chiA Gene Research Articles

Analysis of chitinase gene family in barley and function study of HvChi22 involved in drought tolerance.

Chitinase (Chi) is a pathogenesis-related protein, also reported to play an important role in plant responses to abiotic stress. However, its role in response to abiotic stress in barley is still unclear. In this study, a total of 61 Chi gene family members were identified from the whole genome of wild barley EC_S1. Phylogenetic analysis suggested that these family genes were divided into five groups. Among these genes, four pairs of collinearity genes were discovered. Besides, abundant cis-regulatory elements, including drought response element and abscisic acid response element were identified in the promoter regions of HvChi gene family members. The expression profiles revealed that most HvChi family members were significantly up-regulated under drought stress, which was also validated by RT-qPCR measurements. To further explore the role of Chi under drought stress, HvChi22 was overexpressed in Arabidopsis. Compared to wild-type plants, overexpression of HvChi22 enhanced drought tolerance by increasing the activity of oxidative protective enzymes, which caused less MDA accumulation. Our study improved the understanding of the Chi gene family under drought stress in barley, and provided a theoretical basis for crop improvement strategies to address the challenges posed by changing environmental conditions.

Response analysis of Pinus sibirica to pine wood nematode infection through transcriptomics and metabolomics study.

Pinus sibirica is primarily distributed in Siberia. Owing to its excellent cold resistance and development potential, it has become an important introduced tree species in the Greater Xing'an area of China. Pine wilt disease, triggered by the pine wood nematode (PWN, Bursaphelenchus xylophilus), constitutes a profoundly critical affliction within forest ecosystems. Its incidence has extended to the northeastern region of China in recent years. To explore the potential host status of P. sibirica in the Greater Xing'an area for PWN and to elucidate the responses following inoculation, artificial inoculation, transcriptomics, and metabolomics methods were used. In the artificial inoculation experiments, quantitative analysis of nematode populations within the trees demonstrated that PWN exhibited normal growth and reproductive capabilities within P. sibirica. Subsequently, transcriptome and metabolome sequencing were conducted at four time points before disease onset (3-, 5-, 7-, and 9-days post inoculation). Gene trend analysis and differentially expressed gene screening were employed and the results indicated that genes associated with the flavonoid biosynthesis pathway exhibited predominant enrichment among the up-regulated genes. Metabolome analysis showed that the abundance of flavonoid-related metabolites in P. sibirica increased after inoculation with PWN. Integrated analysis of transcriptome and metabolome revealed that after PWN inoculation in P. sibirica, two chalcone synthase (chs) genes and a chalcone isomerase (chi) gene were significantly upregulated, and the upregulation should accumulate naringenin, pinocembrin, and apigenin to help P. sibirica resist infection of PWN. The results suggested that flavonoid biosynthesis pathway continued to respond after P. sibirica was infected with PWN and played an important role in the interaction between P. sibirica and PWN.

In vitro clonal propagation of nicotiana tabacum with modified medium and studying its gene alteration through bioinformatic analysis

Tobacco has medicinal uses and was used as an insecticide. Also, tobacco holds social and custom noteworthiness in different social orders so hereditary modifications and change ponders can be effectively analyzed within the test plant due to its simple genome courses of action. In this study, we treated tobacco seeds with EMS chemical and analyzed the changes in the DNA mutation compared with the control. Total nine gene sequences were taken such as AP2, CAD, CHI, PIP1, LTP1, QPT, RBOH, UPL5, PR1 to study the DNA alternations, through sequencing and comparison with mutated genes. Gene mutation was determined using the bioinformatics tool namely Bioedit. From the result obtained in bioedit were higher mutation rate was observed in PR1 followed by AP2 and CHI gene. All gene sequences were mutated except RBOH and UPL5. This preliminary study showed the effects of EMS on treating tobacco plants. This genetic alternation study can be used in constructing highly efficient tobacco lines against future TILLING studies.

Reference genome of the nutrition-rich orphan crop chia (Salvia hispanica) and its implications for future breeding.

