Insect Resistance Genes Research Articles

CULTIVARES E CARACTERÍSTICAS DO GRÃO DE MILHO

Maize (Zea mays L) is of global importance as a commodity because it is the main source of energy used in animal feed. The anatomy of corn is divided into endosperm (83%), pericarp (5%), germ (11%) and tip (2%). The starch granules and the protein matrix of the endosperm classifies corn into two types: farinaceous and vitreous. The relationship between hard endosperm (Flint) and floury (Dent) defines the vitreousness of a corn grain. Thus, the greater the vitreousness, the greater the amount of hard endosperm present in the grain. Today on the market there are specific corn cultivars for the production of grain, green corn, sweet corn, white corn (hominy), silage, popcorn, among others. The cultivars varieties are highly heterozygous, with greater productive stability, having greater genetic variability, less uniformity in the product and low productivity, caused by low heterosis, have great rusticity and adaptability, being indicated for low technology systems. Commercial hybrids are the result of the crossing between genetically distinct and homozygous individuals, for maximum heterosis response. Simple hybrids are potentially productive and suitable for systems that employ high technology. Transgenic hybrid corn is obtained by Genetic Engineering from the modification of DNA by introducing genes from other species through recombinant DNA techniques. Transgenics aims at three main targets, which can be classified as: herbicide tolerance; insertion of insect and disease resistance genes; and quality of products. White corn is a special cultivar that is characterized by having toothed and floury grain, it is widely used for the production of hominy, starch (cornstarch), flour and etc. Popcorn is a special cultivar of low productivity with rounded grains, fully vitreous endosperm, and thin film which allows spacing when the grain is heated. Green corn is an alternative to family horticulture because it adds more income, it refers to corn harvested while still in the milky stage, with 70 to 80% moisture. Sweet corn and super sweet corn are also included as green corn, differs from conventional corn in that it is intended for the canning industry and by the presence of mutant alleles that block the conversion of sugars into starch in the endosperm, giving it a sweet character, making sweet corn wrinkled and translucent when dry. Man has selected and developed several maize cultivars with distinct and special characteristics for each cropping system and purpose of use. The present study aims to address through a literature review the anatomical and nutritional characteristics of the maize grain and to address the different cultivars used in Brazilian agriculture.

Identification, Cloning, and Characterization of Two Acupuncture-Injury-Inducing Promoters in Rice

As an important global food crop, rice is damaged by a variety of piercing–sucking pests. Identifying a broad-spectrum promoter induced by the physical signal of sucking pests and applying it to transgenic breeding to mitigate the damage caused by different sucking pests will significantly improve the efficiency of our breeding. This study compared the transcriptome changes in two rice varieties under needle-wounding stress to investigate their differential responses to mechanical damage. The results showed that the insect-susceptible variety TN1 exhibited more differentially expressed genes (DEGs) and greater changes in expression levels after needle treatment, indicating a more active internal gene regulatory network. GO and KEGG enrichment analysis further revealed that TN1 not only exhibited changes in genes related to the extracellular environment, but also mobilized more genes associated with stress response and defense. By screening the differentially expressed genes, we identified two promoters (P1 and P2) with inducible expression characteristics in both the resistant and susceptible rice varieties. These promoters were able to effectively drive the expression of the insect resistance gene OsLecRK1* and enhance the resistance of transgenic plants against the brown planthopper. This study provides promoter resources for the development of insect-resistant transgenic crops.

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.

Optimization of Agrobacterium-mediated transformation conditions for efficient Bacillus thuringiensis (Bt) cotton transformation

