Identification Of Differentially Expressed Genes In Young Spike Of Three Pistils Mutation In Wheat

Wheat(Triticum aestivum L.) is the most important food crop in the world and becomes even more important as the global population increases. Because the arable land is very limited, improving the yield is essential. One way to improve wheat yield potential is to increase grain number per spike. The grain number of per spike is the ultimate embodiment of the floral differentiation, degeneration and grain-setting. The common wheat line three pistils (TP), which selected by Peng from the'trigrain' wheat variety, is a valuable mutant for the wheat breeding. Because the TP mutation has normal spike morphology but produces 3 pistils per floret, it has potential to increase grain number per spike. Homeotic transformation of stamens into pistil-like structures (pistillody) is caused by nuclear-cytoplasm interaction in wheat. The TP mutation is different from the pistillody, because the 3 stamens and 3 pistils are all fertile. Genetic analysis indicated that the three pistils trait is controlled by a single dominant gene Pis1 which was located on chromosome 2DL. Up to now, the genetic basis of the three pistils trait has been clearly, but the sequence information of Pis1 is still unclear. Although the three pistils controlled by a single gene locus, the mutation causes changes of a series of genes at the transcription level and affect various metabolism processes in three pistils plants. So the aim of this study was to identify differentially expressed genes in young spikes of three pistils plants, in order to reveal the molecular mechanism controlling the three pistils characters. The main results are summarized as follows:1. In this study, three pistils (TP) was used as donor parent, and Chinese spring (CS), Chuanmai 28 (CM28), Mianyang 29 (MY29) and Neimai 9 (NM9) were used as recurrent male parent, after 7 times successive backcrossing and then self-crossing for four generations, four near isogentic lines (NILs), CSTP, CM28TP, MY29TP and NM9TP were bred. Genetic similarity and genetic distance of 4 near isogentic lines and their recurrent parents were compared and analysised by SRAP. The results showed:(1) All the 128 pairs of SRAP primers amplified 978 bands,120 pairs (93.8%) of primers were polymorphic and 638 bands (65.2%) were polymorphic. (2) The genetic similarity coefficient of CSTP, CM28TP, MY29TP, NM9TP and their recurrent parents were 0.9346,0.9397,0.9070 and 0.8732, respectively. (3) Cluster analysis revealed that CSTP and CM28TP have least different with the recurrent parent, and was clustered into a small class with the similarity coefficient of 0.93,2. Two forward subtractive cDNA libraries from two pairs of near-isogenic wheat lines, Chuanmai 28 TP (CMTP) and Chinese Spring TP (CSTP), were constructed using suppression subtractive hybridization (SSH). A total of 68 clones in CMTP lines and 197 clones in CSTP lines were identified as potentially overexpressed clones. Thirty-two out of 68 clones in CMTP lines belonged to unknown proteins; while, the remaining 30 clones shared homology to diverse classes of genes involved in protein modulation. and protein synthesis, signal transduction, and ion transporters. Approximately 67% of genes in CSTP lines were either unclassified or had no matches ("no hits") in the database and about 33% of identified genes encoded polypeptides with known functions. Sequence comparisons of cDNA clones between the two forward cDNA libraries revealed that four genes, encoding thioredoxin H, ubiquitin protein ligases, MCM2, and ubiquinol-cytochrome C reductase complex 14 kD proteins, were overexpressed in both libraries. These genes would likely play an important role in determining the three pistils trait in the mutant wheat line.3. The annealing control primer system was used to identify the different expressed genes in three pistils mutation. Using 120 arbitrary ACP primers, we identified 3 differentially expressed genes (DEGs) in young spikes between two near-isogenic lines that are Chuanmai 28 TP (CMTP) and Chinese Spring TP (CSTP) and their recurrent parents. We tentatively designated the three differentially expressed genes as DETP-1, DETP-2 and DETP-3. DETP-1 showed similar function with maize cytoplasmic membrane protein. The cytoplasmic membrane protein involved in cell division in bacteria. DETP-3 is homology to maize endo-1,4-beta-glucanase (EGases). The EGases associated with plant development, cell wall loosening, flowering stem and root expending. DETP-2 showed no significant hit with any sequence found in the database and encodes unknown protein. These genes would likely play an important role in determining the three pistils trait in wheat.4. Full length cDNA sequence of RING domain ubiquitin protein ligase gene TaZFP-1 was acquired by RT-PCR methods. Then, analysis and prediction on the acquired sequences and its amino acids, physicochemical properties, hydrophobicity/hydrophilicity, secondary structure, functional domains, sequence alignment and phylogenetic tree. The results showed that TaZFP-1 gene cDNA was 759 bp in length, encoding 252 amino acids. Molecular weight of the putative TaZFP-1 protein is 27.9 kD with a theoretical PI=6.23. Most amino acids in TaZFP-1 protein are hydrophilic amino acids, so the protein may be a soluble protein. Secondary structure of TaZFP-1 was mainly composed ofα-helices and random coils. Functional domains analysis indicated that the TaZFP-1 is a RING finger domain protein and containing a C3HC4 motif. The molecular evolution threes showed that the TaZFP-1 was clustered into the monocotyledon group and high genetic relationship with O. sativa RING domain E3 ligases.5. The full cDNA sequence of endo-beta-1,4-glucanase gene TaEG was acquired by RT-PCR and RACE methods. Then, analysis and prediction on the acquired sequences and its amino acids, physicochemical properties, hydrophobicity/hydrophilicity, secondary structure, three-dimensional structure, transmembrane domains, sequence alignment and phylogenetic tree. The results showed tha TaEG gene cDNA was 2174 bp in length, encoding 622 amino acids. Molecular weight of the putative TaEG protein is 69.07 kD with a theoretical PI=9.39. Most amino acids in TaEG protein are hydrophilic amino acids, so the protein may be a soluble protein. Secondary structure of TaEG was mainly composed ofα-helices and random coils. Transmembrane domains analysis indicated that TaEG contain a transmembrane domains in 74-96 amino acids, so we prediced the TaEG is a membrane anchored EGase. The cluster analysis also comfirmed that TaEG is a membrane anchored EGase. This study has laid a foundation for further investigate the function of TaEG in wheat pistil development.