Localization of cystathionine β synthase in mice ovaries and its expression profile during follicular development

Background In vitro fertilization （IVF） researches have suggested that cystathionine β synthase （CBS） is involved in oocyte development. However, little is known about the regional and cellular expression patterns of CBS in the ovary. The purpose of this study was to analyze the localization of CBS in mice ovaries and to investigate the expression profile during follicular development. Methods We used in situ hybridization and immunohistochemical analysis to determine CBS expression in the ovaries of female Balb/c mice. Then the follicles were collected from F1 （C57BL×Balb/c） mice and cultured in vitro. With the method of semi-quantitative RT-PCR, we also investigated the expression profile of CBS during follicular development. Results CBS was absent in the oocytes, although it was ubiquitously expressed in the ovary with the strongest expression in follicular cells at all stages. In late antral follicles, CBS expression was markedly higher in granulosa cells located close to the antrum and in cumulus cells around the oocyte. The semi-quantitative RT-PCR showed that CBS mRNA was detected in follicles at all stages in vitro. In cumulus-oocyte complexes superovulated, CBS expression also increased rapidly. Conclusions CBS was located mainly in the follicular cells in the ovaries. The level of CBS expression is high in follicles during folliculogenesis in mice. Differences in the CBS expression profile between oocyte and follicular cells suggest a role for CBS as a mediator in interactions between oocyte and granulosa cells.

Effect of mutations on acetohydroxyacid synthase(AHAS)function in Cyperus difformis L.

Cyperus difformis L.is a troublesome weed in paddy fields and has attracted attention due to its resistance to acetohydroxyacid synthase(AHAS)inhibitors.It was found that the amino acid mutation in AHAS was the primary cause for the resistance of Cyperus difformis.However,the effect of different mutations on AHAS function is not clear in Cyperus difformis.To confirm the effect of mutations on AHAS function,six biotypes were collected,including Pro197Arg,Pro197Ser,Pro197Leu,Asp376Glu,Trp574Leu and wild type,from Hunan,Anhui,Jiangxi and Jiangsu provinces,China and the function of AHAS was characterized.The AHAS in vitro inhibition assay results indicated that the mutations decreased the sensitivity of AHAS to pyrazosulfuron-ethyl,in which the I_(50)(the half maximal inhibitory concentration)of wild type AHAS was 0.04μmol L^(-1)and Asp376Glu,Pro197Leu,Pro197Arg,Pro197Ser and Trp574Leu mutations were 3.98,11.50,40.38,38.19 and 311.43μmol L^(-1),respectively.In the determination of enzyme kinetics parameters,the Km and the maximum reaction velocity(Vmax)of the wild type were 5.18 mmol L^(-1)and 0.12 nmol mg^(-1)min^(-1),respectively,and the Km values of AHAS with Asp376Glu,Trp574Leu,Pro197Leu and Pro197Ser mutations were 0.38-0.93 times of the wild type.The Km value of the Pro197Arg mutation was 1.14times of the wild type,and the Vmax values of the five mutations were 1.17-3.33-fold compared to the wild type.It was found that the mutations increased the affinity of AHAS to the substrate,except for the Pro197Arg mutation.At a concentration of 0.0032-100 mmol L^(-1)branched-chain amino acids(BCAAs),the sensitivity of the other four mutant AHAS biotypes to feedback inhibition decreased,except for the Pro197Arg mutation.This study elucidated the effect of different mutations on AHAS function in Cyperus difformis and provided ideas for further study of resistance development.

