Evaluation of Resistance of Different Kiwifruit Varieties (Lines) to Canker Disease and Brown Spot Disease

Kiwifruit canker and brown spot are significant diseases affecting kiwis,caused by Pseudomonas syringae patho-genic variations(Pseudomonas syringae pv.Actinidiae(Psa))and Corynesporapolytica(Corynespora cassiicola).At present,the research on canker disease and brown spot disease mainly focuses on the isolation and identification of pathogenic bacteria,drug control,resistance gene mining and functional verification.Practice has proved that breeding disease resistant varieties are an effective method to control canker disease and brown spot disease.However,most existing cultivars lack genes for canker and brown spot resistance.Wild kiwifruit resources in nat-ure exhibit extensive genetic diversity due to prolonged natural selection,containing numerous resistance genes.But,due to insufficient understanding of the resistance of most kiwifruit varieties(lines)to canker disease and brown spot disease,some high-quality resources have not been fully utilized.The incidence of canker and brown spot of 18 kiwifruit cultivars(lines)was measured by inoculating isolated branches and leaves,and their resistance to canker and brown spot was analyzed according to the length,disease index,mean diameter,and systematic clustering.The results were as follows:Among 18 different kiwifruit varieties(lines)for canker disease,there were two highly resistant materials,eight disease-resistant materials,four disease-susceptible materials,and two highly susceptible materials.Moreover,regarding brown spot disease,there were one highly resistant material,five dis-ease-resistant materials,four susceptible materials,and three highly susceptible materials.Furthermore,four resources were resistant to both diseases.The outcomes provided a theoretical basis for breeding kiwifruit against canker and brown spot.

Formation mechanisms of ethyl acetate and organic acids in Kluyveromyces marxianus L1-1 in Chinese acid rice soup

This study aims to explore the formation mechanism of ethyl acetate and organic acids in acid rice soup(rice-acid soup)inoculated with Kluyveromyces marxianus L1-1 through the complementary analysis of transcriptome and proteome.The quantity of K.marxianus L1-1 varied significantly in the fermentation process of rice-acid soup and the first and third days were the two key turning points in the growth phase of K.marxianus L1-1.Importantly,the concentrations of ethyl acetate,ethanol,acetic acid,and L-lactic acid increased from day 1 to day 3.At least 4231 genes and 2937 proteins were identified and 610 differentially expressed proteins were annotated to 30 Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways based on the analysis results of transcriptome and proteome.The key genes and proteins including up-regulated alcohol dehydrogenase family,alcohol O-acetyltransferase,acetyl-CoA C-acetyltransferase,acyl-coenzyme A thioester hydrolase,and down-regulated aldehyde dehydrogenase family were involved in glycolysis/gluconeogenesis pathways,starch and sucrose metabolism pathways,amino sugar and nucleotide sugar metabolism pathways,tricarboxylic acid(TCA)cycle,and pyruvate metabolism pathways,thus promoting the formation of ethyl acetate,organic acids,alcohols,and other esters.Our results revealed the formation mechanisms of ethyl acetate and organic acids in rice-acid soup inoculated with K.marxianus L1-1.

Molecular characterization of the SAUR gene family in sweet cherry and functional analysis of PavSAUR55 in the process of abscission

Small auxin up RNA(SAUR) is a large gene family that is widely distributed among land plants. In this study, a comprehensive analysis of the SAUR family was performed in sweet cherry, and the potential biological functions of PavSAUR55 were identified using the method of genetic transformation. The sweet cherry genome encodes 86 SAUR members, the majority of which are intron-less. These genes appear to be divided into seven subfamilies through evolution. Gene duplication events indicate that fragment duplication and tandem duplication events occurred in the sweet cherry. Most of the members mainly underwent purification selection pressure during evolution. During fruit development, the expression levels of Pav SAUR16/45/56/63 were up-regulated, and conversely, those of Pav SAUR12/61were down-regulated. Due to the significantly differential expressions of PavSAUR13/16/55/61 during the fruitlet abscission process, they might be the candidate genes involved in the regulation of physiological fruit abscission in sweet cherry. Overexpression of PavSAUR55 in Arabidopsis produced earlier reproductive growth, root elongation, and delayed petal abscission. In addition, this gene did not cause any change in the germination time of seeds and was able to increase the number of lateral roots under abscisic acid(ABA) treatment. The identified SAURs of sweet cherry play a crucial role in fruitlet abscission and will facilitate future insights into the mechanism underlying the heavy fruitlet abscission that can occur in this fruit crop.

