A rapid and high-throughput system for the detection of transgenic products based on LAMP-CRISPR-Cas12a.

With the increasing acreage of genetically modified crops worldwide, rapid and efficient detection technologies have become very important for the regulation and screening of GM organisms. We constructed a method based on loop-mediated isothermal amplification (LAMP), CRISPR-Cas12a and lateral flow assay (LAMP-CRISPR-Cas12a-LFA). It is an intuitive, sensitive and specific fluorescence detection and test strip system to detect CP4-EPSPS and Cry1Ab/Ac genes in field screening. The LAMP-CRISPR-Cas12a-LFA method has a limit of detection (LOD) of 100 copies based on lateral flow test strips after optimization of the conditions with screened specific primers, and the entire detection process can be completed within 1 h at 61 °C. The system was used to evaluate field test samples and showed high reproducibility after testing products containing CP4-EPSPS and Cry1Ab/Ac genes, and both were detectable. The LAMP-CRISPR-Cas12a-LFA method established in this paper functions as a rapid field detection method. It requires only one portable thermostatic instrument, which renders it compatible with the rapid detection of field samples and useable at experimental workstations, in law enforcement field work, and in local inspection and quarantine departments.

A Rapid and Visual Method for Nucleic Acid Detection of Escherichia coli O157:H7 Based on CRISPR/Cas12a-PMNT.

Rapid, accurate and visual point-of-care testing (POCT) methods for pathogenic bacteria detection are essential for avoiding foodborne diseases caused by pathogens or their toxins. In this study, we proposed a rapid and visual detection method that we named "Cas12aVIP". By combining recombinase polymerase amplification (RPA), a CRISPR/Cas12a system and a cationic-conjugated polythiophene derivative (poly[3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride] (PMNT) mixed with single-stranded DNA (ssDNA)), the solution turned red in the absence of the target DNA based on conformational modifications of the conjugated backbone of PMNT, whereas it displayed yellow, thus realizing the colorimetric detection of DNA. The Cas12aVIP method yielded high specificity and no interference from other nontargeted bacteria. The detection was accomplished in 40 min and the signal could be observed by the naked eye under natural light, presenting great potential for a variety of rapid nucleic acid detection applications without requiring technical expertise or ancillary equipment.

A rapid and high-throughput Helicobacter pylori RPA-CRISPR/Cas12a-based nucleic acid detection system.

BACKGROUND: Helicobacter pylori lives in the human stomach and causes gastric cancer and other gastric diseases. The development of molecular technology has facilitated low-cost, rapid, and high-throughput detection of H. pylori. MATERIALS AND METHODS: The combination of isothermal recombinase polymerase amplification (RPA) and CRISPR-Cas12a was used for early diagnosis and monitoring of H. pylori in clinical settings. The UreB genes from 242 H. pylori strains were subjected to cluster analysis, and we designed corresponding RPA primers and screened 2 sets of CRISPR-derived RNAs (crRNAs) for accurate H. pylori recognition. We then performed specificity and sensitivity validation of seven strains using this RPA-CRISPR/Cas12a method. In addition, the cut-off values of this RPA-CRISPR/Cas12a method based on fluorescence values (i.e., RPA-CRISPR/Cas12a-FT) were determined by comparison with quantitative PCR (qPCR), and further experiments comparing different methods were performed using clinical samples. RESULTS: We developed a rapid detection system based on the combination of RPA and CRISPR-Cas12a, which was applied to the early diagnosis and monitoring of H. pylori in clinical settings. The RPA-CRISPR/Cas12a system was used to detect the UreB gene. We found that the limit of detection (LOD) for the CRISPR/Cas12a method based on the lateral flow dipstick result (i.e., CRISPR/Cas12a-LFD) was 100 copies, the cut-off value was 1.4; and for CRISPR/Cas12a-FT the LOD was 50 copies. This system was used to assess clinical samples and showed high reproducibility with proof-of-concept sensitivity, and the whole detection process was completed within 40 min. CONCLUSION: As a diagnostic method that can detect the UreB gene of H. pylori in gastric tissue samples rapidly, sensitively, visually, and in a high throughput manner, our method provides a new diagnostic option for clinicians. This system is ideal for hospitals or testing sites with limited medical resources.

RNAi-based pest control: Production, application and the fate of dsRNA.