The gene encoding flavonol synthase contributes to lesion mimic in wheat

Lesion mimic often exhibits leaf disease-like symptoms even in the absence of pathogen infection,and is characterized by a hypersensitive-response(HR)that closely linked to plant disease resistance.Despite this,only a few lesion mimic genes have been identified in wheat.In this investigation,a lesion mimic wheat mutant named je0297 was discovered,showing no alteration in yield components when compared to the wild type(WT).Segregation ratio analysis of the F_(2)individuals resulting from the cross between the WT and the mutant revealed that the lesion mimic was governed by a single recessive gene in je0297.Using Bulked segregant analysis(BSA)and exome capture sequencing,we mapped the lesion mimic gene designated as lm6 to chromosome 6BL.Further gene fine mapping using 3315 F_(2)individuals delimited the lm6 within a 1.18 Mb region.Within this region,we identified 16 high-confidence genes,with only two displaying mutations in je0297.Notably,one of the two genes,responsible for encoding flavonol synthase,exhibited altered expression levels.Subsequent phenotype analysis of TILLING mutants confirmed that the gene encoding flavonol synthase was indeed the causal gene for lm6.Transcriptome sequencing analysis revealed that the DEGs between the WT and mutant were significantly enriched in KEGG pathways related to flavonoid biosynthesis,including flavone and flavonol biosynthesis,isoflavonoid biosynthesis,and flavonoid biosynthesis pathways.Furthermore,more than 30 pathogen infection-related(PR)genes exhibited upregulation in the mutant.Corresponding to this expression pattern,the flavonoid content in je0297 showed a significant decrease in the 4^(th)leaf,accompanied by a notable accumulation of reactive oxygen,which likely contributed to the development of lesion mimic in the mutant.This investigation enhances our comprehension of cell death signaling pathways and provides a valuable gene resource for the breeding of disease-resistant wheat.

Relationship Between Polymorphism of Cystathionine beta Synthase Gene and Congenital Heart Disease in Chinese Nuclear Families

Objective To study the relationship between polymorphism of cystathionine beta synthase （CBS） gene and development of congenital heart disease （CHD）. Methods One hundred and twenty-seven CHD case-parent triads were recruited from Liaoning Province as patient group, and 129 healthy subjects without family history of birth defect were simultaneously recruited as control group together with their biological parents. For all subjects the polymorphism of CBS gene G919A locus was examined by PCR-ARMS method, Results The frequencies of three genotypes （w/w, w/m, and m/m） in control group were 27.2%, 58,4%, and 14.4%, respectively, with no significant difference in gender. A significant difference in the allele frequency was found between CHD patients and controls, the wild allele frequency was 67,9% in patients and 55.7% in controls CHD parents＇ genotype distribution was significantly different from that in controls. Further comparison of each type of CHD showed that genotype frequencies in several CHD subtypes were significantly different from those in their corresponding controls. The results of TDT analysis showed that no allele transmission disequilibrium existed in CHD nuclear families. Conclusions CBS gene G919A mutation is associated with the development of CHD, and the mutated allele may decrease the risk of CHD.

Constitutive expression of feedback-insensitive cystathionine γ-synthase increases methionine levels in soybean leaves and seeds

Soybean （Glycine max （L.） Merr.） is a major crop that provides plant-origin protein and oil for humans and livestock. Al- though the soybean vegetative tissues and seeds provide a major source of high-quality protein, they suffer from low con- centration of an essential sulfur-containing amino acid, methionine, which significantly limits their nutritional quality. The level of methionine is mainly controlled by the first unique enzyme of methionine synthesis, cystathione y-synthase （CGS）. Aiming to elevate methionine level in vegetative tissues and seeds, we constitutively over-expressed a feedback-insensitive Arabidopsis CGS （AtD-CGS） in soybean cultivars, Zigongdongdou （ZD） and Jilinxiaoli 1 （JX）. The levels of soluble methionine increased remarkably in leaves of transgenic soybeans compared to wild-type plants （6.6- and 7.3-fold in two transgenic ZD lines, and 3.7-fold in one transgenic JX line）. Furthermore, the total methionine contents were significantly increased in seeds of the transgenic ZD lines （1.5- to 4.8-fold increase） and the transgenic JX lines （1.3- to 2.3-fold increase） than in the wild type. The protein contents of the transgenic soybean seeds were significantly elevated compared to the wild type, suggesting that the scarcity of methionine in soybeans may limit protein accumulation in soybean seeds. The increased protein content did not alter the profile of major storage proteins in the seeds. Generally, this study provides a promising strategy to increase the levels of methionine and protein in soybean through the breeding programs.