Effects of Tsukamurella tyrosinosolvens P9 on growth,physiology and antioxdant enzyme of peanut under drought stress and after re-watering

Background:The plant-growth-promoting rhizobacterium Tsukamurella tyrosinosolvens is a rare strain of actinomycete,in order to recognize and expand the ecological functions of rare actinomycetes.Methods:In this experiment,we studied the effect of Tsukamurella tyrosinosolvens P9 on the drought resistance of peanut by inoculating peanut seedlings in pots and measuring the growth and physiological indicators of peanut under drought stress and re-watering conditions.Results:The results showed that during drought stress,the relative water content of the soil and leaves,chlorophyll content,and stomatal length,width,and aperture were significantly decreased while the levels of malondialdehyde(MDA),H_(2)O_(2) and stomatal density were significantly increased.Peanut growth was also inhibited.However,inoculation with the P9 strain significantly promoted the growth of peanut under drought stress as plant height,fresh weight,root length and root weight were significantly higher compared with the uninoculated drought stress group.In addition,in P9-inoculated plants,the water and chlorophyll contents were significantly higher and the activities of the antioxidant enzymes CAT and SOD were significantly increased(except during the six days of drought treatment).While the stomatal length,width,and aperture were improved,the levels of MDA and H2O2 were significantly decreased.NBT staining showed that inoculation with P9 reduced O^(2−) accumulation under stress.After re-watering,the physiological indexes of inoculated plants recovered more quickly and grew better.Conclusions:The results showed that T.tyrosinosolvens P9 enhanced drought resistance and improves peanut growth by increasing leaf water content,increasing photosynthesis,regulating stomatal closure,and improving antioxidant enzyme activity.

Anti-lipid-oxidation effects and edible safety evaluation of the oil extracted by a supercritical CO_(2) process from coix seed fermented by Monascus purpureus

The physicochemical properties and composition of coix seed oil produced by Monascus purpureus fermentation and supercritical CO_(2)extraction were determined.Anti-lipid-oxidation and edible safety were evaluated using a cholesterol-fish oil model,acute oral toxicity assay,and genetic toxicity assay in vitro and in vivo,respectively.The results show that the extraction oil from fermented coix seed(FCS-O)had good physicochemical quality and abundant active components with physiological function.In particular,γ-tocotrienol,γ-oryzanol,coixenolide and oleic acid concentrations reached 72.83μg/g,745.96μg/g,9.65 mg/g and 316.58 mg/100 g DW,respectively.The FCS-O exhibited higher antioxidant capability in inhibiting lipid oxidation and peroxidation.Compared to the blank control,the concentrations of 7-ketocholestreol and peroxide only were 8.42μg/mL and 16.16 mmol/kg at 168 h of oxidation(P<0.01).In addition,the FCS-O has been confirmed to be a very safe edible oil,with no acute toxicity(LD50>10 g/kg bw,considered actually non-toxic)and no induced mutagenicity,cytotoxicity or genotoxicity.These results serve as a good safety reference for future application of the oil from fermented coix seed.The development and utilization of this kind of oil will be beneficial as a food,food ingredient,nutritional supplement,or natural food antioxidant to promote good health function.

Integrative proteomic-transcriptomic analysis revealed the lifestyles of Lactobacillus paracasei H4-11 and Kluyveromyces marxianus L1-1 under co-cultivation conditions

