Diqiu Liu

Overexpression of the glutathione S-transferase gene from Pyrus pyrifolia fruit improves tolerance to abiotic stress in transgenic tobacco plants

Glutathione S-transferases (GSTs) are ubiquitous enzymes in animals and plants, and they are multifunctional proteins encoded by a large gene family. GSTs are involved in response to the oxidative stress including drought, salt, heavy metals, and so on. Under oxidative stress, the excessive reactive oxygen species (ROS) induce an increase in GST levels, and then the GSTs metabolize the toxic products of lipid peroxidation, damaged DNA and other molecules. Previously, a full-length cDNA of a novel zeta GST gene, PpGST, was characterized from fruit of Pyrus pyrifolia Nakai cv. Huobali. In the present study, a constitutive plant expression vector of PpGST was constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi) to verify the function of PpGST. As a result, the PpGST gene was successfully integrated into the genome of the transgenic tobacco lines and expressed as expected in the transformants through Southern blotting and quantitative reverse transcription-polymerase chain reaction (QRT-PCR) analysis. Growth of T1 generation plants of PpGST transgenic lines and WT under non-stressful conditions was similar, however, the transgenic tobacco lines showed relatively normal growth under drought, NaCl, and cadmium (Cd) stresses. Furthermore, the T1 transgenic tobacco lines showed significantly slower superoxide anion production rate than the WT under abiotic stress. Simultaneously, the MDA content of each T1 transgenic tobacco plant was only slightly increased and significantly lower than that of the WT under drought, salt and Cd stress. Together with the GST activity of the transgenic tobacco lines, which was significantly increased under stressful conditions, as compared with that in WT, overexpression of PpGST in tobacco enhanced the tolerance of transgenic tobacco lines to oxidative damage caused by drought, NaCl, and Cd stresses.

Molecular cloning of a thaumatin-like protein gene from Pyrus pyrifolia and overexpression of this gene in tobacco increased resistance to pathogenic fungi

Thaumatin-like proteins (TLPs) belong to the pathogenesis-related proteins 5 family, and members of TLP gene family accumulate in plants in response to pathogen infection or environmental stress. The biological functions of plant TLPs are believed to be diverse, and they are involved in host defense against both the biotic and abiotic stresses as well as the other physiological processes. Lots of TLPs were well studied as antifungal proteins in vivo or in vitro. To isolate TLP genes involved in self-defense during fruit development, a novel gene PpTLP was isolated from the fruit of Pyrus pyrifolia Nakai cv Huobali which is a kind of local sand pear with high resistance to biotic and abiotic stresses in Yunnan province of China. The nucleotide sequence of PpTLP was highly homologous with TLPs from other plant species, and the protein encoded by PpTLP has 16 conserved cysteine residues and 5 amino acid residues related with antifungal activity of plant TLPs, an arginine residue, a glutamic acid residue, and 3 aspartic acid residues. What is more, the putative protein PpTLP has the highly conserved main-chain conformation and the folding patterns as other plant TLPs with antifungal activity. PpTLP was abundantly expressed in pericarp and leaf of P. pyrifolia Nakai cv Huobali. Furthermore, in order to verify the function of PpTLP, the constitutive plant expression vector of PpTLP was constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi). The Southern blot and quantitative reverse transcription-polymerase chain reaction (QRT–PCR) analyses demonstrated that the PpTLP gene was integrated into the genome of the tobacco transformants and highly expressed in the transgenic lines. The antifungal activity of PpTLP was detected in vitro plates, and the crude protein extract of transgenic tobacco plants inhibited the hyphal growth of Sclerotinia sclerotiorum, Phomopsis sp., Phytophthora parasitica var. nicotianae, and Alternaria sp. in different degrees.

The Use of Fourier Transform Infrared Spectroscopy for Quantification of Adulteration in Virgin Walnut Oil

Currently, the authentication of virgin walnut oil (VWO) has become very important due to the possible adulteration of VWO with cheaper plant oils such as soybean oil (SO), puer tea seed oil (PO), and sunflower oil (SFO). Methods involving Fourier transform infrared (FT-IR) spectroscopy combined with chemometric techniques (partial least square) were developed for quantification of SO, PO, and SFO in VWO. IR spectra of oil samples were recorded at frequency regions of 4000–650 cm−1 on horizontal attenuated total reflectance (HATR) attachment of FT-IR. PLS model correlates the actual and FT-IR estimated values of oil adulterants (SO, PO, and SFO) with coefficients of determination () of 0.9958, 0.9925, and 0.9952, respectively. The obtained RMSEC values of SO, PO, and SFO in VWO are 1.35%, 1.85%, and 1.43% (v/v), respectively. The method, therefore, has potential as a rapid method for quantification of product adulteration.