Chia (Salvia hispanica L.) is one of the most popular nutrition-rich foods and pseudocereal crops of the family Lamiaceae. Chia seeds are a rich source of proteins, polyunsaturated fatty acids (PUFAs), dietary fibers, and antioxidants. In this study, we present the assembly of the chia reference genome, which spans 303.6 Mb and encodes 48,090 annotated protein-coding genes. Our analysis revealed that ~42% of the chia genome harbors repetitive content, and identified ~3 million single nucleotide polymorphisms (SNPs) and 15,380 simple sequence repeat (SSR) marker sites. By investigating the chia transcriptome, we discovered that ~44% of the genes undergo alternative splicing with a higher frequency of intron retention events. Additionally, we identified chia genes associated with important nutrient content and quality traits, such as the biosynthesis of PUFAs and seed mucilage fiber (dietary fiber) polysaccharides. Notably, this is the first report of in-silico annotation of a plant genome for protein-derived small bioactive peptides (biopeptides) associated with improving human health. To facilitate further research and translational applications of this valuable orphan crop, we have developed the Salvia genomics database (SalviaGDB), accessible at https://salviagdb.org.

The effect of osmotic stress, lighting spectrum and temperature on growth and gene expression related to anthocyanin biosynthetic pathway in wild strawberry (Fragaria vesca L.) in vitro

ABSTRACT The goal of this research was to evaluate the effect of light, temperature, sucrose and PEG on the growth of Fragaria vesca in vitro and the expression of regulatory Myb10, WD40 and enzyme-coding genes CHI, CHS, DFR, EGL, F3H and UFGT, which are essential for anthocyanin biosynthesis. We observed plants’ response to osmotic stress, the decrease in growth and microshoot weight. A change in the expression of the investigated genes was evident under the suboptimal concentration of sucrose. The addition of PEG to the medium caused a decrease in microshoot weight and gene expression. Blue + red lights of the LED lighting system significantly affected microshoot growth in vitro. Red and blue + red + UV lights slightly reduced microshoot weight and caused a reddish colour of petioles, which indicate increased anthocyanin synthesis. Moreover, most of the studied genes’ expression tended to increase when shoots were exposed to blue, blue + red and blue + red + UV lights. A temperature of 15°C (vs 22°C) significantly reduced the mean fresh weight of microshoots while increasing CHI and CHS gene expression and decreasing WD40 gene expression. Exposure to a higher temperature (30°C) induced the vitrification of microshoots, although the fresh weight did not differ from that of the control. Gene expression also depended on the duration of exposure. In the case of CHS, gene expression remained the same or increased after exposure for 1 week and then decreased after exposure for 4 weeks.

New insights into the genetic manipulation of the R2R3-MYB and CHI gene families on anthocyanin pigmentation in Petunia hybrida

Several R2R3-MYB genes control anthocyanin pigmentation in petunia, and ANTHOCYANIN-2 (AN2) is treated as the main player in petal limbs. However, the actual roles of R2R3-MYBs in the coloration of different floral tissues in the so called “darkly-veined” petunias are still not clear. The genetic background and expression of AN2 paralogs from various petunias with different color patterns were identified. All “darkly-veined” genotypes have the identical mutation in the AN2 gene, but express a different functional paralog – ANTHOCYANIN-4 (AN4) – abundantly in flowers. Constitutive overexpression of PhAN4 in this petunia resulted not only in a fully colored flower but also in a clearly visible pigmentation in the green tissue and roots, which can be rapidly increased by stress conditions. Suppression of AN4 gene resulted in discolored petals and whitish anthers. Interestingly, when a similar white flower phenotype was achieved by knockout of an essential structural gene of anthocyanin biosynthesis – CHALCONE ISOMERASE-A (CHI-A) – the plant responded directly by upregulating of another paralogs – DEEP PURPLE (DPL) and PURPLE HAZE (PHZ). Moreover, we also found that CHI–B can partially substitute for CHI-A in anthers, but not in vegetative tissues. Further, no significant effects on the longevity of white or enhanced colored flowers were observed compared with the wild type. We concluded that endogenous up-regulation of AN4 leads to the restoration of petal color in the “darkly-veined” phenotypes as a result of the breeding process under human selection, and CHI–B is a backup for CHI-A acitvity in some floral tissues.