Genome-wide identification and function analysis of the sucrose phosphate synthase MdSPS gene family in apple

Sucrose phosphate synthase(SPS)is a rate-limiting enzyme that works in conjunction with sucrose-6-phosphate phosphatase(SPP)for sucrose synthesis,and it plays an essential role in energy provisioning during growth and development in plants as well as improving fruit quality.However,studies on the systematic analysis and evolutionary pattern of the SPS gene family in apple are still lacking.In the present study,a total of seven MdSPS and four MdSPP genes were identified from the Malus domestica genome GDDH13 v1.1.The gene structures and their promoter cis-elements,protein conserved motifs,subcellular localizations,physiological functions and biochemical properties were analyzed.A chromosomal location and gene-duplication analysis demonstrated that whole-genome duplication(WGD)and segmental duplication played vital roles in MdSPS gene family expansion.The Ka/Ks ratio of pairwise MdSPS genes indicated that the members of this family have undergone strong purifying selection during domestication.Furthermore,three SPS gene subfamilies were classified based on phylogenetic relationships,and old gene duplications and significantly divergent evolutionary rates were observed among the SPS gene subfamilies.In addition,a major gene related to sucrose accumulation(MdSPSA2.3)was identified according to the highly consistent trends in the changes of its expression in four apple varieties(‘Golden Delicious’,‘Fuji’,‘Qinguan’and‘Honeycrisp’)and the correlation between gene expression and soluble sugar content during fruit development.Furthermore,the virus-induced silencing of MdSPSA2.3 confirmed its function in sucrose accumulation in apple fruit.The present study lays a theoretical foundation for better clarifying the biological functions of the MdSPS genes during apple fruit development.

Glycogen Synthase Kinase-3β,NLRP3 Inflammasome,and Alzheimer's Disease

Alzheimer’s disease (AD) is the most prevalent cause of dementia worldwide. Because of the progressive neurodegeneration, individual cognitive and behavioral functions are impaired, affecting the quality of life of millions of people. Although the exact pathogenesis of AD has not been fully elucidated, amyloid plaques, neurofibrillary tangles (NFTs), and sustaining neuroinflammation dominate its characteristics. As one of the major tau kinases leading to hyperphosphorylation and aggregation of tau, glycogen synthase kinase-3β (GSK-3β) has been drawing great attention in various AD studies. Another research focus of AD in recent years is the inflammasome, a multiprotein complex acting as a regulator in immunological reactions to exogenous and endogenous danger signals, of which the Nod-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome has been studied mostly in AD and proven to play a significant role in AD development by its activation and downstream effects such as caspase-1 maturation and interleukin (IL)-1β release. Studies have shown that the NLRP3 inflammasome is activated in a GSK-3β-dependent way and that inhibition of the NLRP3 inflammasome downregulates GSK-3β, suggesting that these two important proteins are closely related. This article reviews the respective roles of GSK-3β and the NLRP3 inflammasome in AD as well as their relationship and interaction.

Neuronal nitric oxide synthase/reactive oxygen species pathway is involved in apoptosis and pyroptosis in epilepsy

Dysfunction of neuronal nitric oxide synthase contributes to neurotoxicity,which triggers cell death in various neuropathological diseases,including epilepsy.Studies have shown that inhibition of neuronal nitric oxide synthase activity increases the epilepsy threshold,that is,has an anticonvulsant effect.However,the exact role and potential mechanism of neuronal nitric oxide synthase in seizures are still unclear.In this study,we performed RNA sequencing,functional enrichment analysis,and weighted gene coexpression network analysis of the hippocampus of tremor rats,a rat model of genetic epilepsy.We found damaged hippocampal mitochondria and abnormal succinate dehydrogenase level and Na+-K+-ATPase activity.In addition,we used a pilocarpine-induced N2a cell model to mimic epileptic injury.After application of neuronal nitric oxide synthase inhibitor 7-nitroindazole,changes in malondialdehyde,lactate dehydrogenase and superoxide dismutase,which are associated with oxidative stress,were reversed,and the increase in reactive oxygen species level was reversed by 7-nitroindazole or reactive oxygen species inhibitor N-acetylcysteine.Application of 7-nitroindazole or N-acetylcysteine downregulated the expression of caspase-3 and cytochrome c and reversed the apoptosis of epileptic cells.Furthermore,7-nitroindazole or N-acetylcysteine downregulated the abnormally high expression of NLRP3,gasdermin-D,interleukin-1βand interleukin-18.This indicated that 7-nitroindazole and N-acetylcysteine each reversed epileptic cell death.Taken together,our findings suggest that the neuronal nitric oxide synthase/reactive oxygen species pathway is involved in pyroptosis of epileptic cells,and inhibiting neuronal nitric oxide synthase activity or its induced oxidative stress may play a neuroprotective role in epilepsy.

