Characterization of an Oxidative Stress Inducible Nonspecific Lipid Transfer Protein Coding cDNA and its Promoter from Drought Tolerant Plant <Emphasis Type="Italic">Prosopis juliflora</Emphasis>

Abiotic stress is the principal cause of crop failure worldwide. Prosopis juliflora is a hardy plant reported to be tolerant to drought, salinity, extremes of soil pH, and heavy metal stress. Here, we report the characterization of a cDNA clone for a putative nonspecific lipid transfer protein (Pj LTP1) found abundantly in a drought stressed leaf cDNA library of P. juliflora and its promoter. A multiple sequence analysis of Pj LTP1 with well-established and officially designated nonspecific lipid transfer proteins allergens revealed high similarity at the amino acid level. Northern analysis of Pj LTP1 in P. juliflora leaves under oxidative stress revealed steady upregulation at the time points analyzed. A 929-bp fragment was isolated from the 5′ end of Pj LTP1, and transient reporter gene expression studies revealed it to be a functional promoter. Several cis-acting elements previously reported to function in stress response were found in this promoter. The possible reasons for changes in gene expression during stress in relation to the host plant’s stress tolerance mechanisms are discussed.     

Over-expression of a Rab family GTPase from phreatophyte <Emphasis Type="Italic">Prosopis juliflora</Emphasis> confers tolerance to salt stress on transgenic tobacco

Plant growth and productivity are adversely affected by various abiotic and biotic stress factors. In our previous study, we used Prosopis juliflora, an abiotic stress tolerant tree species of Fabaceae, as a model plant system for isolating genes functioning in abiotic stress tolerance. Here we report the isolation and characterization of a Rab family GTPase from P. juliflora (Pj Rab7) and the ability of this gene to confer salt stress tolerance in transgenic tobacco. Northern analysis for Pj Rab7 in P. juliflora leaf tissue revealed up-regulation of this gene under salt stress under the concentrations and time points analyzed. Pj Rab7 transgenic tobacco lines survived better under conditions of 150 mM NaCl stress compared to control un-transformed plants. Pj Rab7 transgenic plants were found to accumulate more sodium than control plants during salt stress. The results of our studies could be used as a starting point for generation of crop plants tolerant to abiotic stress.     

Activity of defense related enzymes and gene expression in pigeon pea (Cajanus cajan) due to feeding of Helicoverpa armigera larvae

The biochemical and molecular basis of the defense in a mild tolerant (ICPL-332) and susceptible (ICPL-87) cultivars of pigeon pea (Cajanus cajan) due to Helicoverpa. armigera infestation was studied. We found that feeding by the larvae generated H₂O₂ in a localized manner and activity was observed upto 12?h (hrs) of with a sharp decline within 24?h. Similarly, PPO activity was also detected till 12?h of treatment, which decreased after 24?h of feeding by larvae. The activity of trypsin inhibitor was detected in all the treatments when assayed at 12 and 24?h after larval feeding. The expression of defense genes like the Pre-hevein-like protein PR-4 precursor (PR-4), protease inhibitor/seed storage/LTP family protein (Ltp) were significantly up-regulated in ICPL-332 upon infestation after 12?h as compared to ICPL-87, whereas the endo 1, 4 -β-glucanase (Kor-1) gene was expressed in both the cultivars after 24?h of infestation. Both the cultivars varied with respect to the induction of defense-related genes during larval feeding, both the PR-4 and Ltp genes appeared to be important for defense against H. armigera in pigeon pea. Thus, the present study revealed an insight of herbivore-induced biochemical and molecular changes in pigeon pea.     