Cotton is one of the world’s largest fiber-producing crops. It is also known as the important cash crop of Pakistan. Cotton crop products contribute to economy as cottonseed oil, animal feed and the fiber support the textile industries, thus, playing a significant role in uplifting the economy of a country. But over time, bollworm attacks had escalated, severely reducing the yield of cotton and damaging cotton production. Bt (Bacillus thuringiensis) cotton is being widely used nowadays due to its enhanced resistance against cotton bollworms which are responsible for reducing crop production leading to high economic loss. It greatly affects the economy by increasing the cost of production and protection. Using resistant strains of Bt cotton not only makes it easier to combat bollworm attacks but also reduces the substantial expense issues that farmers face. Bt has many toxic proteins among which the two crystal proteins, Cry1Ac and Cry2Ab, are used to develop resistance against bollworms in cotton. This study aimed to optimize the developing transgenic callus containing Cry1Ac and Cry2Ab genes in cotton. The dual gene construct (Cry1Ac + Cry2Ab) provides more resistance to the cotton crop as compared to the single gene construct against whom the most bollworms have developed resistance. The vector was inoculated into the hypocotyl segments and the transformation process was preceded by shifting the hypocotyls at different mediums Agrobacterium tumefaciens strain LBA4404 was used for cotton transformation. The construct-containing vector was introduced into a standard cotton line. A molecular confirmation test was carried out utilizing PCR and gene-specific primers after the formation of the transgenic callus. As a result of the research an indigenous cotton line that expresses double Bt genes for insect resistance was developed.

Salicylic acid inducing the expression of maize anti-insect gene SPI: a potential control strategy for Ostrinia furnacalis

BackgroundDue to being rooted in the ground, maize (Zea mays L.) is unable to actively escape the attacks of herbivorous insects such as the Asian corn borer (Ostrinia furnacalis). In contrast to the passive damage, plants have evolved defense mechanisms to protect themselves from herbivores. Salicylic acid, a widely present endogenous hormone in plants, has been found to play an important role in inducing plant resistance to insects. In this study, we screened and identified the insect resistance gene SPI, which is simultaneously induced by SA and O. furnacalis feeding, through preliminary transcriptome data analysis. The functional validation of SPI was carried out using bioinformatics, RT-qPCR, and heterologous expression protein feeding assays.ResultsBoth SA and O. furnacalis treatment increased the expression abundance of SA-synthesis pathway genes and SPI in three maize strains, and the upregulation of SPI was observed strongly at 6 hours post-treatment. The expression of SPI showed a temporal relationship with SA pathway genes, indicating that SPI is a downstream defense gene regulated by SA. Protein feeding assays using two different expression vectors demonstrated that the variation in SPI protein activity among different strains is mainly due to protein modifications.ConclusionsOur research results indicate that SPI, as a downstream defense gene regulated by SA, is induced by SA and participates in maize's insect resistance. The differential expression levels of SPI gene and protein modifications among different maize strains are one of the reasons for the variation in insect resistance. This study provides new insights into ecological pest control in maize and valuable insights into plant responses to SA-induced insect resistance.

Construction of Host Plant Insect-Resistance Mutant Library by High-Throughput CRISPR/Cas9 System and Identification of A Broad-Spectrum Insect Resistance Gene.

Insects pose significant challenges in cotton-producing regions. Here, they describe a high-throughput CRISPR/Cas9-mediated large-scale mutagenesis library targeting endogenous insect-resistance-related genes in cotton. This library targeted 502 previously identified genes using 968 sgRNAs, generated ≈2000 T0 plants and achieved 97.29% genome editing with efficient heredity, reaching upto 84.78%. Several potential resistance-related mutants (10% of 200 lines) their identified that may contribute to cotton-insect molecular interaction. Among these, they selected 139 and 144 lines showing decreased resistance to pest infestation and targeting major latex-like protein 423 (GhMLP423) for in-depth study. Overexpression of GhMLP423 enhanced insect resistance by activating the plant systemic acquired resistance (SAR) of salicylic acid (SA) and pathogenesis-related (PR) genes. This activation is induced by an elevation of cytosolic calcium [Ca2+ ]cyt flux eliciting reactive oxygen species (ROS), which their demoted in GhMLP423 knockout (CR) plants. Protein-protein interaction assays revealed that GhMLP423 interacted with a human epidermal growth factor receptor substrate15 (EPS15) protein at the cell membrane.Together, they regulated the systemically propagating waves of Ca2+ and ROS, which in turn induced SAR. Collectively, this large-scale mutagenesis library provides an efficient strategy for functional genomics research of polyploid plant species and serves as a solid platform for genetic engineering of insect resistance.

