共享型分析化学混合式教学实践

依托超星SPOC平台建设《分析化学》辽宁省混合式一流课程,并通过辽宁高等教育智慧教育平台开展共享型的教学实践。为建设混合式一流课程,建成了包含课程基本知识、学科发展前沿、相关知识拓展等在内的资源平台,引入案例教学、翻转课堂、课程思政等方式完善教学方法,从知识传授、能力培育和价值养成等维度实现育人目标。为开展共享型的教学实践,与其他高校发展教研共同体、创新教研形态,实现课程内容共建和教学方法共享,构建地方高校分析化学课程共建共享平台。

Heat-inducible SlWRKY3 confers thermotolerance by activating the SlGRXS1 gene cluster in tomato

High temperature stress is one of the major environmental factors that affect the growth and development of plants. Although WRKY transcription factors play a critical role in stress responses, there are few studies on the regulation of heat stress by WRKY transcription factors,especially in tomato. Here, we identified a group I WRKY transcription factor, SlWRKY3, involved in thermotolerance in tomato. First, SlWRKY3 was induced and upregulated under heat stress. Accordingly, overexpression of SlWRKY3 led to an increase, whereas knock-out of SlWRKY3 resulted in decreased tolerance to heat stress. Overexpression of SlWRKY3 accumulated less reactive oxygen species(ROS), whereas knock-out of SlWRKY3 accumulated more ROS under heat stress. This indicated that SlWRKY3 positively regulates heat stress in tomato. In addition,SlWRKY3 activated the expression of a range of abiotic stress-responsive genes involved in ROS scavenging, such as a SlGRXS1 gene cluster.Further analysis showed that SlWRKY3 can bind to the promoters of the SlGRXS1 gene cluster and activate their expression. Collectively, these results imply that SlWRKY3 is a positive regulator of thermotolerance through direct binding to the promoters of the SlGRXS1 gene cluster and activating their expression and ROS scavenging.

Utilizing resequencing big data to facilitate Brassica vegetable breeding:tracing introgression pedigree and developing highly specific markers for clubroot resistance

Clubroot caused by Plasmodiophora brassicae is a devastating disease of Cruciferous crops.Developing cultivars with clubroot resistance(CR)is the most effective control measure.For the two major Brassica vegetable species B.rapa and B.oleracea,several commercial cultivars with unclear CR pedigrees have been intensively used as CR donors in breeding.However,the continuous occurrence of CR-breaking makes the CR pedigree underlying these cultivars one of the breeders'most urgent concerns.The complex intraspecific diversity of these two major Brassica vegetables has also limited the applicability of CR markers in different breeding programs.Here we first traced the pedigree underlying two kinds of CR that have been widely applied in breeding by linkage and introgression analyses based on public resequencing data.In B.rapa,a major locus CRzi8 underlying the CR of the commercial CR donor‘DegaoCR117’was identified.CRzi8 was further shown to have been introgressed from turnip(B.rapa ssp.rapifera)and that it carried a potential functional allele of Crr1a.The turnip introgression carried CRb^(c),sharing the same coding sequence with the CRb that was also identified from chromosome C07 of B.oleracea CR cultivars with different morphotypes.Within natural populations,variation analysis of linkage intervals of CRzi8,PbBa8.1,CRb,and CRb^(c)yielded easily resolved InDel markers(>20 bp)for these fundamental CR genes.The specificity of these markers was tested in diverse cultivars panels,and each exhibited high reliability in breeding.Our research demonstrates the value of the practice of applying resequencing big data to solve urgent concerns in breeding programs.

基于案例分析与翻转课堂的分析化学实验混合式教学实践

设计并实践了新的分析化学实验教学方法和考评方法。将案例分析与翻转课堂相结合,进行线上线下混合式教学。课前教师选择案例并设计问题,学生分组预习;课堂上学生讨论分析为主,教师查漏补缺为辅;课后学生撰写报告上传至线上平台,教师批阅并反馈问题。新的考评方法中,突出对学生表达能力、逻辑思维能力、团队协作能力、实验操作水平、实验台与实验室整洁水平的考查评价。实践表明,新的教学方法有助于提升学生的表达能力和实验操作水平,激发学生学习的积极性和挑战高难度学习任务的热情。新的考评方法为评价学生能力提供了可执行的标准。

