Studies of Transgenic Hybrid Poplar 741 Carrying Two Insect-resistant Genes

Partially modified Bt Cry1Ac gene and the arrowhead proteinase inhibitor (API) gene were used to construct a plant transformation vector pBtiA and this construct was transferred into the genome of the hybrid poplar 741 [ Populus alba L.×( P. davidiana Dode+ P. simonii Carr.)× P. tomentosa Carr.] by Agrobacterium _ mediated transformation. Ten kanamycin resistant plants have been regenerated. Upon insect bioassay using Clostera anachoreta (Fabricius), three of the examined plants were demonstrated to be highly resistant to the testing insects. The mortality of insect larvae on one plant was higher than 90% in 6 days after infestation and the growth of the survival larvae were seriously inhibited. Results of PCR and Southern blot analysis indicated that both Bt Cry1Ac gene and API gene were integrated as a single copy into the genomes of these three plants when Cry1Ac gene fragment was used as the probe. Protein dot blot immunoassay and ELISA analysis revealed that at least the Cry1Ac protein was produced in these three transgenic plants and the expression levels were estimated to be approximately 0.015% of the leaf total soluble protein. This is the first report on insect resistant transgenic hybrid poplar 741 that expresses two insecticidal protein genes.

Selection of Homozygous Cotton Lines Transformed with Two Insect-Resistant Genes

A plant expression vector containing a chimeric Bt29K gene coding for the activated Cry1Ac protein and the arrowhead proteinase inhibitior gene API B were introduced into the cotton cultivar Jihe321 mediated by Agrobactertium tumefaciens. Based on the results of kanamycin resistant testing, PCR detection for both foreign genes and insect bioassay using Heliethis armigera , nine transgenic homozygous cotton lines with insect resistance of more than 90% and better agronomic traits were bred through six generations from the original transgenic plants. Results from insect bioassay and sequence analysis of the PCR products of plants from some homozygous lines indicated that the chimeric Bt29K gene was stably inherited in these transgenic cotton lines. The main agronomic characters of these homozygous cotton lines, such as boll productivity and fibre strength, were better than that of the original cotton cv. Jihe321.

Isolation,Mutagenesis,Identification and Cellulase Properties of a Mulberry Branches Decomposing Fungus from Insect

In order to utilize mulberry branches efficiently and reduce environment pollution, a strain named A1 was isolated from the intestinal tract of Acr/da Chinensis using Congo red staining method on the plate of carboxymethyl cellulose sodium （CMC-Na） medium and mulberry branch powder culture medium. The strain A1 was mutagenized by uhraviolet radiation and the strain Z3 with a significantly improved specific activity of filter paper enzyme was screened. Enzymatic characteristics, degradation ability of cellulose and taxonomic status of A1 and Z3 were also studied in this paper. The results showed that filter paper enzyme specific activity of Z3 increased 74.47% than that of A1 and the property of enzyme production was steady. The optimum pH values of filter paper enzyme produced by A1 and Z3 were 4. 6 and 3.8 respectively and the optimum temperatures were 45 and 50 ℃, respectively. When CMC-Na was used as substrate, Vmax of the two strains was similar while Km of Z3 was lower than that of A1 , which indicated that Z3 had a strong affinity for the substrate. Enzymes produced by Z3 were more resistant to high temperature. Strains A1 and Z3 could grow well on the culture mediums with mulberry branch powder as carbon source, but Z3 grew better than A1. A1 and Z3 were preliminarily identified as AspergiUus according to the morphological identification.

The mRNA Expression Profiles of Five Heat Shock Protein Genes from Frankliniella occidentalis at Different Stages and Their Responses to Temperatures and Insecticides

The westem flower thrips, Frankliniella occidental＆ （Pergande） is a highly invasive pest that is able to exploit many crops across a wide range of environmental conditions. Five full-length cDNAs of heat shock protein （HSP） genes （Fo-HSP90, Fo-HSP70, Fo-HSP60, Fo-HSP40 and Fo-HSP28.9） were cloned from F. occidentalis, and their expression profiles were investigated under conditions of thermal stress and insecticide exposure, and at different stages during development, using real-time quantitative PCR. All five gene sequences showed high similarity to homologs in other species, indicating the conserved fimction of this gene family. HSP60 represents an informative phylogenetic marker at the ordinal taxonomic level within Insecta, but HSP90, which has two homologous copies in Hymenoptera, was not informative. The expression of Fo-HSPs under thermal stress suggests that Fo-HSP90, Fo-HSP70, and Fo-HSP28.9 are inducible by both cold and heat stress, Fo-HSP40 is only heat-inducible, and Fo-HSP60 is thermally insensitive. There were two patterns of cold induction of Fo-HSPs： one is from 0 to 4℃ and the other is around -8℃. All five Fo-HSPs genes were induced by exposure to sublethal concentrations of the insecticide avermectin. The expression of the five Fo-HSPs during different developmental stages suggests that they all play a role in development of F. occidentalis.