Bacillus paralicheniformis 2R5 and its impact on canola growth and N-cycle genes in the rhizosphere.

Chemical fertilization has a negative impact on natural environment. Plant growth-promoting (PGP) rhizobacterial biofertilizers can be a safer alternative to synthetic agrochemicals. In this research, a culture-based method was used to assess the population size of rhizobacteria at the vegetative, flowering, and maturity stages of canola. Rhizobacteria were then isolated from each of the canola growth stages, and their seven PGP traits were determined. The highest abundance of culturable bacteria was found at vegetative stage of the plants. Furthermore, four out of seven PGP traits were produced by the highest % of isolates at vegetative stage. In the greenhouse experiment that included six rhizobacterial strains with best PGP traits, the greatest canola growth promotion ability under sterile conditions was observed after the introduction of Bacillus paralicheniformis 2R5. Moreover, under non-sterile conditions, 2R5 significantly increased canola growth. The presence of trpA, B, C, D, E, F and pstA, S genes in the 2R5 genome could be associated with canola growth promotion abilities. chiA and mbtH genes could contribute to 2R5 antifungal activity against fungal pathogens. Moreover, the introduction of 2R5 significantly increased the abundance of narG, nosZ, nifH, and nirS genes, which can prove that 2R5 strain may be an important member of soil bacterial community.

Chitinase A, a tightly regulated virulence factor of Salmonella enterica serovar Typhimurium, is actively secreted by a Type 10 Secretion System.

As a facultative intracellular pathogen, Salmonella enterica serovar Typhimurium is one of the leading causes of food-borne diseases in humans. With the ingestion of fecal contaminated food or water, S. Typhimurium reaches the intestine. Here, the pathogen efficiently invades intestinal epithelial cells of the mucosal epithelium by the use of multiple virulence factors. Recently, chitinases have been described as emerging virulence factors of S. Typhimurium that contribute to the attachment and invasion of the intestinal epithelium, prevent immune activation, and modulate the host glycome. Here we find that the deletion of chiA leads to diminished adhesion and invasion of polarized intestinal epithelial cells (IEC) compared to wild-type S. Typhimurium. Interestingly, no apparent impact on interaction was detected when using non-polarized IEC or HeLa epithelial cells. In concordance, we demonstrate that chiA gene and ChiA protein expression was solely induced when bacteria gain contact with polarized IEC. The induction of chiA transcripts needs the specific activity of transcriptional regulator ChiR, which is co-localized with chiA in the chitinase operon. Moreover, we established that after chiA is induced, a major portion of the bacterial population expresses chiA, analyzed by flow cytometry. Once expressed, we found ChiA in the bacterial supernatants using Western blot analyses. ChiA secretion was completely abolished when accessory genes within the chitinase operon encoding for a holin and a peptidoglycan hydrolase were deleted. Holins, peptidoglycan hydrolases, and large extracellular enzymes in close proximity have been described as components of the bacterial holin/peptidoglycan hydrolase-dependent protein secretion system or Type 10 Secretion System. Overall, our results confirm that chitinase A is an important virulence factor, tightly regulated by ChiR, that promotes adhesion and invasion upon contact with polarized IEC and is likely secreted by a Type 10 Secretion System (T10SS).

Novel Insights into Anthocyanin Synthesis in the Calyx of Roselle Using Integrated Transcriptomic and Metabolomic Analyses.