Compared with the rice-acid soup inoculated with single starter,the synergistically intensifi ed rice-acid soup inoculated with Lactobacillus paracasei H4-11(L.paracasei H4-11)and Kluyveromyces marxianus L1-1(K.marxianus L1-1)contained more fl avor compounds.Organic acids mainly included L-lactic acid and the main volatile fl avor component was ethyl acetate.Moreover,the signal intensity of astringency and bitterness and the total concentration of volatile sulfur compounds were reduced.The combined analysis results of RNA sequencing(RNA-seq)technology and 4D label-free quantitative(4D LFQ)proteomics explained the fl avor formation pathways in rice-acid soup inoculated with L.paracasei H4-11 and K.marxianus L1-1.In L.paracasei H4-11,L-lactate dehydrogenase,phosphoglucomutase,acetate kinase,alcohol dehydrogenase and acetyl-CoA were up-regulated and D-lactate dehydrogenase and N-Acetyltransferase were down-regulated.In K.marxianus L1-1,Acetyl-CoA,acetaldehyde dehydrogenase,acyl-coenzyme A,N-acetyltransferase,and L-lactate dehydrogenase were up-regulated and hexokinase,alcohol dehydrogenase,and alcohol O-acetyltransferase were down-regulated.The above up-regulation and down-regulation synergistically promoted the formation of characteristic fl avor compounds(mainly L-lactic acid and ethyl acetate).Enzyme-linked immunoassay(ELISA)and parallel reaction monitoring(PRM)quantitative analysis respectively verifi ed that 5 key metabolic enzymes and 27 proteins in L.paracasei H4-11 and K.marxianus L1-1 were associated with the characteristic fl avor of rice-acid soup,as confi rmed by the quantitative results of 4D LFQ.

Effects of Manganese on the Antioxidant System and Related Gene Expression Levels in the “Hong Yang” Kiwifruit Seedlings

To explore how manganese affects the antioxidant system and the expression levels of related genes of“Hong yang”seedlings,the leaves of its tissue cultured seedlings were taken as test materials,and single factor treatment was performed by changing the manganese chloride(MnCl_(2)·4H_(2)O)solution concentration when spraying the leaves.The expression levels of Mn-SOD,POD64 and POD27 genes in leaves were quantitatively analyzed by real-time quantitative PCR(qRT-PCR)at different determination times.Meanwhile,the contents of malondial-dehyde(MDA),hydrogen peroxide(H_(2)O_(2)),the activities of antioxidant enzymes,including catalase(CAT),peroxidase(POD),and superoxide dismutase(SOD).The results showed that the SOD,CAT,POD,ascorbate peroxidase(APX),and reduced glutathione(GSH)activities in leaves were the highest at 12 h post-treatment with 50μM MnCl_(2)·4H_(2)O.Furthermore,the contents of MDA and H_(2)O_(2) in leaves also peaked when the concentration of H_(2)O_(2) is 50μM,which is the minimum value.Additionally at 50μM Mn^(2+),the Mn-SOD and POD27 expression was up-regulated as compared to the control,which promoted the expression of their respective enzyme activities.However,POD64 expression increased with the increasing Mn^(2+) concentration.Therefore,50μM is the optimal concentration of Mn when exogenously applied on“Hong yang”,which improve the antioxidant enzyme activity and regulate the plant’s physiological and biochemical functions.

Integrated transcriptome and metabolome provide insights into flavonoid biosynthesis in'P113',a new purple tea of Camellia tachangensis

Plants typically exhibit the purple phenomenon as a result of an increase in flavonoids and anthocyanins.A new tea germplasm'P113'was recently selected from Camellia tachangensis,which is purple in tender shoots.In the present study,integrated transcriptome and metabolome were used to analyze the flavonoid metabolite components and the genes involved in flavonoid biosynthesis in'P113'.A total of 86 flavonoid metabolites were identified,including 70 significantly differential metabolites(p<0.05)and they were enriched to the three metabolic pathways of ko00941,ko00942 and ko00944 through KEGG pathway analysis.A total of 136 flavonoid involved genes were obtained from transcriptomic study,of which 53 were significantly differentially expressed in developmental shoots.The correlation between metabolite profiling and transcriptome,transcriptome and protein interactions suggested that transcription factor MYB12 and glycosyltransferase UGT78D2 had a good facilitation on purple tender shoots.The metabolic pathways and potential genes that underlie the coloration of the shoots in'P113'are clarified in this study.It also lays the groundwork for identifying potential genes involved in color variation and offers a theoretical framework for the creation and use of distinctive genetic resources and the breeding of new cultivars.