Co-expression of multi-gene in cotton promotes the aggregation of multi-resistance and yield traits

Given the diminishing availability of cultivable lands and environmental degration, it has become imperative to develop cotton varieties that possess resistance against diverse abiotic and biotic stresses, and improved yields. However, conventional breeding techniques such as crossbreeding has proven to be inefficient and time-consuming for generating novel varieties. Transgenic breeding technology offers advantages such as shortened breeding cycle, overcoming reproductive isolation, and precise integration of single or multiple genes into host plants. In this study, we incorporated six genes including the apoptosis inhibitors p35 and IAP, high yield related gene Bn-cs RRM2, drought resistance gene HDG11, glyphosate resistance gene EPSPS, and insect resistance gene Cry1AC into the pBI121 vector, which was then transformed into cotton plants via Agrobacterium-mediated transformation. The overexpression of these genes significantly enhanced plant tolerance to Verticillium wilt, drought, glyphosate, and cotton bollworm, along with high yields in field conditions. Our results suggested that the integration of multiple genes in cotton is a potential strategy for breeding cotton varieties with broad resistance traits.

A sub-chronic feeding study of dual toxin insect-resistant transgenic maize (CEMB-413) on Wistar rats.

Genetically modified (GM) crops expressing insecticidal crystal proteins are widely accepted worldwide, but their commercial utilization demands comprehensive risk assessment studies. A 90-day risk assessment study was conducted on Wistar rats fed with GM maize (CEMB-413) expressing binary insect-resistant genes (cry1Ac and cry2Ab) at low (30%) and high (50%) dose along with a control diet group. The study used fifty Wistar rats randomly distributed in five treatment groups. Our study revealed that compared to controls, GM diet had no adverse effects on animal's health, including body weight, food consumption, clinical pathological parameters, serum hormone levels and histological parameters of testes and ovaries of rats. Differences were observed in transcripts levels of fertility related genes, but these were independent of treatment with GM diet.

Transcriptome analysis revealed differentially expressed genes in rice functionally associated with brown planthopper defense in near isogenic lines pyramiding BPH14 and BPH15.

Although rice has many pests, brown planthopper (BPH) in particular is known to cause substantial damage. The pyramiding application of BPH-resistance genes BPH14 and BPH15 has proven effective in enhancing rice defense against BPH. However, the molecular mechanisms underlying BPH14/BPH15-conferred resistance remain unexplained. In this investigation, we analyzed the transcriptomes of near isogenic lines (NILs) containing either BPH14 (B14), BPH15 (B15), or BPH14/BPH15 (B1415), as well as their recurrent parent (RP) 'Wushansimiao'. In total, we detected 14,492 differentially expressed genes (DEGs) across 12 mRNA profiles of resistant NILs and RP at different feeding stages. In the transcriptomic analysis, 531 DEGs appeared to be common among the resistant NILs compared to RP before and after BPH feeding. These common DEGs were enriched in defense response, phosphorylation, and salt stress response. In addition, 258 DEGs shared only in resistant NILs were obtained among the different feeding stages, which were enriched in oxidative stress response, karrikin response, and chloroplast organization. Considering the expression patterns and relevant research reports associated with these DEGs, 21 were chosen as BPH resistance candidates. In rice protoplasts, the candidate DEG OsPOX8.1 was confirmed to increase reactive oxygen species (ROS) accumulation by chemiluminescence measurement. Our results provide valuable information to further explore the defense mechanism of insect-resistant gene pyramiding lines and develop robust strategies for insect control.

Recent Advances in Molecular Mechanism and Breeding Utilization of Brown Planthopper Resistance Genes in Rice: An Integrated Review.

