Wheat quiescin sulfhydryl oxidase was expressed in Escherichia coli for developing a new biological flour improver. The synthesized wqsox gene was constructed into the vector pMAL-c5x and expressed in E. coli, then the expression conditions of recombinant protein was optimized. The MBP fusion label in recombinant protein was removed by protease digestion after affinity purification. Moreover, enzymatic properties of the purified wQSOX and its effect on bread quality were investigated. The synthesized wqsox gene contained 1 359 bp and encoded 453 amino acids with a deduced molecular weight of 51 kDa. The constructed recombinant vector pMAL-c5x-wqsox could successfully express soluble recombinant protein MBP-wQSOX in E. coli Rosetta gamiB(DE3), and the optimal induced expression conditions for recombinant protein were 25 °C, 0.3 mmol/L IPTG and 6 h. MBP fusion tag was cut out by factor Xa protease and wQSOX was prepared after affinity purification. wQSOX could catalyze the oxidation of DTT, GSH and Cys, accompanying the production of H2O2, and exhibited the highest substrate specificity for DTT. Furthermore, enzymatic properties results demonstrated that the optimal temperature and pH for wQSOX catalyzing oxidation of DTT was 50 °C and 10.0, respectively, and wQSOX presented a good stability under high temperature and alkaline environment. The addition of wQSOX with 1.1 U/g flour significantly (P<0.05) increased 26.4% specific volume of the bread, and reduced 20.5% hardness and 24.8% chewiness of bread crumb compared to the control, indicating a remarkable ability to improve the quality of bread.
Bread ; Escherichia coli/genetics* ; Hydrogen Peroxide ; Oxidoreductases ; Triticum

To identify spatiotemporal expression patterns of vernalization genes in common wheat, we analyzed expression characteristics of several vernalization genes (VRN1, VRN2 and VRN3) in the wheat cultivars 'Chinese spring' and 'Luohan 2' by RT-PCR. The VRN1 gene was expressed at different levels in the leaves and roots at the 3-leaf stage, stems, flag leaves at the grain-filling stage, anthers, ovules, and developing seeds in 'Chinese spring'. Expression of VRN1 increased before flowering date, then decreased after flowering time. Expression of VRN1 was not detected in dry seeds or seeds germination. Expression patterns of VRN1 in 'Luohan 2' were similar to those in 'Chinese spring', except that it was not expressed in roots or in the leaves at the 3-leaf stage in 'Luohan 2'. Expression of VRN2 was only detected in the leaves at the 3-leaf stage and in the embryo buds during seeds germination. The Spatiotemporal expression of VRN3 was similar to that of VRN1, except that VRN3 was not expressed in roots. These results improved our understanding of the molecular regulation of vernalization genes in common wheat.
Flowers ; genetics ; physiology ; Gene Expression Regulation, Plant ; genetics ; Genes, Plant ; genetics ; Triticum ; genetics

Quantitative trait loci (QTL) analysis was conducted in bread wheat for 14 important traits utilizing data from four different mapping populations involving different approaches of QTL analysis. Analysis for grain protein content (GPC) suggested that the major part of genetic variation for this trait is due to environmental interactions. In contrast, pre-harvest sprouting tolerance (PHST) was controlled mainly by main effect QTL (M-QTL) with very little genetic variation due to environmental interactions; a major QTL for PHST was detected on chromosome arm 3AL. For grain weight, one QTL each was detected on chromosome arms 1AS, 2BS and 7AS. QTL for 4 growth related traits taken together detected by different methods ranged from 37 to 40; nine QTL that were detected by single-locus as well as two-locus analyses were all M-QTL. Similarly, single-locus and two-locus QTL analyses for seven yield and yield contributing traits in two populations respectively allowed detection of 25 and 50 QTL by composite interval mapping (CIM), 16 and 25 QTL by multiple-trait composite interval mapping (MCIM) and 38 and 37 QTL by two-locus analyses. These studies should prove useful in QTL cloning and wheat improvement through marker aided selection.
Bread ; Chromosome Mapping ; Quantitative Trait Loci ; genetics ; Triticum ; genetics ; growth & development