Transcriptome-wide identification and expression profiling of Pinus massoniana MYB transcription factors responding to phosphorus deficiency

Myeloblastosis(MYB) proteins constitute one of the largest transcription factor(TF) families in plants and play crucial roles in regulating plant physiological and biochemical processes, including adaptation to diverse abiotic stresses. These TF families contain highly conserved MYB repeats(1 R, R2 R3, 3 R and 4 R) at the N-terminus. Roles for MYB TFs have been reported in response to such stresses as dehydration, salt, cold, and drought. The characterization of Masson pine(Pinus massoniana) MYB TFs are reported, including the analysis of MYB TFs expression in seedlings under controlled conditions and two different phosphate(Pi) deficient treatments. By searching for conserved MYB motifs in full transcriptomic RNA sequencing data for P. massoniana, 59 sequences were identified as MYB TFs. Conserved domainstructures and comparative functional and phylogenetic relationships of these MYB TFs with those in Arabidopsis were assessed using various bioinformatics tools. Based on microarray analysis, P. massoniana MYB genes exhibited different expression patterns under the two Pi deficiency conditions. Genes encoding MYB TFs that showed increased expression under critical Pi deficiency were identified, and some MYBs were differentially expressed only under conditions of severe Pi starvation. These results are useful for the functional characterization of MYB TFs that may be involved in the response to Pi deficiency and play divergent roles in plants.

Genome-level diversification of eight ancient tea populations in the Guizhou and Yunnan regions identifies candidate genes for core agronomic traits

The ancient tea plant,as a precious natural resource and source of tea plant genetic diversity,is of great value for studying the evolutionary mechanism,diversification,and domestication of plants.The overall genetic diversity among ancient tea plants and the genetic changes that occurred during natural selection remain poorly understood.Here,we report the genome resequencing of eight different groups consisting of 120 ancient tea plants:six groups from Guizhou Province and two groups from Yunnan Province.Based on the 8,082,370 identified high-quality SNPs,we constructed phylogenetic relationships,assessed population structure,and performed genome-wide association studies(GWAS).Our phylogenetic analysis showed that the 120 ancient tea plants were mainly clustered into three groups and five single branches,which is consistent with the results of principal component analysis(PCA).Ancient tea plants were further divided into seven subpopulations based on genetic structure analysis.Moreover,it was found that the variation in ancient tea plants was not reduced by pressure from the external natural environment or artificial breeding(nonsynonymous/synonymous=1.05).By integrating GWAS,selection signals,and gene function prediction,four candidate genes were significantly associated with three leaf traits,and two candidate genes were significantly associated with plant type.These candidate genes can be used for further functional characterization and genetic improvement of tea plants.

Characteristics of traditional Chinese acidic rice soup(rice-acid)prepared with different fermentation methods

Rice-acid,a Chinese traditional acidic rice soup(rice-acid),is widely accepted by consumers due to its unique flavor and anti-oxidation,anti-aging and immunity enhancement functions.This study confirmed that L-lactic acid and malic acid were the main organic acids in rice-acid.Low-temperature rice-acid samples produced by enterprises had the highest signal intensity of sour taste.The total content of free amino acids in different fermented rice-acid samples were in the range of 0.003-0.468 mg/g.42 key volatile flavor compounds were identified in rice-acid.8 volatile compounds with a higher contribution to the aroma of rice-acid were respectively acetic acid,1-octen-3-ol,2-heptanol,ethyl acetate,propyl propionate,hexanal,nonanal,and2,3-butanedione.The interaction between lactic acid bacteria(3.00×10^(3)-7.02×10^(6) CFU/mL)and yeasts(5.04×10^(4)-2.25×10^(8) CFU/mL)affected the formation of taste and aroma components in rice-acid.The physicochemical characteristics including titratable acidity,pH,reducing sugars,amino acid nitrogen,gammaaminobutyric acid showed significant differences between low-temperature fermentation samples and hightemperature fermentation samples.In addition,relationships linking all data through Pearson coefficient correlation were also reported.In summary,the study can be used to improve the quality of rice-acid products.

Population Structure Analysis and Genome-Wide Association Study of Tea(Camellia sinensis(L.)Kuntze)Germplasm in Qiannan,China,Based on SLAF-Seq Technology

