Abstract:Protein Disulfide Isomerase （PDI）， a member of the thioredoxin superfamily， is an oxidoreductase widely exists in animals， plants and microorganisms. In plants， the formation and isomerization of protein disulfide bonds， which are mainly catalyzed by disulfide bond proteins， are crucial in the correct assembly and folding of proteins. In this study， based on the sequence similarity to the PDI gene family in Arabidopsis thaliana， 21 PDI genes were identified and found on nine chromosomes of maize. The phylogenetic tree analysis showed that the maize PDI gene family members were divided into four evolutionary branches and 11 developmental groups， and within each group the gene structure and conserved motifs were similar. The analysis of cis-acting elements in the promoters identified cis-acting elements associating to stress， plant hormones and endosperm specific expression. By quantifying the transcriptional profiles， the high expression was detected in embryo， endosperm and grain. In endosperm， a downward trend followed by upward from 10 to 20 days after pollination was observed. Biodiversity analysis revealed that was diversity of ZmPDIL1-1 gene. The result of subcellular localization showed that ZmPDIL1-1 was located to the endoplasmic reticulum. Collectively， this study provided reference for the functional analysis of PDI gene family in maize.

Abstract:Non-specific lipid transfer proteins （nsLTPs） play a crucial role in the transport and secretion of lipids in plants. In this study， two type II nsLTPs were cloned， namely nsLTP2-1 and nsLTP2-2， and analyzes it with functional analysis from Lavandula angustifolia. The analysis of sequence characteristics revealed that the nsLTP2-1 and nsLTP2-2 genes putatively encode 119 and 117 amino acids， respectively， which exhibit conserved lipid transfer proteins （LTP） domains and 8 highly conserved cysteine residues. The phylogenetic analysis revealed that these genes are located in separate branches and share the closest genetic relationship with Perilla frutescens， a member of the Labiatae family. The analysis of gene expression showed that both genes exhibit high expression levels in flower buds， with minimal expression observed in leaves， stems， and petals. However， notable disparities in expression were observed in the calyx， with nsLTP2-1 and nsLTP2-2 demonstrating higher expression levels in mature and young calyxes， respectively. The expression of these genes in flower buds and leaves was found to be stimulated by heavy light， while their expression in flower buds was also induced by abscisic acid. Additionally， the expression of nsLTP2-1 and nsLTP2-2 in leaves was found be triggered by methyl jasmonate and ethylene， respectively. The subcellular localization of the yellow fluorescent protein （EYFP） fusion protein demonstrated that both nsLTPs were localized on the cell membrane and cell wall， indicating they may be related to the transport of secondary metabolites. Following the overexpression of nsLTP2-1 and nsLTP2-2， tobacco leaves were subjected to Nile red staining， after 485-543 nm excitation light excitation， the fluorescence of leaf glandular hair was more than that of wild type. This observation suggests a potential significance of the nsLTPs investigated in this study with regards to lipid synthesis and transport. These findings provide a fundamental basis for elucidating the role of lavender lipid transfer protein in lipid and terpenoid transportation.

Abstract:EIN3/EILs （Ethylene inductive proteins/ethylene inductive 3-like） protein family function as the main members of ethylene signal transduction pathway and play an important regulatory role in plant growth and development and stress response. In this study， ZmEIL2 was isolated from maize inbred line B73 based on sequence homology （GeneBank ID： KJ727458.1）. This gene carries a 1788 bp coding sequence （CDS） that encodes for 595 amino acid residues with a relative molecular mass of 63.81 kD and a theoretical isoelectric point of 6.34. ZmEIL2 contains an EIN3 domain which is specific to EIN3/EILs family. Phylogenetic tree analysis showed that ZmEIL2 protein had the closest evolutionary relationship with SbEIL1 protein in Sorghum bicolor and was relatively distant from EIL proteins in Arabidopsis and soybean. The subcellular localization assay showed that ZmEIL2 protein was localized to the cell membrane and nucleus. Tissue specific expression analysis showed that ZmEIL2 gene was highly expressed in bract， followed by ear， tassel and silks， and the lowest expression was detected in mature leaves. The transcripts of ZmEIL2 gene were inducible under abiotic stresses treatments such as dehydration， PEG， ABA， high salt， heat and cold， particularly under PEG， ABA or high salt treatments. The expression level of ZmEIL2 gene in leaves was significantly higher than that in stems and roots. These results laid a theoretical foundation for further deciphering the molecular mechanism of ZmEIL2 gene in responses to abiotic stresses.