Over half of the world's population relies on rice as their staple food. The brown planthopper (Nilaparvata lugens Stål, BPH) is a significant insect pest that leads to global reductions in rice yields. Breeding rice varieties that are resistant to BPH has been acknowledged as the most cost-effective and efficient strategy to mitigate BPH infestation. Consequently, the exploration of BPH-resistant genes in rice and the development of resistant rice varieties have become focal points of interest and research for breeders. In this review, we summarized the latest advancements in the localization, cloning, molecular mechanisms, and breeding of BPH-resistant rice. Currently, a total of 70 BPH-resistant gene loci have been identified in rice, 64 out of 70 genes/QTLs were mapped on chromosomes 1, 2, 3, 4, 6, 8, 10, 11, and 12, respectively, with 17 of them successfully cloned. These genes primarily encode five types of proteins: lectin receptor kinase (LecRK), coiled-coil-nucleotide-binding-leucine-rich repeat (CC-NB-LRR), B3-DNA binding domain, leucine-rich repeat domain (LRD), and short consensus repeat (SCR). Through mediating plant hormone signaling, calcium ion signaling, protein kinase cascade activation of cell proliferation, transcription factors, and miRNA signaling pathways, these genes induce the deposition of callose and cell wall thickening in rice tissues, ultimately leading to the inhibition of BPH feeding and the formation of resistance mechanisms against BPH damage. Furthermore, we discussed the applications of these resistance genes in the genetic improvement and breeding of rice. Functional studies of these insect-resistant genes and the elucidation of their network mechanisms establish a strong theoretical foundation for investigating the interaction between rice and BPH. Furthermore, they provide ample genetic resources and technical support for achieving sustainable BPH control and developing innovative insect resistance strategies.

A dual-RPA based lateral flow strip for sensitive, on-site detection of CP4-EPSPS and Cry1Ab/Ac genes in genetically modified crops

Traditional transgenic detection methods require high test conditions and struggle to be both sensitive and efficient. In this study, a one-tube dual recombinase polymerase amplification (RPA) reaction system for CP4-EPSPS and Cry1Ab/Ac was proposed and combined with a lateral flow immunochromatographic assay, named "Dual-RPA-LFD", to visualize the dual detection of genetically modified (GM) crops. In which, the herbicide tolerance gene CP4-EPSPS and the insect resistance gene Cry1Ab/Ac were selected as targets taking into account the current status of the most widespread application of insect resistance and herbicide tolerance traits and their stacked traits. Gradient diluted plasmids, transgenic standards, and actual samples were used as templates to conduct sensitivity, specificity, and practicality assays, respectively. The constructed method achieved the visual detection of plasmid at levels as low as 100 copies, demonstrating its high sensitivity. In addition, good applicability to transgenic samples was observed, with no cross-interference between two test lines and no influence from other genes. In conclusion, this strategy achieved the expected purpose of simultaneous detection of the two popular targets in GM crops within 20 min at 37 ℃ in a rapid, equipment-free field manner, providing a new alternative for rapid screening for transgenic assays in the field.

Transposon Polymorphism and Its Potential Impacts on Brown Planthopper (Nilaparvata lugens Stål) Resistance in Rice (Oryza sativa L.)

The brown planthopper (BPH) is a major pest in rice cultivation, significantly affecting both yield and quality; accordingly, exploring and utilizing anti-herbivory genes to enhance rice’s inherent resistance to BPH can be an effective strategy for mitigating infestation. The effects of transposon insertion polymorphisms (TIPs) on rice’s resistance to insect pests have not been reported. In this study, through the identification of transposon insertion sites in susceptible and resistant rice varieties, a total of six possible candidate insect resistance genes were potentially located. Among them, a segment of the LTR/Copia transposon insertion was verified in the promoter of LOC_Os04g02720, which carries a cis-acting element binding site in rice involved in the abscisic acid reaction. Quantitative analysis showed a significant difference of the gene expression between insect-resistant and insect-susceptible varieties (p < 0.05). This study provides insights into the functional analysis of transposons and population transposon polymorphisms, whereas the identification of candidate insect resistance genes offers a theoretical foundation for the development of insect-resistant rice varieties.