Neuroprotective effects of human bone marrow mesenchymal stem cells against cerebral ischemia are mediated in part by an anti-apoptotic mechanism

Transplantation of human bone marrow mesenchymal stem cells(hMSCs) stands as a potent stroke therapy, but its exact mechanism remains unknown. This study investigated the anti-apoptotic mechanisms by which hMSCs exert neuroprotective effects on cerebral ischemia. Primary mixed cultures of rat neurons and astrocytes were cultured and exposed to oxygen-glucose deprivation. A two-hour period of "reperfusion" in standard medium and normoxic conditions was allowed and immediately followed by hMSCs and/or Bcl-2 antibody treatment. Cell viability of primary rat neurons and astrocytes was determined by 3-(4,5-dimethylthianol-2-yl)-2,5 diphenyl tetrazolium bromide and trypan blue exclusion methods. hMSC survival and differentiation were characterized by immunocytochemistry, while the concentration of Bcl-2 in the supernatant was measured by enzyme-linked immunosorbent assay to reveal the secretory anti-apoptotic function of hMSCs. Cultured hMSCs expressed embryonic-like stem cell phenotypic markers CXCR4, Oct4, SSEA4, and Nanog, as well as immature neural phenotypic marker Nestin. Primary rat neurons and astrocytes were protected from oxygen-glucose deprivation by hMSCs, which was antagonized by the Bcl-2 antibody. However, Bcl-2 levels in the supernatants did not differ between hMSCand non-treated cells exposed to oxygen-glucose deprivation. Neuroprotective effects of hMSCs against cerebral ischemia were partially mediated by the anti-apoptotic mechanisms. However, further studies are warranted to fully elucidate this pathway.

A CCAAT-binding factor,SlNFYA10,negatively regulates ascorbate accumulation by modulating the D-mannose/L-galactose pathway in tomato

Ascorbic acid(AsA),an important antioxidant and growth regulator,and it is essential for plant development and human health.Specifically,humans have to acquire AsA from dietary sources due to their inability to synthesize it.The AsA biosynthesis pathway in plants has been elucidated,but its regulatory mechanism remains largely unknown.In this report,we biochemically identified a CCAAT-box transcription factor(SlNFYA10)that can bind to the promoter of SlGME1,which encodes GDP-Man-3’,5’-epimerase,a pivotal enzyme in the D-mannose/L-galactose pathway.Importantly,SlNFYA10 simultaneously binds to the promoter of SlGGP1,a downstream gene of SlGME1 in the Dmannose/L-galactose pathway.Binding assays in yeast and functional analyses in plants have confirmed that SlNFYA10 exerts a negative effect on the expression of both SlGME1 and SlGGP1.Transgenic tomato lines overexpressing SlNFYA10 show decreased levels of SlGME1 and SlGGP1 abundance and AsA concentration in their leaves and fruits,accompanied by enhanced sensitivity to oxidative stress.Overall,SlNFYA10 is the first CCAAT-binding factor identified to date to negatively regulate the AsA biosynthetic pathway at multiple sites and modulate plant responses to oxidative stress.

Response of coralline algae Porolithon onkodes to elevated seawater temperature and reduced pH

Coralline algae(CA),a type of primary calcifying producer presented in coastal ecosystems,are considered one of the highly sensitive organisms to marine environmental change.However,experimental studies on coralline algae responses to elevated seawater temperature and reduced pH have documented either contradictory or opposite results.In this study,we analysed the growth and physiological responses of coralline algae Porolithon onkodes to the elevated temperature(30.8°C)and reduced pH(7.8).The aim of this analysis was to observe the direct and combined effects,while elucidating the growth and photosynthesis in this response.It was demonstrated that the algae thallus growth rate and photosynthesis under elevated temperature were depressed by 21.5%and 14.9%respectively.High pCO2 enhanced the growth and photosynthesis of the thallus at ambient temperature,while they were deceased when both temperature and pCO2 were elevated.CA is among the most sensitive organisms to ocean acidification(OA)because of their precipitate high Mg-calcite.We hypothesize that coralline algae could increase their calcification rate in order to counteract the effects of moderate acidification,but offset by the effect of elevated temperature.Accordingly,our results also support the conclusion that global warming(GW)is a stronger threat to algal performance than OA.Our findings are also proposed that coralline algae may be more resilient under OA than GW.