Roselle (Hibiscus sabdariffa L.) is an annual herbaceous plant of the genus Hibiscus in family Malvaceae. Roselle calyxes are rich in anthocyanins, which play important roles in human health. However, limited information is available on anthocyanin biosynthesis in the roselle calyx. In this study, transcriptomic and metabolomic analyses were performed to identify the key genes involved in anthocyanin biosynthesis in the roselle calyx. Three roselle cultivars with different calyx colors, including FZ-72 (red calyx, R), Baitao K (green calyx, G), and MG5 (stripped calyx, S), were used for metabolomic analyses with UPLC-Q-TOF/MS and RNA-seq. Forty-one compounds were quantified, including six flavonoids and 35 anthocyanins. The calyx of FZ-72 (red calyx) had the highest contents of anthocyanin derivatives such as delphinidin-3-O-sambubioside (955.11 μg/g) and cyanidin-3-O-sambubioside (531.37 μg/g), which were responsible for calyx color, followed by those in MG5 (stripped calyx) (851.97 and 330.06 μg/g, respectively). Baitao K (green calyx) had the lowest levels of these compounds. Furthermore, RNA-seq analysis revealed 114,415 differentially expressed genes (DEGs) in the calyxes at 30 days after flowering (DAF) for the corresponding cultivars FZ-72 (R), Baitao K (G), and MG5(S). The gene expression levels in the calyxes of the three cultivars were compared at different flowering stages, revealing 11,555, 11,949, and 7177 DEGs in R vs. G, R vs. S, and G vs. S, respectively. Phenylpropanoid and flavonoid biosynthesis pathways were found to be enriched. In the flavonoid pathway, 29, 28, and 27 genes were identified in G vs. R, G vs. S, and S vs. R, respectively. In the anthocyanin synthesis pathway, two, two, and one differential genes were identified in the three combinations; these differential genes belonged to the UFGT gene family. After joint analysis of the anthocyanin content in roselle calyxes, nine key genes belonging to the CHS, CHI, UFGT, FLS, ANR, DFR, CCoAOMT, SAT, and HST gene families were identified as strongly related to anthocyanin synthesis. These nine genes were verified using qRT-PCR, and the results were consistent with the transcriptome data. Overall, this study presents the first report on anthocyanin biosynthesis in roselle, laying a foundation for breeding roselle cultivars with high anthocyanin content.

Role of polyamide microplastic in altering microbial consortium and carbon and nitrogen cycles in a simulated agricultural soil microcosm

Microplastics (MPs) are persistent organic pollutants globally, with a continuous increase in MP wastes near and away from the regions of human activities. Studies to date aimed to explore the impact of MPs on ecosystems, but the area of research could not go beyond environmental pollution caused by MPs. To address the menace of MPs, scientists need to pay enough attention to the biogeochemical cycles, microbial communities, and functional microorganisms. Hence, this study aimed to evaluate the impact of adding 0.3% (mass ratio) [low-concentration (LC) group] and 1% [high-concentration (HC) group] of polyamide (PA) MP to the soil microenvironment with regard to the aforementioned parameters. PA MP decreased the soil microbial diversity (Shannon and Simpson indices, P < 0.05). At the phylum level, PA MP increased the abundance of Acidobacteria, Firmicutes, and Crenarchaeota (P < 0.05); at the genus level, it enhanced that of Geobacter, Thiobacillus, Pseudomonas, and Bradyrhizobium (P < 0.01) while decreased that of Bacillus, Flavisolibacter, Geothrix, and Pseudarthrobacter (P < 0.05). PA MP affected the carbon (C) cycle. PA MP accelerated the soil C fixation by enhancing the abundance of the genes accA and pccA. The LC PA MP accelerated organic C degradation and methane metabolism by changing the abundance of mnp, chiA, mcrA, pmoA, and mmoX genes, while the HC PA MP inhibited them with increasing the experimental time. Regarding the effects of PA on the nitrogen (N) cycle, the PA MP promoted N assimilation and ammonification by increasing the abundance of the genes gdh and ureC, the impact of PA MP on N fixation and denitrification depended on its concentration and treating time.This study showed that PA MP impacted the microbial consortium, it also affected the C and N cycles and its effect depended on its concentration and the treating time.