Application and Development of Bt Insect Resistance Genes in Rice Breeding

As pests are an important factor in reducing crop yields, pest control is an important measure in preventing reductions in crop yields. With the aim of ending the use of chemical pesticides, biological control and genetically modified methods are now considered more reasonable pest control strategies. The bacterium Bacillus thuringiensis (Bt) can produce crystal proteins that have specific toxicity to lepidopteran insects, and so it has been applied as a microbial insecticide in the control of crop pests for several decades. With the development of plant genetic engineering, Bt genes encoding insecticidal crystal protein have been introduced into many crop species for pest control. This article indicates that, after years of experiments and research, Bt transgenic rice is close to becoming a commercial insect-resistant rice, and many studies have shown that transgenic rice has pronounced abilities in the control of pests such as yellow stem borers (Scirpophaga incertulas, YSB), striped stem borers (Chilo suppressalis, SSB), and rice leaf rollers (Cnaphalocrocis medinalis, RLR); moreover, it does not obviously differ from non-transgenic rice in terms of safety. This paper suggests that transgenic Bt rice has application potential and commercial value.

Achieving High Expression of Cry in Green Tissues and Negligible Expression in Endosperm Simultaneously via rbcS Gene Fusion Strategy in Rice.

To allay excessive public concern about the safety of transgenic foods, and to optimize insect-resistant genes expression to delay the evolution of resistance in pests, we developed a promising strategy to fuse the GOI (gene of interest) with OsrbcS (rice small subunit of ribulose bisphosphate carboxylase/oxygenase) in transgenic rice, which acted as a carrier, driven by the OsrbcS native promoter to sequester its expression in green tissues. Using eYFP as a trial, we reported a high-level accumulation of eYFP in green tissue and almost none in the seed and root of the fused construct compared to the non-fused construct. After applying this fusion strategy in insect-resistant rice breeding, recombinant OsrbcS-Cry1Ab/Cry1Ac expressed rice plants conferred high resistance to leaffolders and striped stem borers, among which two single-copy lines possessed normal agronomic performance in the field. Specifically, Cry1Ab/Cry1Ac protein levels in single-copy construct transgenic lines ranged from 1.8 to 11.5 µg g-1 in the leaf, higher than the Actin I promoter-driven control, T51-1, about 1.78 µg g-1 in the leaf, but negligible (only 0.00012-0.00117 µg g-1) in endosperm by ELISA analysis. Our study provided a novel approach to creating Cry1Ab/Cry1Ac-free endosperm rice with a high level of insect-resistant protein in green tissues through the simultaneous usage of the OsrbcS promoter and OsrbcS as a fusion partner.

GmERF54, an ERF Transcription Factor, Negatively Regulates the Resistance of Soybean to the Common Cutworm (Spodoptera litura Fabricius)

Soybean is attacked by various herbivorous insect pest species during the whole course of its life cycle in the field. It is important for soybean production to improve insect resistance by identifying and utilizing soybean endogenous insect-resistant genes. The ethylene-responsive transcription factor (ERF) plays a significant role in plant biotic and abiotic stresses; however, few studies focus on its role in insect resistance in soybean. Here, based on our previous common cutworm (CCW)-induced soybean transcriptome data, a soybean ERF gene GmERF54 was cloned, which responded to CCW feeding. Transcriptional analysis revealed that GmERF54 was ubiquitous in all soybean tissues and was expressed differently in insect-resistant and insect-susceptible soybean cultivars. RNA interference of GmERF54 increased the resistance to CCW, while the overexpression of GmERF54 decreased the resistance to CCW in transgenic soybean hairy roots compared with their controls. GmERF54 was localized to the nucleus, had transcriptional activation activity, and interacted with AP2/ERF GmPLT2. Several putative hormone response elements were predicted in the promoter sequence of GmERF54. Four putative elements were only found in the GmERF54 promoter sequence of insect-resistant cultivar Wanxianbaidongdou (WX), but not in the insect-susceptible cultivar Nannong 99-10 (99-10). GmERF54 promoter sequences of WX and 99-10 were cloned into the pCAMBIA1381z vector containing the β-glucuronidase (GUS) gene to generate GmERF54WX:GUS and GmERF5499-10:GUS recombinant vectors, respectively. GUS staining of soybean hairy roots containing GmERF54WX:GUS and GmERF5499-10:GUS showed that GmERF54 was induced by CCW attack and both MeJA (methyl jasmonate) and IAA (indole-3-acetic acid) treatments. Alleles in insect-resistant and insect-sensitive cultivars responded to these inductions differently. Overall, our results reveal that GmERF54 may be involved in the regulation of soybean resistance to CCW.