Secretive Expression of Insect Antifungal Peptide-Encoded Genes in Pichia pastoris and Activity Assay of the Products

The antifungal peptides, drosomycin （Drs） and its isoform drosomycin-like C （Drs-lC） from Drosophila melanogaster and Thanatin from Podisus maculiventris, have potent activity with broad spectrum against filamentous fungi. Secretive expression of these genes in yeasts makes it possible to utilize the supernatants of yeast culture as protective reagents on fruit, vegetable, food and other agricultural products. So the study of effective secretion by yeast expression system is of great importance. Three genes, Drs, Drs-lC, and Thanatin, were cloned into pPICZαA and the recombinant vectors, pPICZαA-Drs, pPICZαA-Drs-lC, and pPICZαA-Thanatin were transformed into Pichia pastoris by the electric transfer method. The recombinant P. pastoris, which was screened by phenotype selection and PCR amplification, was induced to express antifungal peptide by methanol. The expressive products of the three recombinants showed antifungal activity against 5 out of 6 test fungi strains, and the products of Thanatin also had strong activity against the tested bacteria. The three antifungal peptide genes, Drs, Drs-lC, and Thanatin, were constructed into yeast P. pastoris. The expressed peptides were successfully secreted into the culture medium and exhibited potent activities against the test strains.

Construction of Plant Expression Vector Carrying Two Insecticidal Genes and Obtain Insect resistant Transgenic Tobacco Plants

A plant expression vector carrying both pea lectin gene and Soybean trypsin inhibitor gene has been constructed and transferred into tobacco via Agrobacterium mediated transformation. Transgenic plants are further confirmed by ELISA, PCR and PCR Southern assays. Results of bioassays show that transgenic plants display notably inhibitory effects to larvae development and survival of Heliothis armigera Hubner.

Breeding and Identification of Insect-Resistant Rice by Transferring Two Insecticidal Genes,sbk and sck

The plasmid of pCDMARUBA-Hyg, which contained two insect-resistance genes, sbk （modified from CrylA（c）） and sck （modified from CpTI）, was transformed into an Agrobacterium EHA105 for infection of the calli of a super japonica rice Nanjing 45. Primarily, using polymerase chain reaction （PCR） detection with the primers of sbk and sck genes, 42 positive transgenic plants that were marker-free and contained the two target genes were selected from 97 regenerated plants. Results of southern-blotting indicated that 23, 11, 5, 2 and 1 plants had one, two, three, four and five copies of the transformed genes, respectively. Analysis of reverse transcription PCR （RT-PCR） and Bt gene testing paper showed that 28 T3 generation plants derived from four transgenic plants having a single copy were insect-resistant. Feeding experiment with rice stem borer revealed that the insect resistance was greatly increased with the larva mortality ranging from 94% to 100%. In addition, among the transgenic plants, three T3 transgenic plants possessed some desirable characteristics for breeding and production, such as plant height, seed-setting rate, 1000-grain weight and larva mortality. The mechanism of insect resistance of Bt .qene and its application in rice transgenic research were also briefly discussed.

Insect resistance management in Bacillus thuringiensis cotton by MGPS(multiple genes pyramiding and silencing)

The introduction of Bacillus thuringiensis(Bt)cotton has reduced the burden of pests without harming the environment and human health.However,the efficacy of Bt cotton has decreased due to field-evolved resistance in insect pests over time.In this review,we have discussed various factors that facilitate the evolution of resistance in cotton pests.Currently,different strategies like pyramided cotton expressing two or more distinct Bt toxin genes,refuge strategy,releasing of sterile insects,and gene silencing by RNAi are being used to control insect pests.Pyramided cotton has shown resistance against different cotton pests.The multiple genes pyramiding and silencing(MGPS)approach has been proposed for the management of cotton pests.The genome information of cotton pests is necessary for the development of MGPS-based cotton.The expression cassettes against various essential genes involved in defense,detoxification,digestion,and development of cotton pests will successfully obtain favorable agronomic characters for crop protection and production.The MGPS involves the construction of transformable artificial chromosomes,that can express multiple distinct Bt toxins and RNAi to knockdown various essential target genes to control pests.The evolution of resistance in cotton pests will be delayed or blocked by the synergistic action of high dose of Bt toxins and RNAi as well as compliance of refuge requirement.