The limitations of conventional pesticides have raised the demand for innovative and sustainable solutions for plant protection. RNA Interference (RNAi) triggered by dsRNA has evolved as a promising strategy to control insects in a species-specific manner. In this context, we review the methods for mass production of dsRNA, the approaches of exogenous application of dsRNA in the field, and the fate of dsRNA after application. Additionally, we describe the opportunities and challenges of using nanoparticles as dsRNA carriers to control insects. Furthermore, we provide future directions to improve pest management efficiency by utilizing the synergistic effects of multiple target genes. Meanwhile, the establishment of a standardized framework for assessment and regulatory consensus is critical to the commercialization of RNA pesticides.

Isolation, Classification, and Growth-Promoting Effects of Pantoea sp. YSD J2 from the Aboveground Leaves of Cyperus Esculentus L. var. sativus.

Plant growth-promoting (PGP) bacteria are an environmental-friendly alternative to chemical fertilizers for promoting plant development. We isolated and characterized a PGP endophyte, YSD J2, from the leaves of Cyperus esculentus L. var. sativus. Specific PGP characteristics of this strain, such as phosphate solubilization ability, potassium-dissolving ability, siderophore and indole-3-acetic acid (IAA) production, and salt tolerance, were determined in vitro. In addition, positive mutants were screened using the atmospheric and room-temperature plasma (ARTP) technology, with IAA level and organic phosphorus solubility as indices. Furthermore, the effect of the positive mutant on biomass production and antioxidant abilities of greengrocery seedling was evaluated and the genome was mined to explore the underlying mechanisms. The strain YSD J2 showed a good performance of PGP characteristics, such as the production of indole acetic acid and siderophores, solubilization ability of phosphate, and potassium-dissolving ability. It was recognized through 16S rRNA sequencing together with morphological and physiological tests and confirmed as Pantoea sp. The strain exposed to a mutation time of 125 s by ARTP had the highest IAA and organic phosphate (lecithin) concentrations of 10.34 mg/L and 16.52 mg/L, 42.06% and 34.15% higher than those of the initial strain. Inoculation of mutant strain YSD J2 significantly increased plant growth attributes and the activities of peroxidase and superoxide dismutase, respectively, but decreased the content of malondialdehyde significantly compared with the control. Furthermore, genome annotation and functional analysis were performed through whole-genome sequencing and PGP-related genes were identified. Our results indicated that the YSD J2 with PGP characteristics is a potential candidate for the development of biofertilizers.

Weighted gene co-expression network analysis unveils gene networks regulating folate biosynthesis in maize endosperm.

Folates are essential elements for human growth and development, and their deficiency can lead to serious disorders. Waxy maize is a rich source of folates; however, the regulatory mechanism underlying folate biosynthesis in the endosperm remains unclear. Here, we examined changes in the folate content of maize endosperm collected at 15, 18, 21, 24, and 27 days after pollination (DAP) using liquid chromatograph-mass spectrometry and identified genes related to folate biosynthesis using transcriptome sequencing data. The results showed that 5-methyl-tetrahydrofolate and 5,10-methylene tetrahydrofolate were the main storage forms of folates in the endosperm, and their contents were relatively high at 21-24 days. We also identified 569, 3183, 4365, and 5513 differentially expressed genes (DEGs) in different days around milk stage. Functional annotation revealed 518 transcription factors (TFs) belonging to 33 families exhibiting specific expression in at least one sampling time. The key hub genes involved in folate biosynthesis were identified by weighted gene co-expression network analysis. In total, 24,976 genes were used to construct a co-expression network with 29 co-expression modules, among which the brown and purple modules were highly related to folate biosynthesis. Further, 187 transcription factors in the brown and purple modules were considered potential transcription factors related to endosperm folate biosynthesis. These results may improve the understanding of the molecular mechanism underlying folate biosynthesis in waxy maize and lead to the development of nutritionally fortified varieties. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02974-7.

Comparative Phylogenomic Analysis Reveals Evolutionary Genomic Changes and Novel Toxin Families in Endophytic Liberibacter Pathogens.