Analysis of the Genetic Stability of Insect and Herbicide Resistance Genes in Transgenic Rice Lines: A Laboratory and Field Experiment

A lack of stability in the expression of Bacillus thuringiensis genes (CRY) and the dialaninophosphate resistance gene (BAR) in transgenic rice plants can lead to the loss of important characters. The genetic stability of transgenic expression in high-generation lines is thus critically important for ensuring the success of molecular breeding efforts. Here, we studied the genetic stability of resistance to insect pests and herbicides in transgenic rice lines at the molecular and phenotypic levels in a pesticide-free environment. Southern blot analysis, real-time polymerase chain reaction, and enzyme-linked immunosorbent assays revealed high stability in the copy numbers and expression levels of CRY1C, CRY2A, and BAR in transgenic lines across different generations, and gene expression levels were highly correlated with protein expression levels. The insecticide resistance of the transgenic rice lines was high. The larval mortality of Chilo suppressalis was 50.25% to 68.36% higher in transgenic lines than in non-transgenic control lines. Percent dead hearts and percent white spikelets were 16.66% to 22.15% and 27.07% to 33.47% lower in transgenic lines than in non-transgenic control lines, respectively. The herbicide resistance of the transgenic rice lines was also high. The bud length and root length ranged were 2.53 cm to 4.20 cm and 0.28 cm to 0.73 cm higher in transgenic lines than in non-transgenic control lines in the budding stage, respectively. Following application of the herbicide Basta, the chlorophyll content of the transgenic lines began to recover 2 d later in the seedling and tillering stages and 3 d later in the booting and heading stages, by contrast, the chlorophyll content of the non-transgenic lines did not recover and continued to decrease. These findings revealed high genetic stability of the resistance to insect pests and herbicides across several generations of transgenic rice regardless of the genetic background.

Identification and Evaluation of Insect and Disease Resistance in Transgenic Cry1Ab13-1 and NPR1 Maize

PCR detection, quantitative real-time PCR (q-RTPCR), outdoor insect resistance, and disease resistance identification were carried out for the detection of genetic stability and disease resistance through generations (T2, T3, and T4) in transgenic maize germplasms (S3002 and 349) containing the bivalent genes (insect resistance gene Cry1Ab13-1 and disease resistance gene NPR1) and their corresponding wild type. Results indicated that the target genes Cry1Ab13-1 and NPR1 were successfully transferred into both germplasms through tested generations; q-PCR confirmed the expression of Cry1Ab13-1 and NPR1 genes in roots, stems, and leaves of tested maize plants. In addition, S3002 and 349 bivalent gene-transformed lines exhibited resistance to large leaf spots and corn borer in the field evaluation compared to the wild type. Our study confirmed that Cry1Ab13-1 and NPR1 bivalent genes enhanced the resistance against maize borer and large leaf spot disease and can stably inherit. These findings could be exploited for improving other cultivated maize varieties.

Genome-Wide Identification of Brassica napus PEN1-LIKE Genes and Their Expression Profiling in Insect-Susceptible and Resistant Cultivars.

Recently, it has been reported that a gene (PEN1) in Arabidopsis thaliana is highly resistant to Plutella xylostella. We screened all the homologous genes of PEN1 in Arabidopsis thaliana and found that the motif of these genes was very conserved. At present, few insect resistance genes have been identified and characterized in Brassica napus. Therefore, we screened all the homologous genes containing this motif in the Brassica napus genome and systematically analyzed the basic information, conserved domain, evolutionary relationship, chromosomal localization and expression analysis of these genes. In this study, 12 PEN1 homologous genes were identified in the Brassica napus genome, which is more than the number in Arabidopsis thaliana. These genes are unevenly distributed on the 12 chromosomes in Brassica napus. Furthermore, all the PEN1 homologous genes contained light responsiveness elements, and most of the genes contained gibberellin-responsive elements, meJA-responsive elements and abscisic-acid-responsive elements. The results will provide a theoretical basis for screening insect resistance genes from the genome of Brassica napus and analyzing the molecular mechanism of insect resistance in Brassica napus.