Optimization of production conditions for antioxidant peptides from walnut protein meal using solid-state fermentation

Solid-state fermentation (SSF) of walnut protein meal (WPM) by Bacillus subtilis was optimized for the maximum degree of hydrolysis (DH) and reducing power (RP) using response surface methodology (RSM). Optimum fermentation conditions were fermentation time of 82.01 h, inoculum concentration of 10.40, and water content of 1.50 mL/g. Optimized values were 41.80% and 0.78 for DH and RP, respectively. Walnut peptides (WP) were ultrafiltration membrane fractionated. The WPs-II (molecular weight <5 kDa) fraction showed the highest RP value. WPs-II showed good DPPH free radical scavenging and Fe2+ chelating activities, higher than for GSH (l-glutathione reduced) at the same concentration. Amino acid composition analysis of WPs-II showed that Asp, Glu, and Arg were the major amino acids playing important roles in the antioxidant activity. WPs is an efficient antioxidant suitable for use as a food additive and as a pharmaceutical agent.

Application of Fourier Transform Infrared Spectroscopy for the Oxidation and Peroxide Value Evaluation in Virgin Walnut Oil

Recent developments in Fourier transform infrared spectroscopy-partial least squares (FTIR-PLSs) extend the application of this strategy to the field of the edible oils and fats research. In this work, FT-IR spectroscopy was used as an effective analytical tool to determine the peroxide value of virgin walnut oil (VWO) samples undergone during heating. The spectra were recorded from a film of pure oil between two disks of KBr for each sample at frequency regions of 4000–650 cm−1. Changes in the values of the frequency of most of the bands of the spectra were observed and used to build the calibration model. PLS model correlates the actual and FT-IR estimated value of peroxide value with a correlation coefficient of 0.99, and the root mean square error of the calibration (RMSEC) value is 0.4838. The methodology has potential as a fast and accurate way for the quantification of peroxide value of the edible oils.

Characterization and functional analysis of a novel PGIP gene from Pyrus pyrifolia Nakai cv Huobali

Polygalacturonase-inhibiting proteins (PGIPs) are multifunctional proteins related to plant autoimmunity and belong to the plant extracellular leucine-rich repeat (eLRR) protein superfamily. PGIPs play a role in host defense in many plants. In the present study, a novel PGIP gene, PpPGIP was isolated from Pyrus pyrifolia Nakai cv Huobali. The nucleotide sequence of PpPGIP was highly homologous with PGIPs from other plant species and the protein encoded by PpPGIP has several conserved LRR domains. The putative protein PpPGIP was closely clustered with several PGIPs from horticultural plants on the phylogenetic tree. The constructed homology model of PpPGIP indicated that the main-chain conformation and the folding patterns of PpPGIP were highly similar to structural features of PvPGIP2 from Phaseolus vulgaris. The expression levels of PpPGIP in healthy tissue and organ of ‘Huobali’ were analyzed with RT-PCR, and PpPGIP accumulated a little in young leaves, but PpPGIP was expressed abundantly in the pericarp of ‘Huobali’ fruits. Furthermore, in order to verify the function of PpPGIP, the constitutive plant expression vector of PpPGIP was constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi). The Southern blot and real-time PCR analyses demonstrated that the PpPGIP gene was integrated into the genome of the tobacco transformants and highly expressed in the transgenic lines. The antifungal activity of PpPGIP was detected in vitro plates, and the crude protein extract of transgenic tobacco plants inhibited the hyphal growth of Phomopsis sp., Alternaria sp., Penicillium sp., and Aspergillus niger in different degrees.

A bZIP transcription factor, LrbZIP1, is involved in Lilium regale Wilson defense responses against Fusarium oxysporum f. sp. lilii

The basic leucine zipper (bZIP) proteins are ubiquitous in plants and play important roles in plant defense responses. In this study, based on an expressed sequence tag from a suppression subtractive hybridization cDNA library of Lilium regale Wilson during Fusarium oxysporum f. sp. lilii infection, a novel bZIP transcription factor gene LrbZIP1 was isolated from L. regale root using the rapid amplification of cDNA ends method. The predicted protein of LrbZIP1 with 142 amino acid residues contains a basic domain signature and a leucine zipper motif. The quantitative reverse transcription-PCR (qRT-PCR) analysis showed that the transcription level of LrbZIP1 was higher in roots of L. regale than in young stems and leaves. Moreover, the expression of LrbZIP1 was up-regulated in the incompatible interaction between L. regale and F. oxysporum f. sp. lilii as well as after treatments with stress-related signaling molecules. To verify the function of LrbZIP1, a constitutive expression vector of LrbZIP1 was constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi). The results of Southern blotting and qRT-PCR analyses demonstrated that the LrbZIP1 was integrated into genome of the tobacco transformants and highly expressed. Under normal conditions, the T1 transgenic tobacco lines showed higher antioxidant enzyme activities and transcription levels of several resistance-related genes than the wild type. Moreover, the T1 transgenic tobacco plants showed strong resistance to F. oxysporum f. sp. lilii infection.

