Induction of P450 genes in Nilaparvata lugens and Sogatella furcifera by two neonicotinoid insecticides

Nilaparvata lugens and Sogatella furcifera are two primary planthoppers on rice throughout Asian countries and areas. Neonicotinoid insecticides, such as imidaclo- prid （IMI）, have been extensively used to control rice planthoppers and IMI resistance consequently occurred with an important mechanism from the over-expression of P450 genes. The induction of P450 genes by IMI may increase the ability to metabolize this insecticide in planthoppers and increase the resistance risk. In this study, the induction of P450 genes was compared in S. furcifera treated with IM] and nitromethyleneimidazole （NMI）, in two planthopper species by IMI lethal dose that kills 85% of the population （LD85）, and in N. lugens among three IMI doses （LD15, LD50 and LD85）. When IMI and NMI at the LD85 dose were applied to S. furcifera, the expression changes in most P450 genes were similar, including the up-regulation of nine genes and down-regulation of three genes. In terms of the expression changes in 12 homologous P450 genes between N. lugens and S. furcifera treated with IMI at the LD85 dose, 10 genes had very similar patterns, such as up-regulation in seven genes, down-regulation in one gene and no significant changes in two genes. When three different IMI doses were applied to N. lugens, the changes in P450 gene expression were much different, such as up-regulation in four genes at all doses and dose-dependent regulation of the other nine genes. For example, CYP6AY1 could be induced by all IMI doses, while CYP6ER1 was only up-regulated by the LDs0 dose, although both genes were reported important in IMI resistance. In conclusion, P450 genes in two planthopper species showed similar regulation patterns in responding to IMI, and the two neonicotinoid insecticides had similar effects on P450 gene expression, although the regulation was often dose-dependent.

Genetic mutations associated with chemical resistance in the cytochrome P450 genes of invasive and native Bemisia tabaci （Hemiptera： Aleyrodidae） populations in China

Bemisia tabaci （Gennadius） is a species complex, and its two most damaging biotypes B and Q are globally distributed pests. Despite increasing biological and economic impacts, little is known about the evolutionary mechanisms that favor their competition with native populations. Here, we investigated the genetic mutations in the P450 gene of the invasive B, Q biotypes and the native Cv population. Four mutations associated with chemical resistance, Pro-Leu, Ala-Ser, Ser-Phe and Trp-Leu, were found in the cytochrome P450 CYP6C and CYP9F genes of the B and Q biotypes. Bioassay results also revealed that both the B and Q biotypes have about 12-47 times more resistance to acephate, beta- cypermethrin, methomyl, and 5-7 times more resistance to imidacloprid insecticide than Cv population. Our results provide a molecular approach for better understanding and monitoring the pesticide resistances of invasive and native B. tabaei populations in China.

Allotetraploidization event of Coptis chinensis shared by all Ranunculales

Coptis chinensis Franch.,also named Chinese goldthread is a member of Ranunculaceae in the order Ranunculales and represents an important lineage of early eudicots with traditional medicinal value.In our study,by using syntenic analysis combined with phylogenomic analysis of C.chinensis and four other representative genomes from basal and core eudicots,we confirmed that the WGD event in C.chinensis was shared by Aquilegia coerulea and Papaver somniferum L.and quickly occurred after Ranunculales diverged from other eudicots,likely a Ranunculales common tetraploidization(RCT).The synonymous nucleotide substitutions at synonymous sites distribution of syntenic blocks across these genomes showed that the evolutionary rate of the P.somniferum genome is faster than that of the C.chinensis genome by approximately 13.7%,possibly due to Papaveraceaes having an additional special tetraploidization event(PST).After Ks correction,the RCT dated to 115—130 million years ago(MYA),which was close to the divergence of Ranunculaceaes and Papaveraceaes approximately115.45—130.51 MYA.Moreover,we identified homologous genes related to polyploidization and speciation and constructed multiple sequence alignments with different reference genomes.Notably,the event-related subgenomes in the basal genomes all showed genomic fractionation bias,suggesting a likely allopolyploid nature of the RCT,PST and T-Alpha and T-Beta events in Tetracentron sinense.In addition,we detected that the sixteen P450 subfamilies were markedly expanded in the genomes of Ranunculales,and most of them were related to the RCT and PST events.We constructed a new platform for Early Eudicot Comparative Genomic Research(http://www.cgrpoee.top/index.html)to store more information.In summary,our findings support the WGD of C.chinensis shared by Ranunculales,which is likely an allotetraploidization event.This present effort offered new insights into the evolution of key polyploidization events and the genes related to secondary metabolites during the diversification of early eudicots.