Metformin promotes angiogenesis and functional recovery in aged mice after spinal cord injury by adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway

Treatment with metformin can lead to the recovery of pleiotropic biological activities after spinal cord injury.However,its effect on spinal cord injury in aged mice remains unclear.Considering the essential role of angiogenesis during the regeneration process,we hypothesized that metformin activates the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway in endothelial cells,thereby promoting microvascular regeneration in aged mice after spinal cord injury.In this study,we established young and aged mouse models of contusive spinal cord injury using a modified Allen method.We found that aging hindered the recovery of neurological function and the formation of blood vessels in the spinal cord.Treatment with metformin promoted spinal cord microvascular endothelial cell migration and blood vessel formation in vitro.Furthermore,intraperitoneal injection of metformin in an in vivo model promoted endothelial cell proliferation and increased the density of new blood vessels in the spinal cord,thereby improving neurological function.The role of metformin was reversed by compound C,an adenosine monophosphate-activated protein kinase inhibitor,both in vivo and in vitro,suggesting that the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway likely regulates metformin-mediated angiogenesis after spinal cord injury.These findings suggest that metformin promotes vascular regeneration in the injured spinal cord by activating the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway,thereby improving the neurological function of aged mice after spinal cord injury.

Prostaglandin F_(2α)synthase promotes oxaliplatin resistance in colorectal cancer through prostaglandin F_(2α)-dependent and F_(2α)-independent mechanism

BACKGROUND Oxaliplatin(Oxa)is the first-line chemotherapy drug for colorectal cancer(CRC),and Oxa resistance is crucial for treatment failure.Prostaglandin F_(2α)synthase(PGF 2α)(PGFS),an enzyme that catalyzes the production of PGF_(2α),is involved in the proliferation and growth of a variety of tumors.However,the role of PGFS in Oxa resistance in CRC remains unclear.AIM To explore the role and related mechanisms of PGFS in mediating Oxa resistance in CRC.METHODS The PGFS expression level was examined in 37 pairs of CRC tissues and paracancerous tissues at both the mRNA and protein levels.Overexpression or knockdown of PGFS was performed in CRC cell lines with acquired Oxa resistance(HCT116-OxR and HCT8-OxR)and their parental cell lines(HCT116 and HCT8)to assess its influence on cell proliferation,chemoresistance,apoptosis,and DNA damage.For determination of the underlying mechanisms,CRC cells were examined for platinum-DNA adducts and reactive oxygen species(ROS)levels in the presence of a PGFS inhibitor or its products.RESULTS Both the protein and mRNA levels of PGFS were increased in the 37 examined CRC tissues compared to the adjacent normal tissues.Oxa induced PGFS expression in the parental HCT116 and HCT8 cells in a dosedependent manner.Furthermore,overexpression of PGFS in parental CRC cells significantly attenuated Oxainduced proliferative suppression,apoptosis,and DNA damage.In contrast,knockdown of PGFS in Oxa-resistant HCT116 and HCT8 cells(HCT116-OxR and HCT8-OxR)accentuated the effect of Oxa treatment in vitro and in vivo.The addition of the PGFS inhibitor indomethacin enhanced the cytotoxicity caused by Oxa.Treatment with the PGFS-catalyzed product PGF_(2α)reversed the effect of PGFS knockdown on Oxa sensitivity.Interestingly,PGFS inhibited the formation of platinum-DNA adducts in a PGF_(2α)-independent manner.PGF_(2α)exerts its protective effect against DNA damage by reducing ROS levels.CONCLUSION PGFS promotes resistance to Oxa in CRC via both PGF_(2α)-dependent and PGF_(2α)-independent mechanisms.