Liberibacter pathogens are the causative agents of several severe crop diseases worldwide, including citrus Huanglongbing and potato zebra chip. These bacteria are endophytic and nonculturable, which makes experimental approaches challenging and highlights the need for bioinformatic analysis in advancing our understanding about Liberibacter pathogenesis. Here, we performed an in-depth comparative phylogenomic analysis of the Liberibacter pathogens and their free-living, nonpathogenic, ancestral species, aiming to identify major genomic changes and determinants associated with their evolutionary transitions in living habitats and pathogenicity. Using gene neighborhood analysis and phylogenetic classification, we systematically uncovered, annotated, and classified all prophage loci into four types, including one previously unrecognized group. We showed that these prophages originated through independent gene transfers at different evolutionary stages of Liberibacter and only the SC-type prophage was associated with the emergence of the pathogens. Using ortholog clustering, we vigorously identified two additional sets of genomic genes, which were either lost or gained in the ancestor of the pathogens. Consistent with the habitat change, the lost genes were enriched for biosynthesis of cellular building blocks. Importantly, among the gained genes, we uncovered several previously unrecognized toxins, including new toxins homologous to the EspG/VirA effectors, a YdjM phospholipase toxin, and a secreted endonuclease/exonuclease/phosphatase (EEP) protein. Our results substantially extend the knowledge of the evolutionary events and potential determinants leading to the emergence of endophytic, pathogenic Liberibacter species, which will facilitate the design of functional experiments and the development of new methods for detection and blockage of these pathogens. IMPORTANCELiberibacter pathogens are associated with several severe crop diseases, including citrus Huanglongbing, the most destructive disease to the citrus industry. Currently, no effective cure or treatments are available, and no resistant citrus variety has been found. The fact that these obligate endophytic pathogens are not culturable has made it extremely challenging to experimentally uncover the genes/proteins important to Liberibacter pathogenesis. Further, earlier bioinformatics studies failed to identify key genomic determinants, such as toxins and effector proteins, that underlie the pathogenicity of the bacteria. In this study, an in-depth comparative genomic analysis of Liberibacter pathogens along with their ancestral nonpathogenic species identified the prophage loci and several novel toxins that are evolutionarily associated with the emergence of the pathogens. These results shed new light on the disease mechanism of Liberibacter pathogens and will facilitate the development of new detection and blockage methods targeting the toxins.

A sensitive immunosensor based on graphene-PAMAM composites for rapid detection of the CP4-EPSPS protein in genetically modified crops.

A simple electrochemical immunosensor based on nitrogen-doped graphene and polyamide-amine (GN-PAM) composites was proposed for the detection of the CP4-EPSPS protein in genetically modified (GM) crops. In this immunosensor, the amplification of the detection signal was realized through antibodies labeled with gold nanoparticles (AuNPs). The electrochemical responses of the immunosensor were linear (R2 = 0.9935 and 0.9912) when the GM soybean RRS and maize NK603 content ranged from 0.025% to 1.0% and 0.05% to 1.5%, respectively. The limits of detection for the GM soybean RRS and maize NK603 were as low as 0.01% and 0.03%, respectively. The immunosensor also exhibited high specificity, and satisfactory stability, reproducibility, and accuracy. Our findings indicated that the constructed immunosensor provides a new approach for the sensitive detection of the CP4-EPSPS protein. Notably, the sensor may be applied to other proteins or pathogenic bacteria by simply changing the antibodies, and may also be used for multi-component analysis.

Development of a lateral flow test strip for simultaneous detection of BT-Cry1Ab, BT-Cry1Ac and CP4 EPSPS proteins in genetically modified crops.

A colloidal gold immunochromatographic strip (ICS) for simultaneous detection of multiple transgenic proteins, including CP4 EPSPS, BT-Cry1Ab and BT-Cry1Ac, was developed in this study. The sensitivity of the strip to the target protein was 5 ng/mL for CP4 EPSPS, 100 ng/mL for BT-Cry1Ab and Cry1Ac, respectively. Parallel analysis for maize, soybean, sugar beet and cotton showed the strip could detect 1% of transgenic content in crops containing BT-Cry1Ab and Cry1Ac, and, at least, 0.1% of content in crops containing CP4 EPSPS. The detection results for seed samples indicated the multicomponent analysis ICS had good accuracy. The analysis could be completed within 10 min and had the advantages of being high-throughput, easy to operate and visual detection. This is the first report of semi-quantitative ICS for detecting three transgenic proteins simultaneously. The developed approach may provide insights into the development of ICS for analyzing simultaneously multiple components in genetically modified crops.

Comparative analysis reveals the expansion of mitochondrial DNA control region containing unusually high G-C tandem repeat arrays in Nasonia vitripennis.