Multiple-model GWAS identifies optimal allelic combinations of quantitative trait loci for malic acid in tomato

Malic acid(MA)is an important flavor acid in fruits and acts as a mediator in a series of metabolic pathways.It is important to understand the factors affecting MA metabolism for fruit flavor improvement and to understand MA-mediated biological processes.However,themetabolic accumulation of MA is controlled by complex heredity and environmental factors,making it difficult to predict and regulate the metabolism of MA.In this study,we carried out a genome-wide association study(GWAS)on MA using eight milestone models with two-environment repeats.A series of associated SNP variations were identified from the GWAS,and 15 high-confidence annotated geneswere further predicted based on linkage disequilibrium and lead SNPs.The transcriptome data of candidate geneswere explored within different tomato organs as well as various fruit tissues,and suggested specific expression patterns in fruit pericarp.Based on the genetic parameters of population differentiation and SNP distribution,tomato MA content has been more influenced by domestication sweeps and less affected by improvement sweeps in the long-term history of tomato breeding.In addition,genotype×environment interaction might contribute to the difference in domestication phenotypic data under different environments.This study provides new genetic insights into how tomato changed its MA content during breeding and makes available function-based markers for breeding by marker-assisted selection.

Interactions between ShPP2-1, an F-box family gene, and ACR11A regulate cold toleranee of tomato

There is a critical need to identify germplasm resources and genes that promote cold tolerance of tomato because global tomato production is threatened by cold stress.We found that the expression of an F-box gene family member named ShPP2-1 from Solanum habrochaites is cold inducible and studied its contribution to cold tolerance.Overexpression of ShPP2-1 in cultivated tomato(AC)reduced cold tolerance by intensifying damage to cell membranes.To explore the underlying molecular mechanism,we conducted a yeast two-hybrid library screen and found that a protein containing ACT domain repeats named ACR11A interacts with PP2-1.Overexpression of SIACR11A in AC enhanced the cold tolerance of seedlings and germinating seeds.Cold tolerance decreased in tomato plants that overexpressed both of these genes.Additionally,we performed seed germination experiments in the cold with 177 tomato accessions and identified two alleles of SlACR11A that differ in one single-nucleotide polymorphism.We found that one of these alleles,SlACR11A G,is significantly enriched in cold-tolerant tomato plants.Taken together,our fi ndings indicate that the combination of low expression levels of PP2-1 and high expression levels of ACR11A can promote cold tolerance.These genes may therefore serve as direct targets for both genetic engineering and improvement projects that aim to enhance the cold tolerance of tomato.

The loss of function of HEL,which encodes a cellulose synthase interactive protein,causes helical and vine-like growth of tomato

Helical growth is an economical way for plant to obtain resources.The classic microtubule–microfibril alignment model of Arabidopsis helical growth involves restriction of the appropriate orientation of cellulose microfibrils appropriately in the cell walls.However,the molecular mechanism underlying tomato helical growth remains unknown.Here,we identified a spontaneous tomato helical(hel)mutant with right-handed helical cotyledons and petals but left-handed helical stems and true leaves.Genetic analysis revealed that the hel phenotype was controlled by a single recessive gene.Using map-based cloning,we cloned the HEL gene,which encodes a cellulose interacting protein homologous to CSI1 of Arabidopsis.We identified a 27 bp fragment replacement that generated a premature stop codon.Transgenic experiments showed that the helical growth phenotype could be restored by the allele of this gene from wild-type Pyriforme.In contrast,the knockout mutation of HEL in Pyriforme via CRISPR/Cas9 resulted in helical growth.These findings shed light on the molecular control of the helical growth of tomato.