Comprehensive analysis of the potential pathogenesis of COVID-19 infection and liver cancer

BACKGROUND A growing number of clinical examples suggest that coronavirus disease 2019(COVID-19)appears to have an impact on the treatment of patients with liver cancer compared to the normal population,and the prevalence of COVID-19 is significantly higher in patients with liver cancer.However,this mechanism of action has not been clarified.Gene sets for COVID-19(GSE180226)and liver cancer(GSE87630)were obtained from the Gene Expression Omnibus database.After identifying the common differentially expressed genes(DEGs)of COVID-19 and liver cancer,functional enrichment analysis,protein-protein interaction network construction and scree-ning and analysis of hub genes were performed.Subsequently,the validation of the differential expression of hub genes in the disease was performed and the regulatory network of transcription factors and hub genes was constructed.RESULTS Of 518 common DEGs were obtained by screening for functional analysis.Fifteen hub genes including aurora kinase B,cyclin B2,cell division cycle 20,cell division cycle associated 8,nucleolar and spindle associated protein 1,etc.,were further identified from DEGs using the“cytoHubba”plugin.Functional enrichment analysis of hub genes showed that these hub genes are associated with P53 signalling pathway regulation,cell cycle and other functions,and they may serve as potential molecular markers for COVID-19 and liver cancer.Finally,we selected 10 of the hub genes for in vitro expression validation in liver cancer cells.CONCLUSION Our study reveals a common pathogenesis of liver cancer and COVID-19.These common pathways and key genes may provide new ideas for further mechanistic studies.

Identification of hub genes associated with Helicobacter pylori infection and type 2 diabetes mellitus:A pilot bioinformatics study

BACKGROUND Helicobacter pylori(H.pylori)infection is related to various extragastric diseases including type 2 diabetes mellitus(T2DM).However,the possible mechanisms connecting H.pylori infection and T2DM remain unknown.AIM To explore potential molecular connections between H.pylori infection and T2DM.METHODS We extracted gene expression arrays from three online datasets(GSE60427,GSE27411 and GSE115601).Differentially expressed genes(DEGs)commonly present in patients with H.pylori infection and T2DM were identified.Hub genes were validated using human gastric biopsy samples.Correlations between hub genes and immune cell infiltration,miRNAs,and transcription factors(TFs)were further analyzed.RESULTS A total of 67 DEGs were commonly presented in patients with H.pylori infection and T2DM.Five significantly upregulated hub genes,including TLR4,ITGAM,C5AR1,FCER1G,and FCGR2A,were finally identified,all of which are closely related to immune cell infiltration.The gene-miRNA analysis detected 13 miRNAs with at least two gene cross-links.TF-gene interaction networks showed that TLR4 was coregulated by 26 TFs,the largest number of TFs among the 5 hub genes.CONCLUSION We identified five hub genes that may have molecular connections between H.pylori infection and T2DM.This study provides new insights into the pathogenesis of H.pylori-induced onset of T2DM.

Insecticide induction of O-demethylase activity and expression of cytochrome P450 genes in the red imported fire ant (Solenopsis invicta Buren)

The red imported fire ant （Solenopsis invicta） is a global major invasive pest, and has caused significant economic, social and environmental impacts since its invasion to mainland of China in 2004. To date, chemical control has been the most effective measure. However, the long-term use of chemicals would lead to an unexpected rebound. To understand the risks and explore the mechanisms of detoxification or induction to insecticides in S. invicta, the O-demethylase activity and expression of cytochrome P450 genes of workers and queens, and the effects of chlorpyrifos and fipronil exposure in workers were investigated. Biochemical assays showed the O-demethylase activity of cytochrome P450 was significantly higher in workers than in queens （1.66-fold）, and was significantly induced in workers exposed to chlorpyrifos and fipronil, reaching a maximum （3.00- and 1.95-fold） at 48 h and then decreasing dramatically compared to controls （exposed to acetone counter- part）. The relative expression levels of 12 cytochrome P450 expressed sequence tags （ESTs） in workers were significantly higher than in queens （from 2.3- to 36.4-fold）. Multiple cytochrome P450 genes （except 9E4） were co-up-regulated （from 1.5- to 2.86-fold） in workers exposed to fipronil. These results indicated that the increased O-demethylase activity may result from the increased transcription levels of cytochrome P450 related to detoxification of insecticides in S. invicta. It appears that cytochrome P450 plays an important role in enhanced metabolic detoxification of insecticides. At the same time, it also provides the theoretical basis for resistance management and rational usage of insecticides to control S. invicta.