Impact of twenty pesticides on soil carbon microbial functions and community composition

Pesticides are known to affect non-targeted soil microorganisms. Still, studies comparing the effect of multiple pesticides on a wide range of microbial endpoints associated with carbon cycling are scarce. Here, we employed fluorescence enzymatic assay and real-time PCR to evaluate the effect of 20 commercial pesticides, applied at their recommended dose and five times their recommended dose, on soil carbon cycling related enzymatic activities (α-1,4-glucosidase, β-1,4-glucosidase, β-d-cellobiohydrolase and β-xylosidase), and on the absolute abundance of functional genes (cbhl and chiA), in three different South Australian agricultural soils. The effects on cellulolytic and chitinolytic microorganisms, and the total microbial community composition were determined using shotgun metagenomic sequencing in selected pesticide-treated and untreated samples. The application of insecticides significantly increased the cbhl and chiA genes absolute abundance in the acidic soil. At the community level, insecticide fipronil had the greatest stimulating effect on cellulolytic and chitinolytic microorganisms, followed by fungicide metalaxyl-M and insecticide imidacloprid. A shift towards a fungal dominated microbial community was observed in metalaxyl-M treated soil. Overall, our results suggest that the application of pesticides might affect the soil carbon cycle and may disrupt the formation of soil organic matter and structure stabilisation.

Stover and biochar can improve soil microbial necromass carbon, and enzymatic transformation at the genetic level

AbstractTo understand how stover incorporation affects the levels of soil carbon and its biogeochemistry process, we examined soil carbon and amino sugar carbon (AS‐C). Activity of N‐acetylglucosaminidase (NAGase) and the abundance (real‐time quantitative polymerase chain reaction, RT‐qPCR) and communities of the bacterial chitinase‐encoding gene (chiA) were monitored. We implemented a maize cropping system, which included four treatments, i.e., chopped maize stover returned directly (SD), compost produced by maize stover (SC), biochar produced by maize stover (BC), and stover removal (CK). The results showed that BC and SD enhanced soil organic carbon (SOC), microbial biomass carbon:SOC, total AS‐C, glucosamine carbon, galactosamine carbon, NAGase activity, and chiA gene abundance significantly. There were significant positive correlations between the abundance of the soil chiA gene and NAGase activity. Biochar and stover treatments increased the number of total operational taxonomic units (OTUs), unique OTUs, and the chao 1 and ACE richness indices, but decreased the Shannon and Simpson diversity indices. The dominant chiA‐harboring bacterial phyla were Actinobacteria, Proteobacteria, and Bacteroidetes. Some detectable genera in Actinobacteria had high relative abundance, including Streptomyces, Actinoplanes, and Amycolatopsis. PLS‐DA and CCoA revealed that the chiA community in the BC or SD plots was significantly different from that in the CK plots, and differences between BC and SD were not significant. Similar soil properties, diversity, and the abundance of chiA were observed between SC and CK. Greater abundance and diversity of the chiA gene with BC incorporation were associated with higher pH, SOC, and TN concentration. Soil pH was related to the composition and abundances of the chiA‐harboring microbial communities (CCA).

Sequence Analysis and Functional Verification of the Effects of Three Key Structural Genes, PdTHC2'GT, PdCHS and PdCHI, on the Isosalipurposide Synthesis Pathway in Paeonia delavayi var. lutea.