Activity of Brucea javanica oil emulsion against gastric ulcers in rodents

The present study aims to investigate the gastroprotective effect of Brucea javanica oil emulsion(BJOE) in animals. Gastroprotective potential of BJOE was studied on absolute ethanol,aspirin, reserpine and restraint plus water immersion-induced gastric ulcers in mice as well as glacial acetic acid(GAA) and pyloric ligation(PL)-induced gastric ulcers in rats. Except for ulcer scores, total acidity as well as pepsin activity as for the PL-induced gastric ulcer model and ulcer incidence as for the GAA-induced gastric ulcer model were also determined. Histopathological evaluation as for aspirin, reserpine, PL-induced models was conducted. Results showed that BJOE significantly(P < 0.05) reduced ulcer index in the mouse and rat models in a dose-dependent manner. It had significant(P < 0.05) suppressive effect on total activity of gastric juice as well in PL-induced model. Histopathological examination for the stomach samples confirmed the findings in the aspirin, reserpine or PLinduced gastric lesion models, which showed relatively complete mucosa structure and less inflammation. It is concluded that BJOE could be effective on gastric ulcer in rodents and its gastroprotective activity might be related to antioxidant, anti-inflammatory ability and promote gastric mucus secreted. The results may provide beneficial basis for increasing BJOE's clinical indication in future.

Investigation of Linarinic acid and one of its derivatives against cerebral ischemia in mice

The study aims to investigate the effects of(-)-Linarinic acid(LA) and one of its derivatives(LAd) on brain injury induced by ischemia. Malonaldehyde(MDA) is determined as an index for lipid peroxidation both in vitro and vivo. Mice were pre-treated with LA and LAd for 3 d.Thereafter, they were induced to have incomplete cerebral ischemia with both bilateral carotid artery occlusion and hypotension(BCAOH). In the first part of the in vivo experiment, mice were divided into four groups: sham(control), ischemia, ischemia + LA(200 mg/kg, i.g.) and ischemia + LAd(200 mg/kg, i.g.). In the second part, the dose-response of LAd was investigated at 100, 200 and 400 mg/kg i.g., respectively. A modified neurological severity score was developed for evaluating behavioral deficits of the mice with ischemia. Brains of the mice were excised in order to determinate MDA after ischemia for 6 h. Survival time, survival rate, neurological injury score and MDA level in brains were observed. Results were: 1) The data in vitro showed that both LA and LAd could inhibit the generation of MDA. IC50 values obtained by Probit analysis were 2.9 mM for LAd and 4.88 mM for LA;2) BCAOH could significantly shorten the survival span, reduce the survival rate and cause neurological deficits,which were associated with high level of lipid hydroperoxide production in cerebral tissues;3) LAd decreased lipid peroxidation and improved the neurological outcome more than LA.It is concluded that LAd offers a better neuroprotection than LA against brain damage caused by cerebral ischemia.

A 21-bp InDel in the promoter of STP1 selected during tomato improvement accounts for soluble solid content in fruits

Domestication and improvement are important processes that generate the variation in genome and phonotypes underlying crop improvement.Unfortunately,during selection for certain attributes,other valuable traits may be inadvertently discarded.One example is the decline in fruit soluble solids content(SSC)during tomato breeding.Several genetic loci for SSC have been identified,but few reports on the underlying mechanisms are available.In this study we performed a genome-wide association study(GWAS)for SSC of the red-ripe fruits in a population consisting of 481 tomato accessions with large natural variations and found a new quantitative trait locus,STP1,encoding a sugar transporter protein.The causal variation of STP1,a 21-bp InDel located in the promoter region 1124 bp upstream of the start codon,alters its expression.STP1 Insertion accessions with an 21-bp insertion have higher SSC than STP1Deletion accessions with the 21-bp deletion.Knockout of STP1 in TS-23 with high SSC using CRISPR/Cas9 greatly decreased SSC in fruits.In vivo and in vitro assays demonstrated that ZAT10-LIKE,a zinc finger protein transcription factor(ZFP TF),can specifically bind to the promoter of STP1Insertion to enhance STP1 expression,but not to the promoter of STP1Deletion,leading to lower fruit SSC in modern tomatoes.Diversity analysis revealed that STP1 was selected during tomato improvement.Taking these results together,we identified a naturally occurring causal variation underlying SSC in tomato,and a new role for ZFP TFs in regulating sugar transporters.The findings enrich our understanding of tomato evolution and domestication,and provide a genetic basis for genome design for improving fruit taste.