松叶猪毛菜NADP-苹果酸酶基因家族及启动子克隆分析北大核心CSCD

NADP-苹果酸酶(NADP-ME)是C_(4)植物的关键光合酶,在生物和非生物胁迫中发挥了重要的作用。为了进一步研究该酶编码基因的功能,该研究以典型荒漠C_(3)-C_(4)中间型植物松叶猪毛菜为研究对象,在克隆得到NADP-ME家族基因序列的基础上,研究其表达部位及在非生物胁迫下的表达模式,并克隆其启动子序列分析响应非生物胁迫的元件差异。结果表明:(1)成功获得松叶猪毛菜3个NADP-MEs,命名为SaNADP-ME1、SaNADP-ME2和SaNADP-ME4,CDS序列长度分别为1755、1758和1941 bp。(2)SaNADP-ME1主要在根中表达,SaNADP-ME2和SaNADP-ME4主要在叶中表达;在ABA、NaCl、NAHCO_(3)和PEG_(6000)胁迫下松叶猪毛菜幼苗中3个NADP-MEs均可被诱导表达,且SaNADP-ME2和SaNADP-ME4的响应表达模式相似。(3)成功克隆得到SaNADP-ME1、SaNADP-ME2和SaNADP-ME4启动子区域2351、1655和2887 bp。生物信息学分析发现它们都含有基本启动子元件以及响应外界刺激的元件。     

Multiobjective H2/H∞ synthetic gene network design based on promoter libraries

Some current promoter libraries have been developed for synthetic gene networks. But an efficient method to engineer a synthetic gene network with some desired behaviors by selecting adequate promoters from these promoter libraries has not been presented. Thus developing a systematic method to efficiently employ promoter libraries to improve the engineering of synthetic gene networks with desired behaviors is appealing for synthetic biologists.In this study, a synthetic gene network with intrinsic parameter fluctuations and environmental disturbances in vivo is modeled by a nonlinear stochastic system. In order to engineer a synthetic gene network with a desired behavior despite intrinsic parameter fluctuations and environmental disturbances in vivo, a multiobjective H₂/H_∞ reference tracking (H₂ optimal tracking and H_∞ noise filtering) design is introduced. The H₂ optimal tracking can make the tracking errors between the behaviors of a synthetic gene network and the desired behaviors as small as possible from the minimum mean square error point of view, and the H_∞ noise filtering can attenuate all possible noises, from the worst-case noise effect point of view, to achieve a desired noise filtering ability. If the multiobjective H₂/H_∞ reference tracking design is satisfied, the synthetic gene network can robustly and optimally track the desired behaviors, simultaneously.First, based on the dynamic gene regulation, the existing promoter libraries are redefined by their promoter activities so that they can be efficiently selected in the design procedure. Then a systematic method is developed to select an adequate promoter set from the redefined promoter libraries to synthesize a gene network satisfying these two design objectives. But the multiobjective H₂/H_∞ reference tracking design problem needs to solve a difficult Hamilton–Jacobi Inequality (HJI)-constrained optimization problem. Therefore, the fuzzy approximation method is employed to simplify the HJI-constrained optimization problem to an equivalent linear matrix inequality (LMI)-constrained optimization problem, which can be easily solved by selecting an adequate promoter set from the redefined promoter libraries using the LMI toolbox in Matlab.Based on the confirmation of in silico design examples, we can select an adequate promoter set from the redefined promoter libraries to achieve the multiobjective H₂/H_∞ reference tracking design. The proposed method can reduce the number of trial-and-error experiments in selecting an adequate promoter set for a synthetic gene network with desired behaviors. With the rapid increase of promoter libraries, this systematic method will accelerate progress of synthetic biology design.     

Changes in antioxidant enzyme activities in detached leaves of cucumber exposed to chilling

We studied changes in biochemical and physiological status, level of oxidative damage, and antioxidant enzyme activities in detached leaves of cucumber plants (Cucumis sativus L. cv. Pyunggangnaebyungsamchuk) that were exposed to a low temperature (4°C). Chlorophyll fluorescence (F_v/F_m) declined during the chilling treatment, but was slowly restored after the tissues were returned to 25°C. Likewise, the fluorescence quenching coefficient and relative water content decreased during the stress period, but then increased during recovery. In contrast, we detected a significant rise in protein and hydrogen peroxide contents in the chilled leaves, as well as higher activities for superoxide dismutase, ascorbate peroxidase, peroxidase, and glutathione reductase. However, the level of catalase decreased not only during chilling but also after 24 h of recovery. These results indicate that exposure to low temperatures acts as an oxidative stress. Moreover, we propose that a regulating mechanism exists in the detached cucumber leaves and contains an antioxidant defense system that induces active oxygen species, thereby alleviating the effects of chilling stress within 12 h.     