Isosalipurposide (ISP) is the most important yellow pigment in tree peony. In ISP biosynthesis, CHS catalyzes 1-molecule coumaroyl-CoA and 3-molecule malonyl-CoA to form 2′,4′,6′,4-tetrahyroxychalcone (THC), and THC generates a stable ISP in the vacuole under the action of chalcone2′-glucosyltransferases (THC2′GT). In tree peony, the details of the THC2’GT gene have not yet been reported. In this study, the candidate THC2’GT gene (PdTHC2’GT) in Paeonia delavayi var. lutea was screened. At the same time, we selected the upstream CHS gene (PdCHS) and the competitive CHI gene (PdCHI) to study the biosynthesis pathway of ISP. We successfully cloned three genes and sequenced them; subcellular localization showed that the three genes were located in the nucleus and cytoplasm. The overexpression of PdTHC2’GT in tobacco caused the accumulation of ISP in tobacco petals, which indicated that PdTHC2’GT was the key structural gene in the synthesis of ISP. After the overexpression of PdCHS and PdCHI in tobacco, the accumulation of anthocyanins in tobacco petals increased to different degrees, showing the role of PdCHS and PdCHI in anthocyanin accumulation. The analysis of NtCHS and NtCHI of transgenic tobacco lines by qRT-PCR showed that the THC2’GT gene could increase the expression of CHS. THC2’GT and CHI were found to be competitive; hence, the overexpression of THC2’GT could lead to a decrease in CHI expression. The CHS gene and CHI gene could increase the expression of each other. In conclusion, we verified the key structural gene PdTHC2’GT and studied the operation of the genes in its upstream and competitive pathway, providing a new perspective for the biosynthesis of ISP and new candidate genes for the directional breeding of tree peony.

Expression, purification, and basic properties of a novel domain structure possessing chitinase from Escherichia coli carrying the family 18 chitinase gene of Bacillus velezensis strain RB.IBE29.

Bacillus velezensis possesses numerous chitinolytic enzymes; however, not much is known about the role of its chitinase molecules. In this study, the chiA gene, which encodes a novel domain structure possessing family 18 chitinase from B. velezensis RB.IBE29, was expressed successfully in Escherichia coli BL21-CodonPlus (DE3)-RIPL using the pColdII expression vector. The recombinant protein, rBvChiA, was purified using the HisTrap FF column. Purified rBvChiA showed hydrolytic activity against insoluble chitin and bound to chitinous substrates. In addition, the purified recombinant enzyme displayed remarkable inhibition effects on the spore germination of Fusarium falciforme and the egg hatch of root-knot nematodes (Meloidogyne spp.), which are the main causes of black pepper diseases in the Central Highlands region, Vietnam. The current work results might enable further studies to develop novel chitinase A and strain RB.IBE29 as a natural fungicide and nematicide for sustainable black pepper production and other crops in the Central Highlands, Vietnam. This is the second report describing chitinase from B. velezensis based on the experimental data.

Detection of mcr-1 Gene in Undefined Vibrio Species Isolated from Clams.

The increase of antimicrobial resistant strains is leading to an emerging threat to public health. Pathogenic Vibrio are responsible for human and animal illness. The Enterobacteriaceae family includes microorganisms that affect humans, causing several infections. One of the main causes of human infection is related to the ingestion of undercooked seafood. Due to their filter-feeding habit, marine invertebrates, such as clams, are known to be a natural reservoir of specific microbial communities. In the present study, Vibrionaceae and coliforms microorganisms were isolated from clams. A microbial susceptibility test was performed using the disk diffusion method. From 43 presumptive Vibrio spp. and 17 coliforms, three Vibrio spp. with MICs to colistin >512 mg L−1 were found. From the 23 antimicrobial resistance genes investigated, only the three isolates that showed phenotypic resistance to colistin contained the mcr-1 gene. Genotypic analysis for virulence genes in EB07V indicated chiA gene presence. The results from the plasmid cure and transformation showed that the resistance is chromosomally mediated. Biochemical analysis and MLSA, on the basis of four protein-coding gene sequences (recA, rpoB, groEL and dnaJ), grouped the isolates into the genus Vibrio but distinguished them as different from any known Vibrio spp.