Insect mitochondrial DNA (mtDNA) ranges from 14 to 19 kbp, and the size difference is attributed to the AT-rich control region. Jewel wasps have a parasitoid lifestyle, which may affect mitochondria function and evolution. We sequenced, assembled, and annotated mitochondrial genomes in Nasonia and outgroup species. Gene composition and order are conserved within Nasonia, but they differ from other parasitoids by two large inversion events that were not reported before. We observed a much higher substitution rate relative to the nuclear genome and mitochondrial introgression between N. giraulti and N. oneida, which is consistent with previous studies. Most strikingly, N. vitripennis mtDNA has an extremely long control region (7665 bp), containing twenty-nine 217 bp tandem repeats and can fold into a super-cruciform structure. In contrast to tandem repeats commonly found in other mitochondria, these high-copy repeats are highly conserved (98.7% sequence identity), much longer in length (approximately 8 Kb), extremely GC-rich (50.7%), and CpG-rich (percent CpG 19.4% vs. 1.1% in coding region), resulting in a 23 kbp mtDNA beyond the typical size range in insects. These N. vitripennis-specific mitochondrial repeats are not related to any known sequences in insect mitochondria. Their evolutionary origin and functional consequences warrant further investigations.

RPA-Cas12a-FS: A frontline nucleic acid rapid detection system for food safety based on CRISPR-Cas12a combined with recombinase polymerase amplification.

Food analysis to ensure food safety and quality are relevant to all countries. This study aimed to develop a detection technique by combining recombinase polymerase amplification with CRISPR-Cas12a for food safety (termed RPA-Cas12a-FS). Our data showed that this novel method could be detected via fluorescence intensity for the molecular identification of foodborne pathogenic bacteria, genetically modified crops, and meat adulteration. After optimization, the sensitivity and stability of RPA-Cas12a-FS was further enhanced. The RPA-Cas12a-FS system could specifically detect target gene levels as low as 10 copies in 45 min at 37 °C. The RPA-Cas12a-FS system was sensitive both using standard samples in the lab and using samples from the field, which indicated that this detection method was practical. In conclusion, a simple, rapid, and highly sensitive detection method based on CRISPR-Cas12a was developed for molecular identification in the food safety field without requiring technical expertise or ancillary equipment.

Identification and Functional Characterization of a Novel Immunomodulatory Protein From Morchella conica SH.

A novel fungal immunomodulatory protein (FIP) was found in the precious medical and edible mushroom Morchella conica SH, defined as FIP-mco, which belongs to the FIP family. Phylogenetic analyses of FIPs from different origins were performed using Neighbor-Joining method. It was found that FIP-mco belonged to a new branch of the FIP family and may evolved from a different ancestor compared with most other FIPs. The cDNA sequence of FIP-mco was cloned and expressed in the yeast Pichia Pastoris X33. The recombinant protein of FIP-mco (rFIP-mco) was purified by agarose Ni chromatography and determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The protein rFIP-mco could significantly suppress the proliferation of A549 and HepG2 cells at the concentration of 15 and 5 µg/ml, respectively, and inhibited the migration and invasion of human A549 and HepG2 cells at the concentration of 15 and 30 µg/ml respectively in vitro. Further, rFIP-mco can significantly reduce the expression levels of TNF-α, IL-1ß, and IL-6 in the THP1 cells (human myeloid leukemia mononuclear cells). In order to explore the potential mechanism of the cytotoxicity effect of rFIP-mco on A549 and HepG2 cells, cell cycle and apoptosis assay in the two cancer cells were conducted. The results demonstrated that G0/G1 to S-phase arrest and increased apoptosis may contribute to the proliferation inhibition by rFIP-mco in the two cancer cells. Molecular mechanism of rFIP-mco's reduction effect on the inflammatory cytokines was also studied by suppression of the NF-κB signaling pathway. It showed that suppression of NF-κB signaling is responsible for the reduction of inflammatory cytokines by rFIP-mco. The results indicated the prospect of FIP-mco from M. conica SH as an effective and feasible source for cancer therapeutic studies and medical applications.

Production and characterization of the 13 C/15 N single-labeled insecticidal protein Cry1Ab/Ac using recombinant Escherichia coli.