Release of 6-KETO-PGF1α and thromboxane B2 in late appearing cardioprotection induced by the stable PGI analogue: 7-OXO-PGI

We have shown earlier that prostacylin (PGI₂) and its stable analogue: 7-oxo-prostacyclin(7-OXO) may induce a prolonged, late appearing (24–48 h after drug administration), dose dependent protection of the heart from harmful consequences of a subsequent severe ischaemic stress, such as myocardial ischaemia, life-threatening ventricular arrhythmias and early ischaemic morphological changes. In an other study we observed that a similar but shortlived (less than 1 h) cardioprotection, induced by preconditioning brief coronary artery occlusions, is greatly reduced by blockade of the cyclooxygenase pathway, suggesting that prostanoids might play a role in this shortlasting protection.Objective of our present study was to elucidate the importance of some arachidonic acid (AA) metabolites, such as PGI₂ and thromboxane A₂ (TXA₂) in the mechanism of the late appearing, prolonged cardioprotection. Estimation of the metabolites: 6-keto-PGF₁ (6-KETO) and thromboxane B₂ (TXB₂) was made from the perfusate of isolated Langendorff hearts of guinea-pigs pretreated with 50 g/kg 7-OXO, 24 and 48 h before preparation. Pretreatment alone produced a slight, but significant elevation of 6-KETO (from 206±11 to 284±19 pg/ml/min after 24 h, and to 261±18 pg/ml/min after 48 h). No change was seen in TXB₂ production. Global ischaemia for 25 min (followed by 25 min reperfusion) markedly increased the release of both AA metabolites; maximal values were observed in the third min of reperfusion (6-KETO from 206±11 to 1275±55 pg/ml/min and TXB₂ from 29±4 to 172±12 pg/ml/min). All values returned to the preischaemic level by the 25th min of reperfusion. Ischaemic increase in 6-KETO level was significantly higher in the perfusate of hearts from pretreated animals (1507±73 pg/ml/min after 24 h, and 1398±54 pg/ml/min after 48 h) that in those of untreated controls. There was no difference in TXB₂ values. Thus both basal and ischaemic release of PGI₂ increased 24 and 48 h after pretreatment with 7-OXO but not TXA₂ production. Results suggest that endogenous prostanoids might play a role in late appearing cardioprotection.     

Cloning of differential expression fragments in cauliflower after Xanthomonas campestris inoculation

A near isogenic line (NIL) of Brassica oleracea var. botrytis with resistant and susceptible lines C712 and C731, was used in this study. More than 100 differentially expressed cDNA fragments were obtained from black rot resistant cauliflower plants obtained using cDNA-amplified fragment length polymorphism (AFLP) after infection with the pathogen. Thirteen of these fragments were cloned and subjected to reverse Northern blot analysis using both infected and control cDNA pools. Two positive clones, M2 and M6, were isolated. Northern dot blot and Northern blot analyses showed that M2 was constitutively expressed, whereas M6 contained a gene that was differentially expressed during pathogen infection. Moreover, M6 cDNA fragment was also highly expressed 16–24 h after H₂O₂ treatment. Southern blots showed that M6 is a single copy gene in the cauliflower genome, and encodes a protein with 84 % homology to gene on Arabidopsis chromosome 1. The deduced M6 protein has 91 % positive homology with the Arabidopsis 2A6 protein, which regulates ethylene synthesis; 76 % homology with a 1-aminocyclopropane-1-carboxylate oxidase (ACO), the last enzyme in ethylene synthesis; and 70 % homology with an ethylene induced DNA binding factor. These results suggest that M6 gene fragment is a new H₂O₂ downstream defense related gene fragment and can be induced by Xanthomonas campestris pv. campestris and H₂O₂.     