SIRCM1,which encodes tomato Lutescent1,is required for chlorophyll synthesis and chloroplast development in fruits

In plants,chloroplasts are the sites at which photosynthesis occurs,and an increased abundance of chloroplasts increases the nutritional quality of plants and the resultant color of fruits.However,the molecular mechanisms underlying chlorophyll synthesis and chloroplast development in tomato fruits remain unknown.In this study,we isolated a chlorophyll-de fi cient mutant,reduced chlorophyll mutant 1(rcml),by ethylmethanesulfonate mutagenesis;this mutant produced yellowish fruits with altered chloroplast development.MutMap revealed that Solyc08g005010 is the causal gene underlying the rcm1 mutant phenotype.A single-nucleotide base substitution in the second exon of SIRCM1 results in premature termination of its translated protein.SIRCM1 encodes a chloroplast-targeted metalloendopeptidase that is orthologous to the BCM1 protein of Arabidopsis and the stay-green G protein of soybean(Glycine max L.Merr.).Notably,the yellowish phenotype of the lutescent1 mutant can be restored with the allele of SlRCM1 from wild-type tomato.In contrast,knockout of SlRCM1 by the CRISPR/Cas9 system in Alisa Craig yielded yellowish fruits at the mature green stage,as was the case for lutescent1.Amino acid sequence alignment and functional complementation assays showed that SlRCM1 is indeed Lutescent1.These fi ndings provide new insights into the regulation of chloroplast development in tomato fruits.

Toxic Effects of Micro-plastics on Zebrafish Embryos

The large plastic produced by human production and life is degraded into micro-plastics in the environment. Micro-plastics are harmful to organisms and the environment. In order to determine the hazards of micro-plastic pollution, zebrafish embryos were used as the tested organism, and 0.6-1.0 μm polystyrene micro-plastic was used to carry out embryo exposure experiment. The toxic effects of polystyrene plastic particles on zebrafish embryos were investigated comprehensively, and the toxicity was evaluated. The results of this study showed that 0.6-1.0 μm polystyrene micro-plastic had certain toxicity on zebrafish embryos, and it had a lethal effect when the concentration reached 250 mg/L; 25 mg/L 0.6-1.0 μm polystyrene particles can cause cyrtosis and pericardial edema of zebrafish embryos and other non-lethal toxic effects; and 250 and 1 000 mg/L exposure concentration can slow down heart rate. A hazard evaluation index system of polystyrene particles to zebrafish embryos was established, and it is determined that the exposure to 250 mg/L or lower concentration of polystyrene particles has slight hazard.

Effect of shading on ascorbic acid accumulation and biosynthetic gene expression during tomato fruit development and ripening

The purpose of the current study was to determine the effect of leaf shading,fruit shading,and a combination of both,on the accumulation of ascorbic acid(AsA)and the expression levels of AsA biosynthetic genes at the immature green,mature green,breaker,and red ripe stages of Ailsa craig tomato during fruit development.Shading(72%reducing of light intensity)imposed on the leaves significantly reduced AsA content and AsA biosynthetic gene expression in the fruits.Leaf shading,fruit shading,and a combination of both significantly decreased the amount of total AsA and reduced AsA to a range of 18.5%−31.5%at mature green,breaker,and red ripe stages of tomato fruits,with no significant change at the immature green stage of fruits.Moreover,reducing the light intensity in tomato leaves,fruits or both resulted in reduced expression of most AsA biosynthetic genes in the fruits,except for PMM,cAPX,tAPX,and APX7 genes under leaf shading,GPI,PMI,PMM,GP1,GP2,cAPX,and tAPX genes under fruit shading,and PMM,cAPX,APX1,and APX7 genes under both shading.The expression level of GMP,GP1,and GalDH showed an upregulation at the red ripe stage in fruits with leaf shading,and also an up-regulation at the immature green and red ripe stages with both shading.Furthermore,positive correlations between expression of AsA biosynthetic genes and AsA accumulation were recorded under leaf shading,fruit shading,and both types of shading,while a negative correlation was recorded under normal conditions without shading.