Synthetic Cry1Ab/Ac proteins expressed by genetically modified (GM) crops have a high potential to control insect pests without utilizing large amounts of chemical insecticides. Before these crops are used in agriculture, the environmental fate and interactions in the soil must be understood. Stable isotope-labeled Cry1Ab/Ac protein is a highly useful tool for collecting such data. We developed a protocol to produce 13 C/15 N single-labeled Cry proteins. The artificially synthesized gene Cry1Ab/Ac of Bt rice Huahui No. 1, which has been certified by the Chinese government to be safe for human consumption, was subcloned into pUC57, and the expression vector pET-28a-CryAb/Ac was constructed and transformed into Escherichia coli BL21 (DE3) competent cells. Next, 0.2 mM isopropyl thiogalactoside (IPTG) was added to these cells and cultured at 37°C for 4 h to induce the synthesis and formation of inclusion bodies in M9 growth media containing either [U-13 C] glucose (5% 13 C-enriched) or [15 N] ammonium chloride (5% 15 N-enriched). Then, Cry inclusion bodies were dissolved in urea and purified by affinity chromatography under denaturing conditions, renatured by dialysis, and further detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The purities of 13 C/15 N-labeled Cry proteins reached 99% with amounts of 12.6 mg/L and 8.8 mg/L, respectively. The δ 13 C and ä 15 N values of 13 C-labeled Cry protein and 15 N-labeled Cry protein were 3,269‰ and 2,854‰, respectively. A bioassay test revealed that the labeled Cry1Ab/Ac proteins had strong insecticidal activity. The stable isotope-labeled insecticidal Cry proteins produced for the first time in this study will provide an experimental basis for future metabolic studies on Cry proteins in soil and the characteristics of nitrogen (N) and carbon (C) transformations. Our findings may also be employed as a reference for elucidating the environmental behavior and ecological effects of BT plants and expressed products.

Laser Mutagenesis of Phellinus igniarius Protoplasts for the Selective Breeding of Strains with High Laccase Activity.

Phellinus igniarius is a medicinal fungus that utilizes lignin as a nutrient substrate. This fungus has a weak lignin degradation ability and, as a result, a slow growth rate. Laccases are crucial enzymes for lignin degradation in P. igniarius, and thus, the cultivation of strains with high laccase activity is expected to increase the growth rate of P. igniarius. To generate P. igniarius strains with high laccase activity, we performed laser mutagenesis of P. igniarius protoplasts and screened for mutants with high laccase activity. Our results showed that the laser power density and P. igniarius protoplast survival rate exhibited a power-function relationship. The power density threshold value between lethality and growth promotion was 0.24 mW/mm2. Mutagenesis was carried out using a laser beam diameter of 3 mm and an irradiation period of 40 min. After five generations of selection, we identified a high laccase activity strain, termed SJZ2. The laccase activity in SJZ2 during 4 h of fermentation was increased by 36.84% in comparison with the control and ranged from 0.20216 to 0.27664 U. The Km and Vmax of the laccase produced by SJZ2 were 0.21 mmol/mL and 0.53 mmol/L/min, respectively. This study demonstrated the feasibility of laser mutagenesis of P. igniarius protoplasts for the selection of high laccase activity. This study characterized the key factors in the laser mutagenesis process of P. igniarius protoplasts and provided a reference for the application of lasers in biological mutagenesis. Future studies should evaluate the bioactive functionality and stability of this novel strain of P. igniarius, particularly the organoleptic and medical characteristics of the fruiting bodies.

The response of dominant and rare taxa for fungal diversity within different root environments to the cultivation of Bt and conventional cotton varieties.

BACKGROUND: Bacillus thuringiensis (Bt) crops have been cultivated at a large scale over the past several decades, which have raised concern about unintended effects on natural environments. Microbial communities typically contain numerous rare taxa that make up the majority of community populations. However, the response of dominant and rare taxa for fungal diversity to the different root environments of Bt plants remains unclear. RESULTS: We quantified fungal population sizes and community composition via quantitative PCR of ITS genes and 18S rRNA gene sequencing of, respectively, that were associated with Bt and conventional cotton variety rhizosphere soils from different plant growth stages. qPCR analyses indicated that fungal abundances reached their peak at the seedling stage and that the taproots and lateral root rhizospheres of the Bt cotton SGK321 were significantly different. However, no significant differences in population sizes were detected between the same root zones from Bt and the conventional cotton varieties. The overall patterns of fungal genera abundances followed that of the dominant genera, whereas overall patterns of fungal genera richness followed those of the rare genera. These results suggest that the dominant and rare taxa play different roles in the maintenance of rhizosphere microhabitat ecosystems. Cluster analyses indicated a separation of fungal communities based on the lateral roots or taproots from the three cotton varieties at the seedling stage, suggesting that root microhabitats had marked effects on fungal community composition. Redundancy analyses indicated that pH was more correlated to soil fungal community composition than Bt protein content. CONCLUSIONS: In conclusion, these results indicate that dominant and rare fungal taxa differentially contribute to community dynamics in different root microhabitats of both Bt and conventional cotton varieties. Moreover, these results showed that the rhizosphere fungal community of Bt cotton did not respond significantly to the presence of Bt protein when compared to the two conventional cotton varieties.