Dissecting molecular mechanisms underlying ferroptosis in human umbilical cord mesenchymal stem cells:Role of cystathionineγ-lyase/hydrogen sulfide pathway

BACKGROUND Ferroptosis can induce low retention and engraftment after mesenchymal stem cell(MSC)delivery,which is considered a major challenge to the effectiveness of MSC-based pulmonary arterial hypertension(PAH)therapy.Interestingly,the cystathionineγ-lyase(CSE)/hydrogen sulfide(H_(2)S)pathway may contribute to mediating ferroptosis.However,the influence of the CSE/H_(2)S pathway on ferroptosis in human umbilical cord MSCs(HUCMSCs)remains unclear.AIM To clarify whether the effect of HUCMSCs on vascular remodelling in PAH mice is affected by CSE/H_(2)S pathway-mediated ferroptosis,and to investigate the functions of the CSE/H_(2)S pathway in ferroptosis in HUCMSCs and the underlying mechanisms.METHODS Erastin and ferrostatin-1(Fer-1)were used to induce and inhibit ferroptosis,respectively.HUCMSCs were transfected with a vector to overexpress or inhibit expression of CSE.A PAH mouse model was established using 4-wk-old male BALB/c nude mice under hypoxic conditions,and pulmonary pressure and vascular remodelling were measured.The survival of HUCMSCs after delivery was observed by in vivo bioluminescence imaging.Cell viability,iron accumulation,reactive oxygen species production,cystine uptake,and lipid peroxidation in HUCMSCs were tested.Ferroptosis-related proteins and S-sulfhydrated Kelchlike ECH-associating protein 1(Keap1)were detected by western blot analysis.RESULTS In vivo,CSE overexpression improved cell survival after erastin-treated HUCMSC delivery in mice with hypoxiainduced PAH.In vitro,CSE overexpression improved H_(2)S production and ferroptosis-related indexes,such as cell viability,iron level,reactive oxygen species production,cystine uptake,lipid peroxidation,mitochondrial membrane density,and ferroptosis-related protein expression,in erastin-treated HUCMSCs.In contrast,in vivo,CSE inhibition decreased cell survival after Fer-1-treated HUCMSC delivery and aggravated vascular remodelling in PAH mice.In vitro,CSE inhibition decreased H_(2)S levels and restored ferroptosis in Fer-1-treated HUCMSCs.Interestingly,upregulation of the CSE/H_(2)S pathway induced Keap1 S-sulfhydration,which contributed to the inhibition of ferroptosis.CONCLUSION Regulation of the CSE/H_(2)S pathway in HUCMSCs contributes to the inhibition of ferroptosis and improves the suppressive effect on vascular remodelling in mice with hypoxia-induced PAH.Moreover,the protective effect of the CSE/H_(2)S pathway against ferroptosis in HUCMSCs is mediated via S-sulfhydrated Keap1/nuclear factor erythroid 2-related factor 2 signalling.The present study may provide a novel therapeutic avenue for improving the protective capacity of transplanted MSCs in PAH.

Identifi cation of sesquiterpene synthase genes in the genome of Aquilaria sinensis and characterization of anα-humulene synthase

Sesquiterpenes are the major pharmacodynamic components of agarwood,a precious traditional Chinese medicine obtained from the resinous portions of Aquilaria sinensis trees that form in response to environmental stressors.To characterize the sesquiterpene synthases responsible for sesquiterpene production in A.sinensis,a bioinformatics analysis of the genome of A.sinensis identifi ed six new terpene synthase genes,and 16 sesquiterpene synthase genes were identifi ed as type TPS-a in a phylogenetic analysis.The expression patterns for eight of the sesquiterpene synthase genes after treatment with various hormones or hydrogen peroxide were analyzed by real-time quantitative PCR.The results suggest that 100μM methyl jasmonate,ethephon,(±)-abscisic acid or hydrogen peroxide could be eff ective short-term eff ectors to increase the expression of sesquiterpene synthase genes,while 1 mM methyl salicylate may have long-term eff ects on increasing the expression of specifi c sesquiterpene synthase genes(e.g.,As-SesTPS,AsVS,AsTPS12 and AsTPS29).The expression changes in these genes under various conditions refl ected their specifi c roles during abiotic or biotic stresses.Heterologous expression of a novel A.sinensis sesquiterpene synthase gene,AsTPS2,in Escherichia coli produced a major humulene product,so AsTPS2 is renamed AsHS1.AsHS1 is diff erent from ASS1,AsSesTPS,and AsVS,for mainly producingα-humulene.Based on the predicted space conformation of the AsHS1 model,the small ligand molecule may bind to the free amino acid by hydrogen bonding for the catalytic function of the enzyme,while the substrate farnesyl diphosphate(FPP)probably binds to the free amino acid on one side of the RxR motif.Arg450,Asp453,Asp454,Thr457,and Glu461 from the NSE/DTE motif and D307 and D311 from the DDxxD motif were found to form a polar interaction with two Mg^(2+)clusters by docking.The Mg^(2+)-bound DDxxD and NSE/DTE motifs and the free RXR motif are jointly directed into the catalytic pocket of AsHS1.Comparison of the tertiary structural models of AsHS1 with ASS1 showed that they diff ered in structures in several positions,such as surrounding the secondary catalytic pocket,which may lead to diff erences in catalytic products.Based on the results,biosynthetic pathways for specifi c sesquiterpenes such asα-humulene in A.sinensis are proposed.This study provides novel insights into the functions of the sesquiterpene synthases of A.sinensis and enriches knowledge on agarwood formation.

