A systematic identification of cold tolerance genes in peanut using yeast functional screening system

Peanut(Arachis hypogaea L.)is a thermophilic crop,and low temperature leads to a significant reduction in annual yields.Despite a few cold tolerant germplasms or cultivars have been discovered and developed,molecular mechanisms governing peanut cold tolerance is poorly understood.Identification of keys genes involved in cold tolerance is the first step to address the underlying mechanism.In this study,we isolated and characterized 157 genes with potentials to confer cold tolerance in peanut by using a yeast functional screening system.GO(Gene ontology)and KEGG(Kyoto encyclopedia of genes and genomes)enrichment analysis of these genes revealed that ribosome and photosynthesis proteins might play essential roles in peanut cold response.Transcriptome results indicated that 60 cold tolerance candidate genes were significantly induced or depressed by low temperature.qRT-PCR analysis demonstrated that several candidate genes could be also regulated by salt or drought stress.Individual overexpression of two UDP-glycosyltransferases(AhUGT2 and AhUGT268)in transgenic yeast cells could enhance their tolerance to multiple abiotic stress.In conclusion,this study advances our understanding of the mechanisms associated with the cold stress responses in peanut,and offers valuable gene resources for genetic improvement of abiotic stress tolerance in crops.

Yeast Two-Hybrid Screening for Proteins that Interact with the Extracellular Domain of Amyloid Precursor Protein

Alzheimer＇s disease（AD） is a neurodegenerative disorder in which amyloid b plaques are a pathological characteristic. Little is known about the physiological functions of amyloid b precursor protein（APP）. Based on its structure as a type I transmembrane protein, it has been proposed that APP might be a receptor, but so far, no ligand has been reported. In the present study, 9 proteins binding to the extracellular domain of APP were identified using a yeast two-hybrid system. After confirming the interactions in the mammalian system, mutated PLP1,members of the FLRT protein family, and KCTD16 were shown to interact with APP. These proteins have been reported to be involved in Pelizaeus-Merzbacher disease（PMD） and axon guidance. Therefore, our results shed light on the mechanisms of physiological function of APP in AD, PMD, and axon guidance.

Mutagenic and Estrogenic Effects of Organic Compounds in Water Treated by Different Processes: A Pilot Study

Objective In this study, a pilot-scale investigation was conducted to examine and compare the biotoxicity of the organic compounds in effluents from five treatment processes (P1-P5) where each process was combination of preoxidation (O3), coagulation, sedimentation, sand filtration, ozonation, granular activated carbon, biological activated carbon and chlorination (NaClO). Methods Organic compounds were extracted by XAD-2 resins and eluted with acetone and dichlormethane (DCM). The eluents were evaporated and redissolved with DMSO or DCM. The mutagenicity and estrogenicity of the extracts were assayed with the Ames test and yeast estrogen screen (YES assay), respectively. The organic compounds were detected by GC-MS. Results The results indicated that the mutation ratio (MR) of organic compounds in source water was higher than that for treated water. GC-MS showed that more than 48 organic compounds were identified in all samples and that treated water had significantly fewer types and concentrations of organic compounds than source water. Conclusion To different extents, all water treatment processes could reduce both the mutagenicity and estrogenicity, relative to source water. P2, P3, and P5 reduced mutagenicity more effectively, while P1 reduced estrogenicity, most effectively. Water treatment processes in this pilot plant had weak abilities to remove Di-n-butyl phthalate or 1, 2-Benzene dicarboxylic acid.

In Vitro Biossay for Assessing Estrogenic Activity of Sewage Treatment Works

The estrogenic activity of influents and effluents of five municipal sewage treatment works （STWs） in Nanjing was investigated. The water samples were enriched by solid phase extraction, then eluted with different polar solvents, and f＇mally three fractionated extracts were obtained. Estrogen receptor recombinant yeast screen was used to evaluate the estrogenic activity of individual fractionated extracts and their mixture. The results show that the influents and effluents of the two STWs mainly receiving industrial wastewater have no obvious estrogenic activity, and the water samples from the other three STWs mainly receiving domestic wastewater show different degrees of estrogenic activity. The total estrogenic concentration ranged from 171. 52 ng/L EEQ （E2 - equivalent concentration） to 1 008.37 ng/L EEQ in the influents, and from ND （not detected） to 36.3 ng/L EEQ in the effluents. By comparing the results of different fractionated extracts, the methanol and dichloromethane extracts of the water samples show higher estrogenic activity, while the hexane extracts very low. The （xeno）estrogens from domestic wastewater could be removed obviously by activated sludge treatment units. However, the effluents of STWs in Nanjing still have chemical contaminants capable of binding to estrogen receptors.

Assessment of estrogen disrupting potency in animal foodstuffs of China by combined biological and chemical analyses

Food has been documented as one of major routes for human exposure to environmental estrogens（EEs）, but information on the occurrence of EEs in animal foodstuffs is still scarce.This study analyzed estrogenic activity in 16 types of animal foodstuffs（n = 142） collected from four cities（Wuhan, Guangzhou, Wenzhou and Yantai） of China by combined yeast estrogen screen（YES） bioassay and chemical analysis. By bioassay, all samples＇ extracts were found to induce estrogenic activities and the bioassay-derived 17β-estradiol equivalent（EEQbio） ranged from 8.29 to 118.32 ng/g. In addition, the samples were analyzed by liquid chromatography coupled to tandem mass spectrometry for further chemical analysis. 17β-Estradiol was found in all samples in this survey at levels of 0.44 to 15.04 ng/g.All samples had 33.1% detection rate of 17α-ethinylestradiol（EE2）, and the maximum concentration was 2.80 ng/g. Bisphenol A and 4-nonylphenols were detected in 83.8% and83.1% of samples, with concentrations up to 12.56 ng/g and 35.76 ng/g, respectively.However, the concentrations of estrone, diethylstilbestrol and 4-t-octylphenol were found to be below the limit of detection. A comparison of EEQbiomeasured from the YES assay and EEQchemcalculated from chemical analysis showed good correlation（R2= 0.84）. Based on the results, the YES assay can be used as a rapid pre-screening method for monitoring the levels of estrogenic activity in large numbers of animal foodstuffs, and chemical analysis used in combination can be used for the identification of specific EEs.