Development of "Multi-Resistance Rice" by Pyramiding of Insect (Cry1C) and Blast Resistance (Pi1 and Pi2) Genes

[Objective] The paper was to improve the blast resistance of insect-resistant transgenic rice. [Method] The Japonica rice variety Jikang10, a new transgenic variety with exogenous insect-resistant gene Cry1C, was used as the receptor, and Kongyu 131, a traditional breeding variety with broad-spectrum high blast resistance genes Pi1 and Pi2, was used as the donor to breeding new rice varieties. The genes were polymerized by hybridization and multi-generation backcrossing, and the offspring of each generation was screened by molecular marker assisted selection, field identification of multi-resistance against insect pests and diseases and agronomic trait selection. [Result] Four lines SK01, SK02, SK03 and SK04 with better resistances to insect pests and blast and outstanding agronomic traits in field were selected. [Conclusion] The results will lay foundations for breeding new multi-resistance rice varieties in Huanghuai rice region.

Key genes and regulatory networks for diabetic retinopathy based on hypoxia-related genes:a bioinformatics analysis

AIM:To prevent neovascularization in diabetic retinopathy(DR)patients and partially control disease progression.METHODS:Hypoxia-related differentially expressed genes(DEGs)were identified from the GSE60436 and GSE102485 datasets,followed by gene ontology(GO)functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.Potential candidate drugs were screened using the CMap database.Subsequently,a protein-protein interaction(PPI)network was constructed to identify hypoxia-related hub genes.A nomogram was generated using the rms R package,and the correlation of hub genes was analyzed using the Hmisc R package.The clinical significance of hub genes was validated by comparing their expression levels between disease and normal groups and constructing receiver operating characteristic curve(ROC)curves.Finally,a hypoxia-related miRNA-transcription factor(TF)-Hub gene network was constructed using the NetworkAnalyst online tool.RESULTS:Totally 48 hypoxia-related DEGs and screened 10 potential candidate drugs with interaction relationships to upregulated hypoxia-related genes were identified,such as ruxolitinib,meprylcaine,and deferiprone.In addition,8 hub genes were also identified:glycogen phosphorylase muscle associated(PYGM),glyceraldehyde-3-phosphate dehydrogenase spermatogenic(GAPDHS),enolase 3(ENO3),aldolase fructose-bisphosphate C(ALDOC),phosphoglucomutase 2(PGM2),enolase 2(ENO2),phosphoglycerate mutase 2(PGAM2),and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3).Based on hub gene predictions,the miRNA-TF-Hub gene network revealed complex interactions between 163 miRNAs,77 TFs,and hub genes.The results of ROC showed that the except for GAPDHS,the area under curve(AUC)values of the other 7 hub genes were greater than 0.758,indicating their favorable diagnostic performance.CONCLUSION:PYGM,GAPDHS,ENO3,ALDOC,PGM2,ENO2,PGAM2,and PFKFB3 are hub genes in DR,and hypoxia-related hub genes exhibited favorable diagnostic performance.

31 Plant Species against Blood Feeding and Disease Vectors Insects: Beyond Anti-Insect Properties, Unvalued Opportunities and Challenges for Health and Sustainability

Plants with bioactive properties are greatly useful in preventing and controlling blood-sucking and disease-vector invertebrates, particularly in developing countries and low-income communities. Their application is a promising alternative to synthetic compounds whose use remains a health, environmental, and economic challenge. However, many are still unknown and unvalued, while others are becoming ignored and threatened. The main objective of this ethnobotanical study is to identify and characterize indigenous and locally grown plants against blood-sucking and disease-vector insects. Salient opportunities and challenges of using these plants are documented and discussed. Semi-structured interviews, using a prepared questionnaire, were conducted with 228 informants. The consensus index (CI) was calculated to analyze the reliability of the collected information. The identified 31 anti-insect plant species belong to 20 botanical families, four morphological categories, and six habitat types. They can be categorized as insecticidal plants (42% of the total), insect repellent (42% of the total), and both insecticidal and insect repellent (16% of the total). More than 54% of these are still abundant in the study area, while about 35.5% have become rare and difficultly accessible. Based on the numerical importance of related anti-insect plant species, the seven targeted blood-sucking insects range in the following decreasing order: Jiggers (16 species) > Fire Ants (9 species) > Flies (8 plants) > Mosquitoes (4 species) > Fleas (2 species) > Bedbugs (1 species) > lice (0 species). The three most commonly used plants, with the highest confirmation indices, are Tetradenia riparia (ICs = 0.712), Eucalyptus globulus subsp. maidenii (ICs = 0.302), and Solanum aculeastrum (ICs = 0.288). The antimicrobial role of many locally grown anti-insect plants and the multiple other associated valorization possibilities are ignored by most informants. Domesticating, propagating, protecting, and promoting the sustainable use of these plants would be an appropriate route for their conservation and continued availability.