Mutations in Plasmodium knowlesi Kelch protein 13 and the dihydropteroate synthase gene in clinical samples

Objective:To determine the genetic diversity,natural selection and mutations in Plasmodium(P.)knowlesi drug resistant molecular markers Kelch 13 and dhps gene in clinical samples of Malaysia.Methods:P.knowlesi full-length gene sequences Kelch 13 gene(PkK13)from 40 samples and dhps gene from 30 samples originating from Malaysian Borneo were retrieved from public databases.Genetic diversity,natural selection,and phylogenetic analysis of gene sequences were analysed using DNAsp v5.10 and MEGA v5.2.Results:Seventy-two single nucleotide polymorphic sites(SNPs)across the full-length PkK13 gene(63 synonymous substitutions and 9 non-synonymous substitutions)with nucleotide diversity ofπ~0.005 was observed.Analysis of the full-length Pkdhps gene revealed 73 SNPs andπ~0.006(44 synonymous substitutions and 29 non-synonymous substitutions).A high number of haplotypes(PkK13;H=37 and Pkdhps;H=29)with haplotype diversity of Hd~0.99 were found in both genes,indicating population expansion.Nine mutant alleles were identified in PkK13 amino acid alignment of which,7(Asp3Glu,Lys50Gln,Lys53Glu,Ser123Thr,Ser127Pro,Ser149Thr and Ala169Thr)were within the Plasmodium specific domain,2(Val372Ile and Lys424Asn)were in the BTB/POZ domain and no mutation was observed within the kelch propeller domain.The 29 non-synonymous mutations in the Pkdhps gene were novel and only presented in exon 1 and 2.Conclusions:Monitoring the mutations from clinical samples collected from all states of Malaysia along with clinical efficacy studies will be necessary to determine the drug resistance in P.knowlesi.

A nonsynonymous mutation in an acetolactate synthase gene (Gh_D10G1253) is required for tolerance to imidazolinone herbicides in cotton

Background Herbicide tolerance in crops enables them to survive when lethal doses of herbicides are applied to surrounding weeds.Herbicide-tolerant crops can be developed through transgenic approaches or traditional mutagenesis approaches.At present,no transgenic herbicide tolerant cotton have been commercialized in China due to the genetically-modified organism(GMO)regulation law.We aim to develop a non-transgenic herbicide-tolerant cotton through ethyl methanesulfonate(EMS)mutagenesis,offering an alternative choice for weed management.Results Seeds of an elite cotton cultivar Lumianyan 37(Lu37)were treated with EMS,and a mutant Lu37-1 showed strong tolerance to imidazolinone(IMI)herbicides was identified.A novel nonsynonymous substitution mutation Ser642Asn at acetolactate synthase(ALS)(Gh_D10G1253)in Lu37-1 mutant line was found to be the potential cause to the IMI herbicides tolerance in cotton.The Ser642Asn mutation in ALS did not present among the genomes of natural Gossypium species.Cleaved amplified polymorphic sequence(CAPS)markers were developed to identify the ALS mutant allele.The Arabidopsis overexpressing the mutanted ALS also showed high tolerance to IMI herbicides.Conclusion The nonsynonymous substitution mutation Ser642Asn of the ALS gene Gh_D10G1253 is a novel identi-fied mutation in cotton.This substitution mutation has also been identified in the orthologous ALS genes in other crops.This mutant ALS allele can be used to develop IMI herbicide-tolerant crops via a non-transgenic or transgenic approach.