Duyun Maojian tea is a famous tea in China.In this study,the specific-locus amplified fragment(SLAF)sequencing method was used to analyze the population structure and conduct a genome-wide association study(GWAS)of 2 leaf traits of 123 tea plants in Qiannan,China.A total of 462,019 SLAF tags and 11,362,041 single-nucleotide polymorphism(SNP)loci were obtained.The results of phylogenetic tree analysis,cluster analysis,and principal component analysis showed that 123 tea germplasms were clustered into three groups,and the heterozygosity rates for Groups I,II,and II were 0.206,0.224,and 0.34,respectively.Generally,tea germplasms in a production area are clustered in a group,indicating that tea germplasms in different production areas have certain genetic diversity.The traditional Duyun Maojian tea core production areas,TS and DC-SJ,are clustered into Group I and Group II respectively,while the ZY production area is relatively independent in Group III.Furthermore,based on GWAS analysis,11 candidate genes related to leaf apex and 7 candidate genes related to leaf shape were obtained.This study clarified the genetic relationship among eight Duyun Maojian tea production areas and obtained candidate genes related to leaf apex and leaf shape development.The results showed that population structure and candidate genes are an effective basis for the breeding of Duyun Maojian tea germplasm.

Comparative transcriptomic analysis of Rosa sterilis inflorescence branches with different trichome types reveals an R3-MYB transcription factor that negatively regulates trichome formation

Rosa sterilis S.D.Shi is an important economic tree in China that produces fruits with high nutritional and medicinal value.Many of R.sterills’organs are covered with different types of trichomes or prickles that directly affect fruit appearance and plant management.This study used RNA sequencing technology to analyze the transcriptomes of two parts of the inflorescence branch,namely inflorescence stems with flagellated trichomes and pedicels with both flagellated and glandular trichomes.Comparative transcriptomic analysis showed that many transcription factors(TFs)are potentially involved in the formation and development of trichomes.The accumulation of RsETC1,a TF of the R3-MYB family,was significantly higher in inflorescence stems than in pedicels;quantitative reverse transcription PCR(qRTPCR)verified that its expression was significantly higher in inflorescence stems than in pedicels during the first three development stages,indicating its inhibitory action on the initiation of glandular trichomes in R.sterilis.The mRNA level of RsETC1 accumulated to significantly higher levels in trichomeless tissues than in tissues with trichromes,suggesting that this gene may inhibit the formation of trichomes in R.sterilis.Over-expression of RsETC1 in Arabidopsis resulted in glabrous phenotypes,and the expression of trichome-related endogenous genes,except for TTG1,was markedly reduced.In addition,the contents of the phytohormones jasmonic acid(JA),gibberellin A3(GA_(3)),and cytokinins(CKs)in pedicels were significantly higher than those in inflorescence stems,and the expression patterns of the genes related to hormone biosynthesis and signal transduction presented consistent responses,suggesting that the transduction of these hormones might be crucial for trichome initiation and development.These data provide a new perspective for revealing the molecular mechanism of trichome formation in R.sterilis.

Cloning and Characterization of EuGID1 in Eucommia ulmoides Oliver

Gibberellic acid controlled the key developmental processes of the life cycle of landing plants,and regulated the growth and development of plants.In this study,a novel gibberellin receptor gene EuGID1 was obtained from Eucommia ulmoides Oliver.The cDNA of EuGID1 was 1556 bp,and the open reading frame was 1029 bp,which encoded 343 amino acids.EuGID1 had the homology sequence with the hormone-sensitive lipase family.Amino acid sequence alignment confirmed EuGID1 protein had the highest homology with the GID1 protein of Manihot esculenta.EuGID1 was located in the nucleus and cell membrane and had expression in four plant organs.Overexpression of EuGID1 in transgenic Arabidopsis plants promoted plant elongation and increased siliques yield.

Genome-Wide Characterization of the Cellulose Synthase Gene Superfamily in Tea Plants(Camellia sinensis)

The cellulose synthase gene superfamily,including Cellulose synthase A(CesA)and cellulose synthase-like(Csl)gene families,is responsible for the synthesis of cellulose and hemicellulose,respectively.The CesA/Csl genes are vital for abiotic stress resistance and shoot tenderness regulation of tea plants(Camellia sinensis).However,the CesA/Csl gene family has not been extensively studied in tea plants.Here,we identified 53 CsCesA/Csl genes in tea plants.These genes were grouped into five subfamilies(CsCesA,CsCslB,CsCslD,CsCslE,CsCslG)based on the phylogenetic relationships with Arabidopsis and rice.The analysis of chromosome distribution,gene structure,protein domain and motif revealed that most genes in CsCesA,CsCslD and CsCslE subfamilies were conserved,whereas CsCslB and CsCslG subfamily members are highly diverged.The transcriptome analysis showed that most CsCesA/Csl genes displayed tissue-specific expression pattern.In addition,members of CsCslB4,CsCesA1/3/6,CsCslB3/4,CsCslD3,CsCslE1 and CsCslG2/3 subfamilies were up-regulated under drought and cold stresses,indicating their potential roles in regulating stress tolerance in tea plants.Furthermore,the expression levels of CsCslG2_6 and CsCslD3_5 in different tissues and cultivars,respectively,were positively correlated with the cellulose content that is negatively related with shoot tenderness.Thus,these two genes were speculated to be involved in the regulation of shoot tenderness in tea plants.Our findings may help elucidate the evolutionary relationships and expression patterns of the CsCesA/Csl genes in tea plants,and provide more candidate genes responsible for stress tolerance and tenderness regulation in tea plants for future functional research.