Effects of light and hydrogen peroxide on gene expression of newly identified antioxidant enzymes in the harmful algal bloom species Chattonella marina

ABSTRACT

Antioxidant enzymes are essential proteins that maintain cell proliferation potential by protecting against oxidative stress. They are present in many organisms including harmful algal bloom (HAB) species. We previously identified the antioxidant enzyme 2-Cys peroxiredoxin (PRX) in the raphidophyte Chattonella marina. This enzyme specifically decomposes a hydrogen peroxide (H₂O₂). PRX is the only antioxidant enzyme so far identified in C. marina. This study used mRNA-seq, using Trinity assemble and blastx for annotation, to identify a further five antioxidant enzymes from C. marina: Cu Zn superoxide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX) and thioredoxin (TRX). In the gene expression analysis of six enzymes (Cu/Zn-SOD, GPX, CAT, APX, TRX and PRX) using light-acclimated (100 μmol photons m^?2 s^?1) C. marina cells, only PRX gene expression levels were significantly increased by strong light irradiation (1000 μmol photons m^?2 s^?1). H₂O₂ concentration and scavenging activity were also increased and significantly positively correlated with PRX gene expression levels. In dark-acclimated cells, expression levels of all antioxidant enzymes except APX were significantly increased by light irradiation (100 μmol photons m^?2 s^?1). Expression decreased the following day, with the exception of PRX expression. With the exception of CAT, gene expression of antioxidant enzymes was not significantly induced by artificial H₂O₂ treatment, although average gene expression levels were slightly increased in some enzymes. Thus, we suggest that light is the main trigger of gene expression, but the resultant oxidative stress is also a possible factor affecting the gene expression of antioxidant enzymes in C. marina.     

Expression of the mosquitocidal cryIVB gene under the control of different promoters in Bacillus sphaericus 2362 and acrystalliferous Bacillus thuringiensis subsp. israelensis c4Q2-72

The 3.7 kb XbaI fragment harbouring the cryIVB gene which encoded a 130 kDa mosquitocidal toxin protein from Bacillus thuringiensis subsp. israelensis (B.t.i.) was placed downstream to the cat-86 gene promoter (P _cat-86, spore stage specific expression) or bgaB gene promoter (P _bgaB , vegetative stage specific expression). The constructs were subcloned into pBC16 to obtain pBTC3 and pBTC6, respectively. Both plasmids and the other construct, pBTC1 were successfully transferred into B. thuringiensis subsp. israelensis c4Q2-72 and B. sphaericus 2362. Western blot analysis showed that P _bgaB in front of P _cryIVB could enable cells to produce a 130 kDa protein from the vegetative stage (4 h) whereas those with P _cat-86 could not. The positive detection of 130 kDa crystal protein during the vegetative stage (4 h) by Western blot analysis indicated the vegetative-stage-specific expression of P _bgaB , while the 130 kDa crystal protein produced from cryIVB gene under control of P _cat-86 was detected only at 48 h. The strong activity of P _bgaB , together with P _cryIVB within pBTC6 in both bacterial hosts was also shown by the toxicity assay against Aedes aegypti larvae (B.t.i. c4Q2-72, 5.6 ± 3.6 × 10² c.f.u./ml; B. sphaericus 2362, 5.4 ± 2.5 × 10² c.f.u./ml) which were 100-fold and 10-fold more toxic to such larvae when compared with pBTC3 (P _cat-86 together with P _cryIVB ) and pBTC1 (contained only its self promoter) in the same bacterial host strains, respectively. The plasmid pBTC6 is not stable in either Bacillus host.