Identification and Pathogen Stimulation Patterns of Neuronal Nitric Oxide Synthase(nNOS)in Black Rockfish(Sebastes schlegelii)

Neuronal nitric oxide synthase(nNOS)was the producer of nitric oxide(NO)which played important gas messenger molecules in biological process.It also can take effect as immune regulation molecule in organism.Black rockfish(Sebastes schlegelii)is an important economic fish which were widely farmed in East Asia countries.Meanwhile,the pathogenic bacteria such as the Edwardsiella tarda and Vibrio anguillarum in seawater always brought serious obstacles to their healthy growth.In order to explore the expression pattern of n NOS gene under the pathogen stimulation and predict its immune function,the n NOS gene in black rockfish named Ssn NOS was identified.It was 3780 bp in length,located on chromosome 6,and contained 27 coding domain sequence(CDs).According to the phylogenetic analysis,the Ssn NOS showed closest relative to the counterpart gene of swamp eel(Monopterus albus).Meanwhile,analysis of Ssn NOS expression in various healthy tissues showed that Ssn NOS expression level was highest in healthy brain tissues,followed by intestinal tissues.In addition,Ssn NOS showed significant expression changes in response to stimulation by two pathogens.Particular in gill,the expression of Ssn NOS after pathogenic stimulation increased significantly.The Elisa analysis showed the Ssn NOS content in gills was much higher than that in other tissues at all time points.Moreover,the expression patterns of Ssn NOS in brain,intestine and kidney after stimulation by pathogens showed a distinct expression pattern which first down-regulated and then up-regulated.Therefore,the Ssn NOS may be an important signaling molecule for fish to respond rapidly in immune stimulation.

ZmMS39 encodes a callose synthase essential for male fertility in maize(Zea mays L.)

Callose contributes to many biological processes of higher plants including pollen development,cell plate and vascular tissue formation,as well as regulating the transport function of plasmodesmata.The functions of callose synthase genes in maize have been little studied.We describe a maize male-sterile mutant 39(ms39)characterized by reduced plant height.In this study,we confirmed using CRISPR/Cas9 technology that a mutation in Zm00001d043909(ZmCals12),encoding a callose synthase,is responsible for the male sterility of the ms39 mutant.Compared with male-fertile plants,callose deposition around the dyads and tetrads in ms39 anthers was significantly reduced.Increased cell autophagy observed in ms39 anthers may have been due to the premature programmed cell death of tapetal cells,leading to collapse of the anther wall structure.Disordered glucose metabolism in ms39 may have intensified autophagy in anthers.Evaluation of the ms39 gene on maize heterosis by paired-crossed experiment with 11 maize inbred lines indicated that ms39 can be used for maize hybrid seed production.

Correlation between the expressions of metastasis-associated factor-1 in colon cancer and vacuolar ATP synthase

BACKGROUND Clinical prognosis often worsens due to high recurrence rates following radical surgery for colon cancer.The examination of high-risk recurrence factors post-surgery provides critical insights for disease evaluation and treatment planning.AIM To explore the relationship between metastasis-associated factor-1 in colon cancer(MACC1)and vacuolar ATP synthase(V-ATPase)expression in colon cancer tissues,and recurrence rate in patients undergoing radical colon cancer surgery.METHODS We selected 104 patients treated with radical colon cancer surgery at our hospital from January 2018 to June 2021.Immunohistochemical staining was utilized to assess the expression levels of MACC1 and V-ATPase in these patients.RESULTS The rates of MACC1 and V-ATPase positivity were 64.42%and 67.31%,respe-ctively,in colon cancer tissues,which were significantly higher than in paracan-cerous tissues(P<0.05).Among patients with TNM stage III,medium to low differentiation,and lymph node metastasis,the positive rates of MACC1 and V-ATPase were significantly elevated in comparison to patients with TNM stage I-II,high differentiation,and no lymph node metastasis(P<0.05).The rate of MACC1 positivity was 76.67%in patients with tumor diameters>5 cm,notably higher than in patients with tumor diameters≤5 cm(P<0.05).We observed a positive correlation between MACC1 and V-ATPase expression(rs=0.797,P<0.05).The positive rates of MACC1 and V-ATPase were significantly higher in patients with recurrence compared to those without(P<0.05).Logistic regression analysis revealed TNM stage,lymph node metastasis,MACC1 expression,and V-ATPase expression as risk factors for postoperative colon cancer recurrence(OR=6.322,3.435,2.683,and 2.421;P<0.05).CONCLUSION The upregulated expression of MACC1 and V-ATPase in colon cancer patients appears to correlate with clinicopathological features and post-radical surgery recurrence.