Non-conventional expression of recombinant chitinase A originated from Bacillus licheniformis DSM8785, in Saccharomyces cerevisiae INVSc1

Chitinases are glycosyl hydrolases, that cleave the ?-1,4 linkage between N-acetyl glucosamines present in chitin chains. Chitin is the second most abundant polysaccharide on Earth after cellulose, and it is produced in the exoskeleton of crustaceans and insects, and in some parts of the cell walls of fungi. Enzymatic development and the extraction of superior derivatives from chitin wastes ? such as chitooligosaccharides with vast importance in the medical and biofuels industry ? lead to the necessity of creating chitinases using different strains of organisms. In this paper, the chiA gene from the Bacillus licheniformis DSM8785 encoding chitinase A (ChiA) with C-terminal hexahistidine tag was cloned and expressed in the extracellular expression system pYES2 from Saccharomyces cerevisiae INVSc1 as a hyperglycosylated enzyme. The production of recombinant ChiA was successfully confirmed by dot blotting, using anti-His antibodies. The optimal time of expression was identified to be 24 h when galactose was added only at the beginning of fermentation, the chitinase activity starting to decrease after this threshold. Nevertheless, in another experiment, when galactose was added every 24 h for 72 h, the expression continued for the entire period. The purified enzyme was detected, using sodium dodecyl sulphate?polyacrylamide gel electrophoresis (SDS-PAGE), as a heterogeneous diffuse band between 80 and 180 kDa. The molecular mass of the same ChiA enzyme expressed in Pichia pastoris KM71H and Escherichia coli BL21 (DE3) was compared using SDS-PAGE with ChiA expressed in S. cerevisiae INVSc1. The activity of ChiA was determined using the fluorogenic substrate, 4-methylumbelliferyl ?-D-N,N,N-triacetylchitotrioside (4MUTC). Using a bioinformatics simulation, the number of the glycolsylation sites of the ChiA gene sequence and the proximity of these sites to the alpha factor sequence were hypothesized to be a possible reason for which ChiA enzyme was internally expressed.

Effect of restoration types on the community structure of microbes harboring nifH and chiA genes in alpine meadow

Biological nitrogen (N) fixation and organic N degradation are the main sources of soil available N, while microorganisms driving such processes play an important role in soil N supply and the maintenance of soil fertility. In this study, real-time quantitative PCR and amplicon sequencing technology were used to examine the effects of restoration types on the community structure of N2-fixing and chitin-degrading bacteria harboring nifH and chiA genes, respectively, and the gene abundance under four meadows (undisturbed, grazing, fencing, and fencing + reseeding mea-dows) in Qinghai-Tibet Plateau. The results showed that the abundance of nifH and chiA in the four meadows followed the order of undisturbed meadow > grazed meadow > fencing meadow > fencing + reseeding meadow. The abundance of nifH and chiA in the undisturbed meadow was 3.4-6.3 times and 3.3-8.3 times of that in the other three meadows. The α diversity of N2-fixing bacteria in gra-zing, fencing, and fencing + reseeding meadows was significantly higher than that in the undisturbed meadow, while the α diversity of chitin-degrading bacteria was higher in the undisturbed and grazing meadows. Grazing significantly increased the relative abundance of Proteobacteria, but decreased the relative abundance of Cyanobacteria and Actinobacteria. The abundance of nifH and chiA was significantly affected by soil moisture, nutrients, and vegetation characteristics, while the community structure of nifH and chiA was affected by soil moisture, soil organic carbon content, and soil pH. Compared with undisturbed meadow, grazing reduced the potential of N fixation and organic N degradation.The improvement of 10 years grazing prohibition with fencing and reseeding measures on the function of N fixation and organic N degradation was not obvious. The characteristics of functional microbes and their influencing factors should be comprehensively considered during meadow restoration, which might take longer time or take reasonable management measures to restore grazing meadow to undisturbed level.