Soybean CALCIUM-DEPENDENT PROTEIN KINASE17 Positively Regulates Plant Resistance to Common Cutworm (Spodoptera litura Fabricius).

Soybean is frequently attacked by herbivorous pests throughout the growth period. Exploring anti-insect genes to improve insect resistance in soybean is an important soybean breeding goal. Here, we cloned and characterized the gene for a quantitative trait locus (QTL) related to insect resistance, Glyma.06g189600, which encodes CALCIUM-DEPENDENT PROTEIN KINASE17 (GmCDPK17) in soybean. The pairwise sequence alignment analysis revealed that the presumed protein of GmCDPK17 shares 52.06% similarity with that of GmCDPK38, a known negative regulatory gene of insect resistance in soybean. Ectopic expression of GmCDPK17 and GmCDPK38 restored the phenotypes of the Arabidopsis insect-susceptible mutant cpk10 and insect-resistant mutant cpk28, respectively. Moreover, transgenic hairy roots of the soybean cultivar Jack were generated by Agrobacterium-mediated transformation. Overexpression of GmCDPK17 increased soybean hairy root resistance to common cutworm (CCW), while RNA interference of the gene decreased soybean hairy root resistance to CCW. Sequencing data from the cultivated and wild soybeans were used to analyze the genetic diversity of GmCDPK17. This gene was subjected to domestication selection. Six and seven haplotypes (Haps) were identified in cultivated and wild soybeans, respectively. The resistance Hap1 is not widely used in cultivated soybeans and is mainly distributed at low latitudes. Accessions with resistance haplotypes of the GmCDPK17 and GmCDPK38 genes showed high resistance to CCW. Altogether, we revealed a novel positive regulatory insect resistance gene, GmCDPK17, which may further improve insect resistance in soybean.

Differential assembly of root-associated bacterial and fungal communities of a dual transgenic insect-resistant maize line at different host niches and different growth stages.

Transgenic technology has been widely applied to crop development, with genetically modified (GM) maize being the world’s second-largest GM crop. Despite the fact that rhizosphere bacterial and fungal populations are critical regulators of plant performance, few studies have evaluated the influence of GM maize on these communities. Plant materials used in this study included the control maize line B73 and the mcry1Ab and mcry2Ab dual transgenic insect-resistant maize line 2A-7. The plants and soils samples were sampled at three growth stages (jointing, flowering, and maturing stages), and the sampling compartments from the outside to the inside of the root are surrounding soil (SS), rhizospheric soil (RS), and intact root (RT), respectively. In this study, the results of alpha diversity revealed that from the outside to the inside of the root, the community richness and diversity declined while community coverage increased. Morever, the different host niches of maize rhizosphere and maize development stages influenced beta diversity according to statistical analysis. The GM maize line 2A-7 had no significant influence on the composition of microbial communities when compared to B73. Compared to RS and SS, the host niche RT tended to deplete Chloroflexi, Gemmatimonadetes and Mortierellomycota at phylum level. Nitrogen-fixation bacteria Pseudomonas, Herbaspirillum huttiense, Rhizobium leguminosarum, and Sphingomonas azotifigens were found to be enriched in the niche RT in comparison to RS and SS, whilst Bacillus was found to be increased and Stenotrophomonas was found to be decreased at the maturing stage as compared to jointing and flowering stages. The nitrogen fixation protein FixH (clusters of orthologous groups, COG5456), was found to be abundant in RT. Furthermore, the pathogen fungus that causes maize stalk rot, Gaeumannomyces radicicola, was found to be abundant in RT, while the beneficial fungus Mortierella hyalina was found to be depleted in RT. Lastly, the abundance of G. radicicola gradually increased during the development of maize. In conclusion, the host niches throughout the soil-plant continuum rather than the Bt insect-resistant gene or Bt protein secretion were primarily responsible for the differential assembly of root-associated microbial communities in GM maize, which provides the theoretical basis for ecological agriculture.