The Effect of Active Oxygen on the Activity of ACC Synthase Induced by Exogenous IAA

During the course of mungbean (Phaseolus radiatus L.) germination, the rate of ethylene production and the activity of ACC synthase (1_aminocyclopropane_1_carboxylic acid synthase, EC4.4.1.4) began to increase in the 5th day of germination, and reached its peak in the 10th day and then decreased. The ethylene production and the activity of ACC synthase were obviously promoted by 10 μmol/L exogenous IAA (indole_3_acetic acid). The production of superoxide radical (O -· 2) and hydrogen peroxide (H 2O 2) were also promoted by exogenous IAA, suggesting that there was some relationship between active oxygen production and the activity of ACC synthase induced by exogenous IAA. The production of ethylene and the activity of ACC synthase increased dramatically when the seedlings were treated with exogenous O -· 2, whereas the exogenous H 2O 2 had no effects on the production of ethylene and the activity of ACC synthase. Exogenous SOD (superoxide dismutase, one scavenger of O -· 2) could inhibit the production of ethylene and the activity of ACC synthase, but exogenous CAT (catalase) could not. So it was possible that IAA would stimulate the activity of ACC synthase by inducing the production of O -· 2 in germinating mungbean seedlings, and this might be one of the regulating mechanism of ethylene synthesis in higher plants; the production of H 2O 2 induced by IAA was not the cause of the increase of the activity of ACC synthase and the production of ethylene.

cDNA Cloning, Expression and Characterization of Taxadiene Synthase, a Diterpene Cyclase from Taxus chinensis

Taxadiene synthase, a diterpene cyclase, catalyzes the conversion of geranylgeranyl diphosphate (GGPP) to taxadiene, a key intermediate in Taxol biosynthesis in yew. A 2 151 bp cDNA fragment encoding taxadiene synthase of Taxus chinensis (Pilg.) Rehd. was cloned by homology-based PCR and cDNA library screening. The 5′-terminal 611 bp cDNA fragment of taxadiene synthase was isolated by PCR. The two fragments were ligated together and gave a 2*!712 bp cDNA fragment with a 2*!586 bp open reading frame (ORF), encoding 862 amino acid residues including a presumptive plastidial transit peptide. The taxadiene synthase of T. chinensis most closely resembles the one from T. brevifolia (97% identity). Heterologous overexpression of 2.5 kb cDNA fragment from T. chinensis was obtained using a fusion expression vector pET-32a and the Escherichia coli strain BL21trxB. The expressed proteins from E. coli BL21trxB were present as inclusion bodies. After the inclusion bodies were denatured, renatured and refolded, the recombinant enzyme was purified by a single step with a His-binding metal affinity column. The catalytic product of taxadiene synthase of T. chinensis was detected by capillary gas chromatography-mass spectrometry (GC-MS) and identified as taxa-4(5),11(12)-diene.

cDNA Cloning and Sequencing of Squalene Synthase of Artemisa apiacea

[Objective] cDNA from squalene synthase was cloned and sequenced.[Method] A pair of specific primers was designed according to the cDNA gene sequence of squalene synthase published in GenBank.Total RNA was extracted from the cell of Artemisia apiacea.The genes of squalene synthase were amplified by using RT-PCR.It was connected with pMD19-T vector and the cloned fragment sequences were analyzed.[Result] SS gene with the whole length of 1 257 bp was amplified and the fragment encoded 418 amino acids.The homo...