The responsive patterns of phytochrome gene family members to photoperiod and abiotic stresses were comparatively analyzed and the favorable natural variation sites of these genes were identified. This would help understand the mechanism of phytochrome gene family in photoperiod-regulated growth and development and abiotic stress response. In addition, it may facilitate the molecular marker assisted selection of key traits in foxtail millet. In this study, we used RT-PCR to clone three phytochrome genes SiPHYA, SiPHYB and SiPHYC from ultra-late maturity millet landrace variety 'Maosu'. After primary bioinformatics analysis, we studied the photoperiod control mode and the characteristics of these genes in responding to five abiotic stresses including polyethylene glycol (PEG)-simulated drought, natural drought, abscisic acid (ABA), high temperature and NaCl by fluorescence quantitative PCR. Finally, we detected the mutation sites of the three genes among 160 foxtail millet materials and performed haplotype analysis to determine the genes' functional effect. We found that the cloned cDNA sequences of gene SiPHYA, SiPHYB and SiPHYC were 3 981, 3 953 and 3 764 bp respectively, which contained complete coding regions. Gene SiPHYB and SiPHYC showed closer evolutionary relationship. Photoperiod regulated all of the three genes, but showed more profound effects on diurnal expression pattern of SiPHYB, SiPHYC than that of SiPHYA. Under short-day, when near heading, the expression levels of SiPHYA and SiPHYB were significantly lower than that under long-day, indicating their roles in suppressing heading of foxtail millet under long-day. SiPHYB and SiPHYC were responsive to PEG-simulated drought, natural drought, ABA and high temperature stresses together. SiPHYA and SiPHYB responded differently to salt stress, whereas SiPHYC did not respond to salt stress. Re-sequencing of 160 foxtail millet materials revealed that SiPHYB was highly conservative. Two missense mutations of SiPHYA, such as single nucleotide polymorphism (SNP) 7 034 522^C→T and SNP7 036 657^G→C, led to delaying heading and increasing plant height. One missense mutation of SiPHYC, such as SNP5 414 823^G→T, led to shortening heading under short-day and delaying heading under long-day, as well as increasing plant height and panicle length regardless of photo-thermal conditions. Photoperiod showed different regulatory effects on SiPHYA, SiPHYB and SiPHYC. SiPHYB and SiPHYC jointly responded to various abiotic stresses except for the salt stress. Compared with the reference genotype, mutation genotypes of SiPHYA and SiPHYC delayed heading and increased plant height and panicle length.
Gene Expression Regulation, Plant ; Photoperiod ; Phytochrome/metabolism* ; Plant Proteins/metabolism* ; Setaria Plant/metabolism* ; Stress, Physiological/genetics*

OBJECTIVETo identify the function of the gene encoding Neurospora crassa EAA33149.1 protein which has 46.85% similarity with Aspergillus niger phA gene.

METHODSThe bioinformatics analysis was conducted using the prediction algorithms SignalP v3.0, arginine and lysine propeptide cleavage sites in eukaryotic protein sequence prediction algorithms ProP 1.0 server, transmembrane domain prediction algorithms Tmpred and TMHMM v2.0, potential GP I-anchor sites prediction algorithm big-P I Predictor and the subcellular protein location prediction algorithms TargetP v1.01. According to the analysis results, the gene was cloned into Saccharomyces cerevisiae.

RESULTSThe signal peptide, the cleavage site and the secretion pathway were determined, and the expressed recombinant protein with 54,000 displayed phytase activity.

CONCLUSIONThe gene has been identified to encode N. crassa phyA.

Algorithms ; Amino Acid Sequence ; Computational Biology ; methods ; Fungal Proteins ; genetics ; metabolism ; Molecular Sequence Data ; Neurospora crassa ; genetics ; Phytochrome A ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism

Genomic DNA sequence analysis of phytochrome like photoreceptors in a number of bacteria revealed several open reading frames (ORFs) encoding proteins with amino acid sequences homologous to plant phytochromes. The phytochrome like photoreceptors, collectively called bacteriophytochromes, contain an N-terminal domain homologous to the chromophore-binding domain (CBD) of higher plants and a C-terminal domain of histidine kinase domain( HKD). Due to their simple structure, bacteriophytochromes broaden the view of phytochrome evolution and provide us with a simple model to investigate phytochrome-mediated light signal in higher plants. In this report, the bacteriophytochromes from Synechocystis sp. PCC6803 were investigated. The gene cph1 and its fragment cph1 (C-435) were isolated from the Synechocystis sp. PCC6803 genomic DNA by polymerase chain reaction(PCR) using specific primers. Then, the genes were cloned with the vector pBluescript, yielding plasmids pBlu-cphl and pBlu-cph1 ( C-435), before they are subcloned with the vector pET30, using the EcoRV and Xho I restriction sites. pBlu-cph1, pBlu-cph1 (N-435) were cleaved with Sma I and Xho I, and the released genes were ligated to the pET30a fragment. The E. coli [strain BL21 (DE3)] cells containing recombinant pET30a were grown in medium RB at 20 degrees C, and harvested 6 h later after induction with isopropyl thio-beta-D-galactoside (IPTG). Then, reconstitution systems were employed to study the characteristics of the genes. In the reconstitution system, autoassembly of aprotein of phytochrome with PCB was investigated. The chromophore addition was an autocatalytic process. Reconstitution products were red/infrared (R/FR) photochromic, which was similar to that of the phytoehrome in higher plants. How ever, the spectral change ratios (deltaAmax/deltaAmin) of the two fragments differed from each other. It was also shown that PCB was covalently bound to apo-protein via Zn2+ fluoresc ence SDS-PAGE. After irradiation by light of 700 nm, the maximum absorption spectrum o f holo-Cphl was 650nm. The absorption of it after denaturatior in the dark with ur ea in the presence of hydrochloric acid (pH = 2) was 660nm, which was similar with th at of cis-PCB. In addition, after irradiation by light of 650nm, the maximum absorption spectrum of holo-Cph1 was 700nm. The absorption of it after denaturation in the dark with urea in the presence of hydrochloric acid (pH = 2) was 600nm, which was similar with that of trans-PCB. The result showed that the photochromism of phytochrome resulted from the isomerizaation of chromophore (PCB in this report). The reconstitution of Cph1 (C-435) under the same condition supported the conclusion. Fluorescence emission spectrum of the products suggested that bacteriophytochrom e structure with cis-PCB was more stable than that with trans-PCB. The new reconstitution system in this report sets a base for the application of phytochrome as photochromic biomaterials in biosensors. In addition, phytochrome shows great potential in food, cosmetic and biological engineering, etc.
Bacterial Proteins ; biosynthesis ; chemistry ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; radiation effects ; Genetic Vectors ; Photochemistry ; Phytochrome ; biosynthesis ; chemistry ; genetics ; Protein Kinases ; biosynthesis ; chemistry ; genetics ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; Synechocystis ; chemistry