Cultivation of Drought-Tolerant and Insect-Resistant Rice Affects Soil Bacterial, but Not Fungal, Abundances and Community Structures.

The impacts of rice varieties with stacked drought tolerance and insect resistance on soil microbiomes are poorly understood. Hence, the objective of this study was to investigate the effects resulting from the cultivation of the drought-tolerant and insect-resistant rice cultivar, Hanhui3T, on soil physical-chemical properties, and bacterial and fungal community composition. Soil samples of Hanhui3T and conventional rice varieties (Hanhui3 and Zhonghua11) were collected in triplicate at the booting stage, and bacterial and fungal population sizes and community structures were assessed using qPCR and Illumina MiSeq sequencing, respectively. The Bt protein concentration of Hanhui3T was significantly higher than that of Hanhui3 and Zhonghua11, while the pH of Hanhui3T was significantly lower. Bacterial population sizes and community composition were significantly different between Hanhui3T and Hanhui3 (or Zhonghua11), while no similar effects were observed for fungal communities. These differences suggest that the effect of Hanhui3T cultivation on bacterial community composition is stronger than the effect on fungal communities. Moreover, bacterial abundance was positively correlated to soil pH, while bacterial community structure variations were mainly driven by soil pH and Bt protein concentration differences. In conclusion, the abundances and structure of bacterial communities were altered in rhizosphere with Hanhui3T cultivation that changed soil pH and Bt protein concentrations, while fungal communities displayed no such responsiveness.

Analysis and Evaluation of the Characteristic Taste Components in Portobello Mushroom.

To identify the characteristic taste components of the common cultivated mushroom (brown; Portobello), Agaricus bisporus, taste components in the stipe and pileus of Portobello mushroom harvested at different growth stages were extracted and identified, and principal component analysis (PCA) and taste active value (TAV) were used to reveal the characteristic taste components during the each of the growth stages of Portobello mushroom. In the stipe and pileus, 20 and 14 different principal taste components were identified, respectively, and they were considered as the principal taste components of Portobello mushroom fruit bodies, which included most amino acids and 5'-nucleotides. Some taste components that were found at high levels, such as lactic acid and citric acid, were not detected as Portobello mushroom principal taste components through PCA. However, due to their high content, Portobello mushroom could be used as a source of organic acids. The PCA and TAV results revealed that 5'-GMP, glutamic acid, malic acid, alanine, proline, leucine, and aspartic acid were the characteristic taste components of Portobello mushroom fruit bodies. Portobello mushroom was also found to be rich in protein and amino acids, so it might also be useful in the formulation of nutraceuticals and functional food. PRACTICAL APPLICATION: The results in this article could provide a theoretical basis for understanding and regulating the characteristic flavor components synthesis process of Portobello mushroom.

Impact of transgenic Cry1Ac + CpTI cotton on diversity and dynamics of rhizosphere bacterial community of different root environments.

The objective of this study was to characterize the diversity and dynamics of rhizosphere bacterial community, especially the response of dominant and rare bacterial taxa to the cultivation of Bt cotton for different root environments at different growth stages. qPCR analyses indicated that bacterial abundances of the taproots and lateral root rhizospheres of the Bt cotton SGK321 were significantly different at seedling and bolling stages. But no significant differences were detected between the same root zones from Bt and the conventional cotton varieties. Total bacterial genera had similar pattern with dominant genera in abundance, and with rare genera in richness to the changes of bacterial community, respectively. Although the rhizosphere bacterial diversity of the three cotton varieties changed in taproot and lateral root, no significant differences were detected in the same root environments between Bt and conventional cotton. Moreover, Soil pH was more correlated with variations in the bacterial community composition than Bt proteins. In conclusion, these results revealed no indication that rhizosphere bacterial community of Bt cotton had different response to increased Bt protein regarding the same root environment. In particular, dominant and rare bacterial taxa showed the variation in diversity and community composition in different root microhabitats.