Nitrogen is one of the most important nutrient elements for plants and a major limiting factor in plant growth and crop productivity. Glutamine synthase (GS) is a key enzyme involved in the nitrogen assimilation and recycling in plants. So far, members of the glutamine synthase gene family have been characterized in many plants such as Arabidopsis, rice, wheat, and maize. Reports show that GS are involved in the growth and development of plants, in particular its role in seed production. However, the outcome has generally been inconsistent, which are probably derived from the transcriptional and post-translational regulation of GS genes. In this review, we outlined studies on GS gene classification, QTL mapping, the relationship between GS genes and plant growth with nitrogen and the distribution characters, the biological functions of GS genes, as well as expression control at different regulation levels. In addition, we summarized the application prospects of glutamine synthetase genes in enhancing plant growth and yield by improving the nitrogen use efficiency. The prospects were presented on the improvement of nitrogen utility efficiency in crops and plant nitrogen status diagnosis on the basis of glutamine synthase gene regulation.
Arabidopsis ; Genes, Plant ; Glutamate-Ammonia Ligase ; genetics ; Nitrogen ; metabolism ; Oryza ; Plants ; enzymology ; genetics ; Triticum ; Zea mays

Plants with alien genomic components (alien chromosomes / chromosomal fragments / genes) are important materials for genomic research and crop improvement. To date, four strategies based on trait observation, chromosome analysis, specific proteins, and DNA sequences have been developed for the identification of alien genomic components. Among them, DNA sequence-based molecular markers are mainly used to identify alien genomic components. This review summarized several molecular markers for identification of alien genomic components in wheat, cabbage and other important crops. We also compared the characteristics of nine common molecular markers, such as simple sequence repeat (SSR), insertion-deletion (InDel) and single nucleotide polymorphism (SNP). In general, the accuracy of using a combination of different identification methods is higher than using a single identification method. We analyzed the application of different combination of identification methods, and provided the best combination for wheat, brassica and other crops. High-throughput detection can be easily achieved by using the new generation molecular markers such as InDel and SNP, which can be used to determine the precise localization of alien introgression genes. To increase the identification efficiency, other new identification methods, such as microarray comparative genomic hybridization (array-CGH) and suppression subtractive hybridization (SSH), may also be included.
Chromosomes, Plant ; Comparative Genomic Hybridization ; Genome, Plant/genetics* ; Genomics ; Triticum/genetics*

In this study, peaT1 gene was subcloned into the Pichia pastoris expression vector pPIC9K, which contained both the methanol-inducible promoter and the transcription terminator of the AOX1 gene, resulting the plasmid pPIC9K-peaT1. The recombinant plasmid was linearized by Sal I or Bgl II and transformed into P. pastoris GS115 by electroporation method. Recombinant strain was screened by Minimal Dextrose Medium and further confirmed by PCR. The gene was in frame integrated into the Pichia genome through homologous recombination, resulting the recombinant strain. Regulated by the alpha-Factor, promoter of AOX1 gene and termination signal of yeast genomic, the recombinant protein was expressed and secreted into the supernatant. The SDS-PAGE analysis indicated that the apparent molecular weight of target protein was about 35 kD. Bioassay results showed that the inhibition rate of the expressed protein against TMV was 30.37%. The supernatant was collected and then purified by anion exchange chromatography. This protein can promote seedling growth of wheat obviously.
Alternaria ; genetics ; Fungal Proteins ; biosynthesis ; genetics ; pharmacology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Triticum ; drug effects ; growth & development

Leymus racemosus had a high resistant capacity to wheat scab (Fusarum head blight). The transfer of scab resistant gene from L. racemosus to Triticum aestivum is of great significance for broadening the germplasm of wheat resistance. To obtain Triticum aestivum-Leymus racemosus translocation line with scab resistance, we irradiated the pollen of T. aestivum-L. racemosus disomic addition line DA7Lr by ⁶⁰Co-γ-rays 1 200 R (100 R/min) prior to pollinating to emasculation T. aestivum cv. Chinese Spring. One plant with one translocation chromosome was detected in the M1 by GISH. The plant with one translocation chromosome was self-pollinated, and at meiotic metaphase I its progenies with two translocation chromosomes were analyzed for chromosome pairing behavior in their pollen mother cells (PMCs). One rod bivalent was observed at meiotic metaphase I, indicating that the plant with two translocation chromosomes was one translocation homozygote. Sequential GISH-FISH analysis, using Oligo-pAs1-2 and Oligo-pSc119.2-2 as probe, translocation line was confirmed as T6DL·7LrS. The translocation line had higher resistance to wheat scab and feasibility to be used as a new source in wheat breeding resistant to scab disease.
Chromosomes, Plant ; Disease Resistance ; genetics ; In Situ Hybridization, Fluorescence ; Plant Breeding ; Plant Diseases ; genetics ; Poaceae ; genetics ; Pollen ; Translocation, Genetic ; Triticum ; genetics