Identification and Validation of Vascular-Associated Biomarkers for the Prognosis and Potential Pathogenesis of Hypertension Using Comprehensive Bioinformatics Methods

Background: Hypertension, also known as increased blood pressure, is a phenomenon in which blood flows in blood vessels and causes persistently higher-than-normal pressure on the vessel wall. The identification of novel prognostic and pathogenesis biomarkers plays a key role in the management of hypertension. Methods: The GSE7483 and GSE75815 datasets from the gene expression omnibus (GEO) database were used to identify the genes associated with hypertension that were differentially expressed genes (DEGs). The functional role of the DEGs was elucidated by gene body (GO) enrichment analysis. In addition, we performed an immune infiltration assay and GSEA on the DEGs of hypertensive patients and verified the expression of novel DEGs in the blood of hypertensive patients by RT-qPCR. Results: A total of 267 DEGs were identified from the GEO database. GO analysis revealed that these genes were associated mainly with biological processes such as fibroblast proliferation, cell structural organization, extracellular matrix organization, vasculature development regulation, and angiogenesis. We identified five possible biomarkers, Ecm1, Sparc, Sphk1, Thbsl, and Mecp2, which correlate with vascular development and angiogenesis characteristic of hypertension by bioinformatics, and explored the clinical expression levels of these genes by RT-qPCR, and found that Sparc, Sphk1, and Thbs1 showed significant up-regulation, in agreement with the results of the bioinformatics analysis. Conclusion: Our study suggested that Sparc, Sphk1 and Thbs1 may be potential novel biomarkers for the diagnosis, treatment and prognosis of hypertension and that they are involved in the regulation of vascular development and angiogenesis in hypertension.

Mining elite loci and candidate genes for root morphology-related traits at the seedling stage by genome-wide association studies in upland cotton(Gossypium hirsutum L.)

Root system architecture plays an essential role in water and nutrient acquisition in plants,and it is significantly involved in plant adaptations to various environmental stresses.In this study,a panel of 242 cotton accessions was collected to investigate six root morphological traits at the seedling stage,including main root length(MRL),root fresh weight(RFW),total root length(TRL),root surface area(RSA),root volume(RV),and root average diameter(AvgD).The correlation analysis of the six root morphological traits revealed strong positive correlations of TRL with RSA,as well as RV with RSA and AvgD,whereas a significant negative correlation was found between TRL and AvgD.Subsequently,a genome-wide association study(GWAS)was performed using the root phenotypic and genotypic data reported previously for the 242 accessions using 56,010 single nucleotide polymorphisms(SNPs)from the CottonSNP80K array.A total of 41 quantitative trait loci(QTLs)were identified,including nine for MRL,six for RFW,nine for TRL,12 for RSA,12 for RV and two for AvgD.Among them,eight QTLs were repeatedly detected in two or more traits.Integrating these results with a transcriptome analysis,we identified 17 candidate genes with high transcript values of transcripts per million(TPM)≥30 in the roots.Furthermore,we functionally verified the candidate gene GH_D05G2106,which encodes a WPP domain protein 2in root development.A virus-induced gene silencing(VIGS)assay showed that knocking down GH_D05G2106significantly inhibited root development in cotton,indicating its positive role in root system architecture formation.Collectively,these results provide a theoretical basis and candidate genes for future studies on cotton root developmental biology and root-related cotton breeding.