Overexpression of chalcone isomerase A gene in Astragalus trigonus for stimulating apigenin

Apigenin is one of the most studied flavonoids and is widely distributed in the plant kingdom. Apigenin exerts important antioxidant, antibacterial, antifungal, antitumor activities, and anti-inflammatory effects in neurological or cardiovascular disease. Chalcone isomerase A (chiA) is an important enzyme of the flavonoid biosynthesis pathway. In order to enhance the apigenin production, the petunia chi A gene was transformed for Astragalus trigonus. Bialaphos survived plants were screened by PCR, dot blot hybridization and RT-PCR analysis. Also, jasmonic acid, salicylic acid, chitosan and yeast extract were tested to evaluate their capacity to work as elicitors for apigenin. Results showed that yeast extract was the best elicitor for induction of apigenin with an increase of 3.458 and 3.9 fold of the control for calli and cell suspension culture, respectively. Transformed cell suspension showed high apigenin content with a 20.17 fold increase compared to the control and 6.88 fold more than the yeast extract treatment. While, transformed T1 calli derived expressing chiA gene produced apigenin 4.2 fold more than the yeast extract treatment. It can be concluded that the highest accumulation of apigenin was obtained with chiA transgenic cell suspension system and it can be utilized to enhancement apigenin production in Astragalus trigonus.

Identification and characterization of unique 5-hydroxyisoflavonoid biosynthetic key enzyme genes in Lupinus albus.

5-Hydroxyisoflavonoids, no 5-deoxyisoflavonoids, in Lupinus species, are due to lack of CHRs and Type II CHIs, and the key enzymes of isoflavonoid biosynthetic pathway in white lupin were identified. White lupin (Lupinus albus) is used as food ingredients owing to rich protein, low starch, and rich bioactive compounds such as isoflavonoids. The isoflavonoids biosynthetic pathway in white lupin still remains unclear. In this study, only 5-hydroxyisoflavonoids, but no 5-deoxyisoflavonoids, were detected in white lupin and other Lupinus species. No 5-deoxyisoflavonoids in Lupinus species are due to lack of CHRs and TypeII CHIs. We further found that the CHI gene cluster containing both TypeI and TypeII CHIs possibly arose after the divergence of Lupinus with other legume clade. LaCHI1 and LaCHI2 identified from white lupin metabolized naringenin chalcone to naringenin in yeast and tobacco (Nicotiana benthamiana), and were bona fide Type ICHIs. We further identified two isoflavone synthases (LaIFS1 and LaIFS2), catalyzing flavanone naringenin into isoflavone genistein and also catalyzing liquiritigenin into daidzein in yeast and tobacco. In addition, LaG6DT1 and LaG6DT2 prenylated genistein at the C-6 position into wighteone. Two glucosyltransferases LaUGT1 and LaUGT2 metabolized genistein and wighteone into its 7-O-glucosides. Taken together, our study not only revealed that exclusive 5-hydroxyisoflavonoids do exist in Lupinus species, but also identified key enzymes in the isoflavonoid biosynthetic pathway in white lupin.

Noninsect-Based Diet Leads to Structural and Functional Changes of Acidic Chitinase in Carnivora.

Acidic chitinase (Chia) digests the chitin of insects in the omnivorous stomach and the chitinase activity in carnivorous Chia is significantly lower than that of the omnivorous enzyme. However, mechanistic and evolutionary insights into the functional changes in Chia remain unclear. Here we show that a noninsect-based diet has caused structural and functional changes in Chia during the course of evolution in Carnivora. By creating mouse-dog chimeric Chia proteins and modifying the amino acid sequences, we revealed that F214L and A216G substitutions led to the dog enzyme activation. In 31 Carnivora, Chia was present as a pseudogene with stop codons in the open reading frame (ORF) region. Importantly, the Chia proteins of skunk, meerkat, mongoose, and hyena, which are insect-eating species, showed high chitinolytic activity. The cat Chia pseudogene product was still inactive even after ORF restoration. However, the enzyme was activated by matching the number and position of Cys residues to an active form and by introducing five meerkat Chia residues. Mutations affecting the Chia conformation and activity after pseudogenization have accumulated in the common ancestor of Felidae due to functional constraints. Evolutionary analysis indicates that Chia genes are under relaxed selective constraint in species with noninsect-based diets except for Canidae. These results suggest that there are two types of inactivating processes in Carnivora and that dietary changes affect the structure and activity of Chia.