DWARF and SMALL SEED1,a Novel Allele of OsDWARF,Controls Rice Plant Architecture,Seed Size,and Chlorophyll Biosynthesis

Plant architecture is a vital agronomic trait to control yield in rice(Oryza sativa L.).A dwarf and small seed 1(dss1)mutant were obtained from the ethyl methanesulfonate(EMS)mutagenized progeny of a Guizhou glutinous landrace cultivar,Lipingzabianhe.The dss1 mutant displayed phenotypes similar to those of brassinosteroid(BR)deficient mutants,such as dwarfing,dark green and rugose erect leaves,small seeds,and loner neck internode panicles with primary branching.In our previous study,the underlying DSS1 gene was isolated,a novel allele of OsDWARF(OsBR6ox)that encodes a cytochrome P450 protein involved in the BR biosynthetic pathway by MutMap technology.In this work,we confirmed that a Thr335Ile amino acid substitution residing in DSS1/OsDWARF was responsible for the dwarf,panicle architecture,and small seed phenotypes in the dss1 mutants by genetic transformation experiments.The overexpression of OsDWARF in the dss1 mutant background could not only recover dss1 to the normal plant height and panicle architecture but also rescued normal leaf angles,seed size,and leaf color.Thus,the specific mutation in DSS1/OsDWARF influenced plant architecture,seed size,and chlorophyll biosynthesis.

Identification and Characterization of a Novel Yellow Leaf Mutant yl1 in Rice

Leaf-color mutants play an important role in the study of chlorophyll metabolism,chloroplast development,and photosynthesis system.In this study,the yellow leaf 1(yl1)rice mutant was identified from the ethyl methane sulfonate-treated mutant progeny of Lailong,a glutinous japonica rice landrace cultivated in Guizhou Province,China.Results showed that yl1 exhibited yellow leaves with decreased chlorophyll content throughout the growth period.Chloroplast development in the yl1 mutant was disrupted,and the grana lamellae was loosely packed and disordered.RNA sequencing and real-time quantitative polymerase chain reaction(qRT-PCR)analysis revealed that the chlorophyll synthesis-related genes OsCHLH,OsCHLM,OsCHLG,PORB,and YGL8,as well as the chloroplast development-related genes FtsZ,OsRpoTp,and RbcL,were down-regulated in the yl1 mutant.Genetic analysis revealed that the yellow leaf phenotype of yl1 was controlled by recessive nuclear gene.By employing the MutMap method,the mutation responsible for the phenotype was mapped to a 6.17 Mb region between 17.34 and 23.51 Mb on chromosome 3.Two non-synonymous single-nucleotide polymorphisms(SNPs)located in the gene locus LOC_Os03g31210 and LOC_Os03g36760 were detected in this region.The two SNPs were further confirmed by PCR and Sanger sequencing.The expression patterns of the two candidate genes indicated that LOC_Os03g36760 showed greater potential for functional verification.Subcellular protein localization revealed that the encoded product of LOC_Os03g36760 was localized in the nucleus,cytoplasm,and plasma membrane.These results will be useful for further characterization and cloning of the yl1 gene,and for research on the molecular mechanisms controlling biogenesis and chloroplast biochemical processes.