The PR10 gene family is highly expressed in Lilium regale Wilson during Fusarium oxysporum f. sp. lilii infection

Pathogenesis-related (PR) proteins play key roles in plant responses to pathogens and abiotic stresses. In this study, nine novel PR genes were isolated from Lilium regale Wilson, which is a wild lily species of China with high-level resistance to the soilborne fungal pathogen Fusarium oxysporum f. sp. lilii, and homology analysis classified them into the PR10 family. These novel LrPR10s were clustered together with PR10s from monocotyledons in a phylogenetic tree, moreover, phylogenetic analysis divided the nine LrPR10s into two groups. The main-chain conformation and folding patterns of the LrPR10s were highly conserved with other plant PR10s. The expression patterns of the nine LrPR10s in L. regale during normal development were examined by QRT-PCR, and the transcription levels of the LrPR10s were relatively high in roots. Furthermore, QRT-PCR analysis indicated that the expression levels of LrPR10-1, LrPR10-2, LrPR10-5, LrPR10-6, and LrPR10-7 in L. regale roots were up-regulated by two or more stress-related signaling molecules including salicylic acid, jasmonic acid, ethylene, and H2O2, while the other four LrPR10s were repressed by these four signaling molecules. In addition, five members of the LrPR10 gene family including LrPR10-2, LrPR10-4, LrPR10-5, LrPR10-6, LrPR10-7, and LrPR10-9 were strongly induced by F. oxysporum in resistant L. regale compared with the susceptible Lilium Oriental hybrid ‘Siberia’. The other four LrPR10s were down-regulated by F. oxysporum infection. In summary, our results indicate that the members of PR10 gene family are involved in L. regale defense responses against F. oxysporum f. sp. lilii.

STUDY ON THE INTERACTION BETWEEN THEASINESIN AND BOVINE SERUM ALBUMIN BY FLUORESCENCE METHOD

Theasinesin (TS), a polymer of epigallocatechin gallate, is the main active component of tea polyphenols. Several studies indicate that tea polyphenols have extensive pharmacology activity. However, there is little research about the transportation and metabolism of tea polyphenols in vivo. Serum albumin is a most important protein serving as a depot protein and as a transport protein for many drugs and other bioactive small molecules. This study observed the interaction between TS and bovine serum albumin (BSA) by fluorescence and absorption spectroscopy. The results showed that both static and dynamic quenching occurred in the fluorescence quenching of BSA by TS. The binding sites number is 1.1845 and the binding sites may close to the tyrosine residues. The thermodynamic parameters ΔH°, ΔG°, ΔS° at temperatures 310 K were calculated 1.7 KJ, −35.4 KJ, and 0.12 KJ. The negative sign of free energy (ΔG°) means that the interaction process is spontaneous. The positive enthalpy (ΔH°) and entropy (ΔS°) values of the interaction of TS and BSA indicate that the binding is mainly entropy-driven and the enthalpy is unfavorable for it, the hydrophobic forces playing a major role in the reaction. A distance of 4.037 nm was found between donor (BSA) and acceptor (TS), obtained according to the Főrster theory of non-radiation energy transfer, which indicates that the energy transfer from BSA to TS occurs with high probability. The results of synchronous fluorescence spectra and UV–vis absorption spectra showed that the peptide strands of BSA molecules extended more and the hydrophobicity decreased with the addition of TS.

MAPK-mediated auxin signal transduction pathways regulate the malic acid secretion under aluminum stress in wheat ( Triticum aestivum L.)

An isobaric tags for relative and absolute quantitative (iTRAQ)-based quantitative proteomic approach was used to screen the differentially expressed proteins during control treatment (CK), aluminum (Al) and Al+ indole-3-acetic acid (IAA) treatment of wheat lines ET8 (Al-tolerant). Further, the the expression levels of auxin response factor (ARF), Aux/IAA, Mitogen activated protein kinase (MAPK) 2c, and MAPK1a were analyzed. Results showed that 16 proteins were determined to be differentially expressed in response to Al and IAA co-treatment compared with Al alone. Among them, MAPK2c and MAPK1a proteins displayed markedly differential expression during the processes. The expression of ARF2 was upregulated and Aux/IAA was downregulated by Al, while both in concentration- and time-dependent manners. Western-blot detection of MAPK2c and MAPK1a indicated that Al upregulated MAPK2c and downregulated MAPK1a in both concentration- and time-dependent manners. Exogenous IAA could promote the expression of MAPK2c, but inhibit the expression of MAPK1a in the presence/absence of Al. These findings indicated that IAA acted as one of the key signaling molecule controls the response mechanism of wheat malic acid efflux to Al stress through the suppression/activation of Aux/IAA and ARFs, and the activity of MAPK2c and MAPK1a were positively or negatively regulated.