Maize (Zea mays L.), wheat (Triticum aestivum L.) and rice (Oryza sativa L.) are three staple crops and accordingly it is very meaningful to optimize the condition of their protoplasts isolation. The concentration of the enzyme, the time of isolation and centrifugal force in protoplast isolation were investigated to find their effects on protoplast yield and viability using leaves of maize (Zong 3), wheat (Chinese Spring) and rice (Nipponbare). The results show that the concentration of the enzyme and the time of isolation affected the protoplast yield significantly. Although the yield of protoplast was increased with high concentration of enzyme and long incubated time, it led to too much cells breakdown. The orthogonal experimental design results show that the best condition of maize protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 7 h, 100 x g 2 min and the protoplasts yield was 7x106 cells/g fresh weight (FW); the best condition of wheat protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 5 h, 100 x g 2 min and the protoplasts yield was 6 x 10(6) cells/g FW; the best condition of rice protoplast isolation was Cellulase R-10 2.0%, Macerozyme R-10 0.7%, 50 r/min 7 h, 1 000 x g 2 min and the protoplasts yield was 6x10(6) cells/g FW. The vitalities were more than 90% using fluorescein diacetate staining method. 50%-80% transformation efficiency was obtained when protoplasts were transformed by green fluorescent protein using PEG-Ca2+ method.
Cell Culture Techniques ; methods ; Oryza ; chemistry ; genetics ; Plant Leaves ; enzymology ; Protoplasts ; cytology ; Triticum ; chemistry ; genetics ; Zea mays ; cytology ; genetics

Using a modified TAIL-PCR technique, the 5' -flanking region of the X gene in wheat was successfully isolated. Two novel modifications of the TAIL-PCR were introduced here: using a battery of random 10-mers as the short arbitrary primers instead of three degenerate 16-mers; using 29 degrees C instead of 44 degrees C as the annealing temperature for the low-stringency cycle; increasing five high-stringency cycles and reducing five low-stringency cycles; and using single primers for the third round of product identification. Isolated 5' -flanking region was fused to the GUS gene, and tested for expression in Arabidopsis plants. Histochemical analysis of the transgenic plants showed the report gene was driven by isolated 5'-flanking region. Modified TAIL-PCR technique could isolate rapidly the promoter of any gene from organisms with large genomes.
Base Sequence ; Genes, Plant ; genetics ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Promoter Regions, Genetic ; genetics ; Triticum ; genetics ; metabolism

Psathyrostachys juncea is a potential source of useful genes, such as the barley yellow dwarf virus resistance, salt tolerance and drought tolerance, for wheat improvement. Conventional sexual hybridization between wheat and Psathyrostachys juncea is very difficult to occur as the two are sexual incompatible. Somatic hybridization is a promising technique for creating hybrids across the sexual border. Here we report a fusion system for somatic hybridization of wheat using PEG method. Mixed protoplasts of two wheat (Triticum aestivum L. cv. Jinan 177) culture cells (cha9 and 176) were used as the recipients to fuse with the donors, the protoplasts of Psathyrostachys juncea (Fisch.) Nevski irradiated with ultraviolet light (UV) at an intensity of 380 microW/cm2 for 1 min or 2 min. Sixteen clones were generated in the combination I, (wheat 176 + wheat cha9 + P. juncea 1 min UV treatment) and five of the hybrid clones could differentiate to green plants. All the regenerated clones were confirmed as somatic hybrids by cytological, isozyme, chromosome and random amplified polymorphic DNA (RAPD) analysis. Chloroplast genome of the hybrids was analyzed using 7 pairs of wheat-specific chloroplast microsatellite (SSR) primers. Three clones were obtained from the combination II (wheat 176 + wheat cha9 + P. juncea 2 min UV treatment), and all browned slowly and died in 3 months. This result indicated that the mixed wheat cells was helpful to the formation and regeneration of hybrid callus and the dosage of the UV had significant effect on the development of the fusion products.
Chromosomes, Plant ; Hybridization, Genetic ; Poaceae ; genetics ; growth & development ; Random Amplified Polymorphic DNA Technique ; Regeneration ; Triticum ; genetics ; growth & development