genes were upregulated and381were downregulated, among them,40%DEGs were unknown. The classification of DEGs by GO category and KEGG pathway analysis revealed that the DEGs were participated in numerous pathways, involved in16cell components including plastid, cytoplasm, nucleus, etc.;10molecular function including transcription factor, antioxidant, signal transducer, enzyme regulator, etc.;13biological process including metabolic process, signal transduction, response to stress, gene expression, etc.(3) Validating the reliability of the sequencing data using Quantitative real-time PCR (qRT-PCR) analyze. Six up-regulated genes and five down-regulated genes were selected at random for qRT-PCR assays to analyse the expression levels. The M.26leaf samples with cold treatment and total RNA ware abstracted. The qRT-PCR relative expressions were compared with the high-throughput Illumina sequencing analysis results. The change tendencies showed similar results between the qRT-PCR and the sequencing methods, suggesting that the sequencing methods were valid.(4) Isolation of a novel anti-stress gene and characterization of its encoded protein MdTLP7(Tubby-like protein7). We had previously developed differential gene expression profile of apple under chilling and identified an obesity-like gene, TLP7, which was up-regulated more than1000times, indicating its possible role in plant abiotic stress tolerance. This gene was cloned from the apple cDNA library. It is1245-bp long and its encoded protein TLP7has415amino acids and consists of leading sequence, F-box domain and Tubby domain.(5) Identification of the stress-resistant function of MiTLP7. Construction of prokaryotic expression vector, MdTLP7was transformed into E.coli. Survival test on solid and liquid medium was carried out under a variety of stress conditions including4℃,50℃,0.5M NaCl and0.5M KC1. In result, the quantity of the cell carrying MdTLP7gene was significantly more than that of empty vector. The results above revealed that MdTLP7confers strong stress-tolerance to E. coli cell against salt and temperature stresses.(6) Analyze of the functional region. According to the characterization of M/TLP7, serial deletion from both N-terminus and C-terminus of MdTLP7was performed. After recombinant expression, the result showed that leading sequence and F-box domain were not essential for anti-stress function, while deletion of the Tubby domain caused M/TLP7protein reduce the ability of anti-stress function. In Tubby domain,120-310amino acid residues played vital roles in stress tolerance. The result of homology modeling showed that the Tubby domain structured by a β barrels and intermediate a helix.120-310residues formed the P barrel, indicating that the β barrel of the Tubby domain played crucial roles in stress tolerance, while a helix not essential for the anti-stress function.