Minerals in edible insects:a review of content and potential for sustainable sourcing

In response to the rapid increase in world population and subsequent demands for food,edible insects represent an alternative food source for humans that is rich in proteins,amino acids and minerals.Entomophagy is a tradition in many countries including China and Thailand,and edible insects have attracted a lot of attention in Western World due to their suitable nutrient composition,high mineral content(e.g.,Fe,Zn,Ca,Mg)and potential use as a supplement in human diet.In this study,we surveyed mineral content in seven insect orders and 67 species of mass produced and wild-harvested edible insects.The total content of essential elements in edible insects was very high in Tenebrio molitor,Bombyx mori,and Zonocerus variegatus.The heavy metal content(summarized for eight species)was below the maximum limit allowed for safe consumption.Sustainable supply of minerals derived from insect biomass is complicated due to the high variations of mineral content in insects and the potential of its change due to processing.

Twenty-Three Years of Insect Pollinator-Dependent Crop Studies in Agro-Ecological Zones of Cameroon (2011-2020)

Cultivation of pollinator-dependent crops has expanded globally, increasing our reliance on insect pollination. This essential ecosystem service is provided by a wide range of managed and wild pollinators whose abundance and diversity are thought to be in decline, threatening sustainable food production. In Cameroon, several studies on pollinator-dependent crops carried out in different agro-ecological zones (AEZ) have been published in national and international journals, in order to present the importance and impact of flowering insects on fruit and seed yields of plant species. We proposed to produce a review article highlighting the different flowering insects and their importance for different plants according to AEZ, without however focusing on the quality of the journal (predator or non-predator) and how the different insects were identified (scientific names given in the publications). Thus, from 1997 to 2020, we collected 116 published papers from which only 26 were kept for this review. The results show that Hymenoptera, including the Apidae, followed by Megachilidae, are the most excellent pollinators of plant species in Cameroon, and they are present in different agro-ecological zones. The majority of publications focused on bees, particularly the honeybee Apis mellifera.

A Review on Characteristics, Extraction Methods and Applications of Renewable Insect Protein

Due to the expected rise in the world population,an increase in the requirements for quality and safety of food and feed is expected,which leads to the growing demand for new sources of sustainable and renewable protein.Insect protein is gaining importance as a renewable material for several reasons,reflecting its potential contribu-tions to sustainability,resource efficiency,and environmental conservation.Some insect species are known to be able to efficiently convert organic waste into high-value products such as protein,requiring less land and water compared to traditional livestock.In addition,insect farming produces fewer greenhouse gas emissions,contri-buting to mitigating climate change.Insects are considered as a major potential alternative to animal or plant protein due to their many nutritional benefits,including high protein,mineral,and vitamin contents.On average,the protein content of insects ranges between 35%and 60%dry weight,which exceeds plant protein sources,such as cereal,soybeans,and lentils.As the acceptance of insect protein grows and technologies advance,the food and feed industries continue to explore and expand their applications,offering consumers diverse and sustainable pro-tein choices.In this review,we discuss the recentfindings relating to insect protein focusing on its characteristics,extraction methods,applications,and opportunities along with some trade-offs and uncertainties.

CRISPR-Cas9-mediated construction of a cotton CDPK mutant library for identification of insect-resistance genes

Calcium-dependent protein kinases(CDPKs)act as key signal transduction enzymes in plants,especially in response to diverse stresses,including herbivory.In this study,a comprehensive analysis of the CDPK gene family in upland cotton revealed that GhCPKs are widely expressed in multiple cotton tissues and respond positively to various biotic and abiotic stresses.We developed a strategy for screening insect-resistance genes from a CRISPR-Cas9 mutant library of GhCPKs.The library was created using 246 single-guide RNAs targeting the GhCPK gene family to generate 518 independent T0 plants.The average target-gene coverage was 86.18%,the genome editing rate was 89.49%,and the editing heritability was 82%.An insect bioassay in the field led to identification of 14 GhCPK mutants that are resistant or susceptible to insects.The mutant that showed the clearest insect resistance,cpk33/74(in which the homologous genes GhCPK33 and GhCPK74 were knocked out),was selected for further study.Oral secretions from Spodoptera litura induced a rapid influx of Ca2+in cpk33/74 leaves,resulting in a significant increase in jasmonic acid content.S-adenosylmethionine synthase is an important protein involved in plant stress response,and protein interaction experiments provided evidence for interactions of GhCPK33 and GhCPK74 with GhSAMS1 and GhSAM2.In addition,virus-induced gene silencing of GhSAMS1 and GhSAM2 in cotton impaired defense against S.litura.This study demonstrates an effective strategy for constructing a mutant library of a gene family in a polyploid plant species and offers valuable insights into the role of CDPKs in the interaction between plants and herbivorous insects.