Screening Of Differential Expression Genes From Wheat-Thinopyrum Alien Addition Line SN6306 Induced By Drought Stress Simulated By PEG-6000

In our country, wheat is the major food source, and the material of food industry, which planting area and yield is only inferior to rice and maize. So, identification and cloning of wheat drought resistance genes to be used for wheat improvement have attracted increasing research focus. In this study, differentially expressed wheat genes responsive to drought were screened using the annealing control primer-based Gene Fishing? for germplasm SN6306, which is more drought-tolerant than its parent, Yannong 15. The results are list as follows:(1) Drought Resistance Determination of Germplasm SN6306 at the Germination StageThree wheat cultivars(SN6306, Luohan 2, and YN15) were used to identify the drought resistance of germplasm SN6306 at the germination stage. Seeds of three wheat cultivars were dealed with sterile distilled water or 20% PEG-6000. We found that germplasm SN6306 is more drought-tolerant than Yannong 15 by measuring the longest root length, coleoptile length, and plantule length and calculating the damage rate.(2) Drought Resistance Determination of Germplasm SN6306 at the Seedling StageThree wheat cultivars(SN6306, Luohan 2, and YN15) were used for this part of the study. By calculating the survival rate we found that survival rates were significantly different between SN6306, YN15, and Luohan2. Survival rates were highest for SN6306, followed by Luohan2, and YN15. By the end of the 6th day, the survival rate of three wheat cultivars is 52.5%, 43.13% and 26.25%, respectively.(3) Identification of Drought- induced Genes in Germplasm SN6306 Leaves Using Annealing Control Primer-based Gene Fishing?We got 111 differentially expressed genes(DEGs) that were predominantly expressed or upregulated during drought conditions by using Annealing Control Primer-based Gene Fishing?. According to their putative physiological functions, these identified genes are involved in several processes and/or pathways, including photosynthesis, protein synthesis and degradation, osmoprotectant synthesis, reactive oxygen scavenging, signal transduction, transcription, transmembrane transport and ion homeostasis, and stress response, which may contribute to drought-stress tolerance in SN6306.(4) Cloning and Sequence Analysis of Ta XTHIn the Gene Fishing-PCR result, a novel gene fragment, encoding Ta XTH, was further studied. The full DNA sequence of Ta XTH is 2224 bp and contained three exons and 2 introns and its open reading frame(ORF) length was 936 bp, which encodes 311 amino acids. It has 76% identities with the homologous protein from Brachypodium distachyon. Based on the results of multiple sequence alignment and polygenetic tree analysis, it was inferred that Ta XTH belongs to XTH gene and had the typical conservative DEIDFEFLG motif.(5) Cloning and expression Analysis of Ta TPPA 553 bp c DNA fragment was obtained us ing Gene Fishing? with clone number 52 and identity to the trehalose-6-phosphate phosphatase(TPP). Ta TPP encodes a protein of 373 amino acids, which the length of ORF is 1122 bp.The multiple alignments showed that TPP proteins displayed high sequence identity with other TPP proteins. The phylogenetic analysis of the same TPP sequences indicated that Ta TPP was closely related to the TPPs of Aegilops tauschii and Brachypodium distachyon. Semi-quantitative RT-PCR was used to analyze the expression patterns of Ta TPP in SN6306 leaves. As shown in Fig. 7, under simulated drought stress, Ta TPP was faintly expressed at 0 h, peaked at 12 h, and declined gradually until reaching basal levels at 48 h.(6) Constructing plant expression vectorThe overexpression vectors o f Ta TPP have been constructed successfully, which called p Ubi-TPP and p RTPP respectively.