Abstract:Soluble inorganic pyrophosphatase regulates the homeostasis of inorganic pyrophosphate (PPi) in the cytoplasm through hydrolysis. In this study, seven genes encoded for soluble inorganic pyrophosphatase in maize (ZmPPases) were cloned and identified on chromosomes 2, 4, 5, 6, 8, 10, respectively. The relative molecular mass of ZmPPases was varied from 22.8 kDα to 25.6 kDα with the isoelectric point of 4.84 to 6.24. Secondary structure analysis of ZmPPases revealed alpha helix and beta strand motifs. Phylogenetic analysis together with the homozygous proteins in maize, rice, Sorghum, and Arabidopsis suggested three phylogenetic branches of soluble inorganic pyrophosphatases. ZmPPases were localized in the cell membrane and nucleus by transient expression in N. benthamiana. The ZmPPase genes were inducible under salt and drought stress treatments, whereas ZmPPase2.1 (Zm00001d051564) was down-regulated under salt stress treatment. Collectively, this study reported a comprehensive analysis of the sequence and expression of ZmPPases family members, which might provide a theoretical basis for further functional research.

Abstract:In order to study the functional basis of Phage shock protein A (PspA), the full-length coding sequence (CDS) of the PspA gene was isolated based on sequence homolog and its expression pattern of PspA from N. flagelliforme under drought stress was studied by RT-qPCR. We further generated a plasmid pCAM35s-GFP-PspA which were subjected for a subcellular localization analysis and a transformation in A. thaliana. PspA contained a 777-bp full-length coding sequence, and this gene was significantly up-regulated in N. flagelliforme upon drought stress treatment. A transient expression of PspA in N. benthamiana suggested a sublocalization on the plasma membrane. Moreover, by transforming this gene in A. thaliana, the transgenic plants showed low copies of the PspA gene using Southern hybridization and expressed the recombination protein by Western blot analysis. The transgenic plants expressing the PspA gene, if compared to the wild type plants, were found to be highly resistant against drought stress. Collectively, the results provided a foundation for further exploring the biological function of the N. flagelliforme PspA gene that interplays with drought stress.

Abstract:Lipoxygenase is a key enzyme involved in the pathway of fatty acid metabolism, and plays an important role in the growth and development of plants, responses to environmental stresses and the synthesis of aroma components. In this study, the red callus from the leaves of Zihong 3 was used for PCR amplification of the MdLOX1a gene and its partial promoter sequence. Sequencing analysis revealed that MdLOX1a has an open reading frame of 2592 bp encoding for 863 deduced amino acids, with a predicted protein molecular weight of 97.69 KDa and the isoelectric point of 5.14. The MdLOX1a gene was found to be localized on chromosome 9 of the apple genome, consisting of 8 exons and 7 introns. Phylogenetic tree analysis with amino acid sequence indicated that MdLOX1a and Pb9S-LOX5 were assigned within the same branch. In addition, we obtained the MdLOX1a promoter sequence with a length of 1058 bp, which contained several cis-acting elements in response to various stresses. Subcellular localization revealed that MdLOX1a was localized on both the cell membrane and the nucleus. The tissue disparity analysis showed that the expression level of MdLOX1a gene was abundant in the pericarp, followed by flower and pulp. The expression of MdLOX1a gene gradually increased with fruit ripening, and its transcription has been induced under continuous light treatment. The expression of MdLOX1a gene was significantly reduced after low temperature (16 ℃) and exogenous ABA treatment at different concentrations. The overexpression of MdLOX1a showed that the transgenic lines showed the induction of the aromatic components in relative to the control lines, suggesting that MdLOX1a might associate with the synthesis of aroma components.