Indoor Toxicity Determination of Several Pesticides to Ceratovacuna lanigera( Zehntner)

Toxicity of 96% chlorpyrifos TC, 98% imidacloprid TC, 93% thiamethoxam TC and mixed pesticides of chlorpyrifos with other two agents （D ＋ B, D ＋ S） to C. lanigera （Zehntner） were determined under laboratory conditions by leaf dipping method. The results showed that all of the pesticides had high toxicity with the order of thiamethoxam TC 〉 imidacloprid TC 〉 D ＋ B 〉 chlorpyrifos TC 〉 D ＋ S. The results provided theoretic basis for selecting suitable insecti- cides to control the C. lanigera （Zehntner）.

Interference of Quinolinate Phosphoribosyltransferase Gene QPT Affects Agronomic Traits and Leaf Quality in Nicotiana tabacum L.

Quinolinate phosphoribosyltransferase(QPRTase),a key enzyme in ensuring nicotinic acid is available for the synthesis of defensive pyridine alkaloids in Nicotiana species,also plays an important role in nicotinamide adenine dinucleotide(NAD)biosynthesis.In this study,the morphological traits,the quality characteristics and photosynthetic parameters in QPT-overexpressing/interfering tobacco plants were investigated,respectively.Results showed that the interference of QPT gene not only reduced significantly the morphological traits including plant height,stem girth,leaf number and leaf length,etc.at 20 days after transplanting(DAT),but the flowering period was delayed 10-15 d in interfered tobaccos compared with the overexpressed,control and wild-type counterparts.However,at 40 DAT and 60 DAT,only three indexes(plant height,stem girth and leaf number)in QPT-interfering plants appeared significant difference in comparison with other three types of tobacco lines.Meanwhile,the determination results from nicotine,sugar,K+and Cl-content showed the nicotine content in interfered plants was always significantly lower than that in overexpressed plants,control and the wild-type ones respectively whatever toppling or not.At the same time,the toppling treatment also caused the increasement of K+content among the four different tobacco lines,but the maximum increase amplitude of K+content was found in QPT-overexpressing tobaccos while the minimum appeared in QPT-interfering plants.Finally,QPT-interference in transgenic tobaccos likewise affected the photosynthesis by reducing net photosynthetic rate(Pn),stomata conductance(Gs)and transpiration rate(Tr),while there was no significant difference between QPT-overexpressing plants and the controls and the wild-types.

A CRISPR/Cas9-based visual toolkit enabling multiplex integration at specific genomic loci in Aspergillus niger

Aspergillus niger is a highly versatile fungal strain utilized in industrial production.The expression levels of recombinant genes in A.niger can be enhanced by increasing the copy number.Nevertheless,given the prolonged gene editing cycle of A.niger,a“one-step”strategy facilitating the simultaneous integration of recombinant genes into multiple genomic loci would provide a definitive advantage.In our previous study,a visual multigene editing system(VMS)was designed to knock out five genes,employing a tRNA-sgRNA array that includes the pigment gene albA and the target genes.Building upon this system,hybrid donor DNAs(dDNAs)were introduced to establish a clustered regularly interspaced short palindromic repeats(CRISPR)-based multiplex integration toolkit.Firstly,a CRISPR-Cas9 homology-directed repair(CRISPR-HDR)system was constructed in A.niger by co-transforming the CRISPR-Cas9 plasmid(with a highly efficient sgRNA)and the dDNA,resulting in precise integration of recombinant xylanase gene xynA into the target loci(theβ-glucosidase gene bgl,the amylase gene amyA,and the acid amylase gene ammA).Subsequently,the length of homology arms in the dDNA was optimized to achieve 100%editing efficiency at each of the three gene loci.To achieve efficient multiplex integration in A.niger,the CRISPR plasmid pLM2 carrying a sgRNA-tRNA array was employed for concurrent double-strand breaks at multiple loci(bgl,amyA,ammA,and albA).Hybrid dDNAs were then employed for repair,including dDNA1-3(containing xynA expression cassettes without selection markers)and dDNAalbA(for albA knockout).Among the obtained white colonies(RLM2′),23.5%exhibited concurrent replacement of the bgl,amyA,and ammA genes with xynA(three copies).Notably,the xynA activity obtained by simultaneous insertion into three loci was 48.6%higher compared to that obtained by insertion into only the bgl locus.Furthermore,this multiple integration toolkit successfully enhanced the expression of endogenous pectinase pelA and Candida antarctica lipase CALB.Hence,the combined application of VMS and the CRISPR-HDR system enabled the simultaneous application of multiple selection markers,facilitating the rapid generation in the A.niger cell factories.