Cyclin gene SlCycB1 alters plant architecture in association with histone H3.2 in tomato

Cell cycle regulation plays a critical role in plant growth and development.In this study,the role of a tomato cell cycle gene SlCycB1 has been characterized.Expression analysis revealed that SlCycB1 was mostly expressed in stem,root,and leaves,with relative lower expression in flower and fruit.Tomato plants overexpressing SlCycB1 exhibited a reduction in cell number and increased cell size leading to the growth retardation.Furthermore,yeast two-hybrid analysis and bimolecular fluorescence complementation revealed that SlCycB1 interacted with histone H3.2,an essential component of the nucleosome.Histone H3.2 was transcriptionally up-regulated in the SlCycB1 overexpressing tomato lines.Furthermore,the overexpression of histone H3.2 in transgenic plants showed similar phenotypes to SlCycB1 overexpressing lines.Based on these findings,we concluded that SlCycB1 overexpression altered tomato architecture in association with histone H3.2.

Protective effect of 4-methyl-2-{[4-(toluene-4-sulfonyl)-thiomorpholine-3-carbonyl]-amino}-pentanoic acid isopropyl ester against cerebral ischemia

To investigate the effects of 4-methyl-2-{[4-(toluene-4-sulfonyl)-thiomorpholine-3-carbonyl]-amino}-pentanoic acid isopropyl ester(HD5-6)against cerebral ischemia in rodents,the models with global and focal ischemia were induced by bilateral common carotid artery occlusion plus hypotension(BCAOH)and permanent cerebral artery occlusion(p-MCAO)in mice(n=10–12 per group in BCAOH;n=8 per group in p-MCAO)and rats(n=10-11 in each group).HD5-6 prolonged lifetime and improved neurological function.Neurological deficits score in HD5-6(30 mg/kg)decreased significantly.Malonaldehyde(MDA)in HD5-6-treated mice with ischemia considerably dropped.The infarction volume of the HD5-6-treated rats with MCAO-induced ischemia decreased significantly in the high dose group(P<0.05,i.g.and P<0.01,i.v.).Immunohistochemistry showed that Brain derived neurotrophic factor(BDNF)in the ipsilateral hemisphere increased and Vascular endothelial growth factor(VEGF)decreased with HD5-6 treatment.HD5-6 has protective effects against experimental cerebral ischemia in rodents and the action mechanism may involve anti-oxidation and neurogenesis.

Six Thallus Surface Types of Coralline Algae with Descriptions of Two New Records of Amphiroa beauvoisii and Neogoniolithon setchellii in Sanya reef,China

Coralline algae are globally distributed calcifying species and play critical ecological roles to marine ecosystems by contributing significantly to their structural complexity and diversity.Thallus surface types of historical samples in Sanya coral reef reserve were studied based on the scanning electron microscope(SEM)method.Our results show six thallus surface types within the study area:Corallina-type,Jania-type,Leptophytum-type,Phymatolithon-type,Pneophyllum-type,and Spongites-type.The Phymatolithon-type is the dominant surface type in Sanya reefs.Two new record species in the region are described:Amphiroa beauvoisii and Neogoniolithon setchellii.Although thallus surface types provide useful diagnostics characters for distinguishing coralline algae at tribe or subfamily level,species identification needs to refer to the reproductive features.This is the first surface study of coralline algae in the South China Sea.This result provides the baseline data needed for the monitoring and management of reef-building organisms of coral reef in China.

Regulation of invertase and sucrose for improving tomato fruit flavor:A review

Tomato(Solanum lycopersicum L.)is a commercially farmed vegetable belonging to the Solanaceae family,the third most important vegetable after potato(Solanum tuberosum L.)and onion(Allium cepa L.).It is cultivated for its fresh fruits and processed paste,with over 153 million metric tons of global production.However,modern tomato cultivars have limited sugars,acids,and volatiles allelic diversity as flavor has generally been less prioritized in breeding programs.Invertase is an essential regulator of flavor and sugar metabolism in tomato.Genetic control of tomato flavor is still incomplete without a clear understanding of the roles of invertase and sucrose metabolism.This review provides an overview of our current understanding of the invertase mode of action in sucrose metabolism,their evolutionary and functional divergence in the tomato genome,role in stress response,genetic and hormonal control of fruit flavor and quality.We summarized the primary roles of invertase in sugar metabolism and fruit flavor.