Abstract:F-box proteins, widely existing in eukaryotes, were involved in cell cycle regulation, apoptosis and multiple hormone signal transduction. Recently, it was showed that F-box proteins mediated the responses to abiotic and biotic stresses, which is crucial to maintain the normal growth and development for plant. Drought and other abiotic stresses seriously affected plant growth and development. It is urgent to solve the problem which enhances the drought resistance of plant at present. The Grapevine, one of important fruit trees, was widely cultivated among China and world. Drought stress severely affected the plant growth and dramatically reduced the fruit quality of grapevine. Therefore, it has a great meaning to isolate the drought-responsive genes and analyze its functions for stress-resistant improvement in grapevine. According to the analysis of drought treated grapevine transcriptome, we isolated 11 F-box genes obviously up-regulated under drought stress. Pfam software analysis showed these F-box genes encode amino acid sequences having a complete F-box domain. Among them, VvF-box5 exhibited higher expression level compared with other genes and VvF-box5 is located on the 19th chromosome. There are 1 824 bp in opening reading frame of the VvF-box5, including 5 exons and 4 introns according to gene sequence analysis. VvF-box5 protein contains 1 F-box domain in its N terminal and a fibrinogen-like domain（FBD） and two leucine-rich repeat（LRR）in its C terminal. Protein secondary structure prediction analysis showed that the VvF-box5 contain 15 α-helixs and 14 β-pleated sheets. The promoter element analysis showed that the promoter of VvF-box5 contains multiple stresses-responsive elements, such as GA response element GARE-motif, MeJA response element CGTCA-motif, Drought stress related components ethylene-responsive element (ERE), heat stress-responsive element (HSE) and low temperature responsive element (LTR), light response cis elements ACE, Box 4 and Sp1 and other element related to cell cycle regulation and development. Real-time qRT-PCR showed that VvF-box5 responded to drought, salt, ABA and JA. Subcellular localization showed that VvF-box5 was mainly located in nucleus in onion epidermis cells. Overexression of VvF-box5 clearly improved the drought tolerance in transgenic plants. In addition, the fusion protein 6×His-VvF-box5 was induced and expressed in prokaryotic expression system and purified by Ni-NTA Resin, laying foundation for studying VvF-box5 functions.

Abstract:Improving the efficiency of nitrogen absorption in maize has important significance. In consideration of the characteristics of transportation of NO3- in CLC protein family, this study clones the CLC family genes ZmCLCa of maize by homology cloning method. Based on bioinformatics analysis, we find that the protein has a voltage-gated chlorine ion channel structure domain and subcellular localization result shows that the protein is located on the cell membrane. Under 200 mmol/L KNO3 processing conditions, the content of NO3- in transgenic arabidopsis of strain is obviously higher than that in wild arabidopsis. Therefore, ZmCLCa gene likely plays an important role in the process of nitrogen absorption of maize..

Abstract:A plant protoplast system can be used to study transient gene expression, protein subcellular localization, protein-protein interaction and protein activity as well as gene function. The application of heterologous protoplast systems to the expressed proteins may exhibit aberrant trait. To avoid this mistaking, it is necessary to establish and apply the host protoplast systems. In wheat, the PEG-mediated gene protoplast transformation is hampered by the release of nucleases from the protoplasts, which leads to extensive degradation of plasmid DNAs. In this study, in order to get the high efficiency of the wheat protoplast transformation, means including inhibiting the nucleases activity and enlarging the plasmid quantity were used. The results showed that the protoplast transformation efficiency could be improved through double-fold usage of the plasmid quantity and inhibiting the nucleases activity by low transformation temperate. Consequently, the wheat protoplast transformation efficiency was raised to 85 %. Moreover, this wheat protoplast transformation system was used to study the expression protein subcellular localization of 2 wheat disease-related genes. This research has reference value for the future relevant work

Abstract:Superoxide dismutase (SOD) is a major antioxidant enzymes, which is important for plants to cope with adversity stress and anti-aging effect. A cDNA of Cu/Zn-SOD gene defining by the EST sequence assembly from Microarray data, was cloned from the photo-thermosensitive male sterile wheat (PTGMS) line BS366. The length of ORF sequence is 495bp, which encoded a 164-amino-acid polypeptide. Protein sequence contained a typical Cu/Zn-SOD gene family functional domain and 3 dimensional structures. Besides, the protein was located in the cytoplasm by ubcellular localization analysis. Evolution and clustering analysis showed that the Cu/Zn-SOD protein of wheat shared 89% and 94% sequence similarities with the Cu/Zn-SOD protein of Brachypodium distachyon and Hordeum vulgare, respectively. The methods of quantitative real-time PCR (qRT-PCR) were utilized to analyze the expression patterns in different wheat tissues and abiotic stresses. It was showed that the Cu/Zn-SOD gene expressed in root, stem, leaf, pistil, stamen and glume, belonging to constitutive expression. The highest expression level was found in chlorenchyma such as leaf, stem and glume. In addition, the gene expression was induced by a variety of stressesrevealing that the Cu/Zn-SOD gene of wheat might involve in multiple stress regulation pathway. To illuminate the regulation of Cu/Zn-SOD in wheat fertility, expression profile of Cu/Zn-SOD in PTGMS line BS366 pollen during fertility transformation was performed. The results showed that the relative expression of Cu/Zn-SOD at microsporocyte stage and meiosis stage in fertile environment were 8 times and 16 times higher than control, respectively, while there was no significant changes in sterility environment, suggesting that Cu/Zn-SOD might be involved in the regulation of fertility transformation of wheat PTGMS line BS366. This information lays a foundation for further functional study of Cu/Zn-SOD in wheat.