Association between Maternal Drug Use and Cytochrome P450 Genetic Polymorphisms and the Risk of Congenital Heart Defects in Offspring

Objective This study aimed to assess the associations between maternal drug use,cytochrome P450(CYP450)genetic polymorphisms,and their interactions with the risk of congenital heart defects(CHDs)in offspring.Methods A case-control study involving 569 mothers of CHD cases and 652 controls was conducted from November 2017 to January 2020.Results After adjusting for potential confounding factors,the results show that mothers who used ovulatory drugs(adjusted odds ratio[a OR]=2.12;95% confidence interval[CI]:1.08-4.16),antidepressants(a OR=2.56;95%CI:1.36-4.82),antiabortifacients(a OR=1.55;95%CI:1.00-2.40),or traditional Chinese drugs(a OR=1.97;95%CI:1.26-3.09)during pregnancy were at a significantly higher risk of CHDs in offspring.Maternal CYP450 genetic polymorphisms at rs1065852(A/T vs.A/A:OR=1.53,95%CI:1.10-2.14;T/T vs.A/A:OR=1.57,95%CI:1.07-2.31)and rs16947(G/G vs.C/C:OR=3.41,95%CI:1.82-6.39)were also significantly associated with the risk of CHDs in offspring.Additionally,significant interactions were observed between the CYP450 genetic variants and drug use on the development of CHDs.Conclusions In those of Chinese descent,ovulatory drugs,antidepressants,antiabortifacients,and traditional Chinese medicines may be associated with the risk of CHDs in offspring.Maternal CYP450 genes may regulate the effects of maternal drug exposure on fetal heart development.

Response of Cytochrome P450 Expression to Maize Volatiles in Helicoverpa armigera (Hner)

The 7-ethoxycoumarin O-deethylase （ECOD） activities of cytochrome P450s and differential expression of six cytochrome P450 genes induced by the volatiles from both damaged and undamaged maize plants were investigated in the cotton bollworm, Helicoverpa armigera （Hiabner）. The ECOD activity changed with time of exposure to maize volatiles. At 36 h after cotton bollworm larvae exposure to maize volatiles, the ECOD activities in cotton bollworm damaged and artificially damaged groups were 2.36 and 4.53 times higher than the control group respectively. The relative expression levels of CYP4S1, CYP6B2 and CYP6B7 in the cotton bollworm were significantly increased in artificially damaged plant group, which was 2.93, 5.09 and 10.66 times higher than that in the control group, respectively. The expression levels of CYP6B2, CYP6B6, CYP9A12, and CYP9A14 were much lower in the larvae exposure to volatiles from both healthy and pest damaged maize seedlings than in the control group at 12 h after larvae exposure to maize volatiles. For the cotton bollworm damaged maize group, the expression of CYP4S1 and CYP9A14 increased.

Characterization and expression analysis of genes encoding Taxol biosynthetic enzymes in Taxus spp.

Taxol(Paclitaxel),an important anticancer drug,is derived at very low yields from Taxus(yew)species that grow very slowly.In the present study,thirteen genes that encode enzymes involved in Taxol biosynthesis in Taxus spp.were analyzed with bioinformatics methods,and their expression levels in different tissues and after cold and hormone treatments were also analyzed.The results indicated that many cis-elements related to abiotic stresses and hormones were found in the promoter sequences of the 8 genes involved in Taxol biosynthesis.Moreover,the 13 enzymes encoded by the target genes were located in different organelles and had many phosphorylation sites in the response proteins.The 13 genes were expressed highly either in roots or in stems,with lower transcripts in needles,and they were highly expressed after treatment with cold,gibberellin,methyl jasmonate or coronatine,consistent with predictions based on the bioinformatics analysis.These results suggest that the factors such as hormones and abiotic stresses stimulate taxane biosynthesis in yews,providing an important way to sustainably generate taxanes from yew trees or their cell cultures to improve Taxol yields.

Relation of cytochrome P450 2C19 gene 681G>A single nucleotide polynmrphism to clopidogrel resistance after PCI in Chinese

Objectives Clopidogrel is a prodrug that has to be converted to an active metabolite by hepatic cytochrome P450(CYP) isoenzymes to inhibit platelet aggregation.Individualvariability of platelet inhibition by clopidogrel suggests a possibility for genetic factors having a significant influence on clopidogrel responsiveness.In this study,we sought to determine the association between the single nucleotide polymorphism of CYP 2C19 681G】A and the occurrence of clopidogrel resistance(CR) in Chinese.Methods The study enrolled 614 hospitalized patients who underwentsuccessful percutaneouscoronary intervention with drug-eluting stents were received the treatmentwith dual antiplatelet regimen(aspirin plus clopidogrel).All patients received loading doses of 600 mg clopidogrel and 300 mg aspirin.20μmol/L ADP-induced platelet aggregation ratio(PAR ) was assessed 24 h after clopi- dogrel administration.The maximum residual PAR≥70%was defined as CR.Genomic DNA was extracted from whole blood samples according to standard protocols,the single nucleotide polymorphism of the CYP2C19 681G】A was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in all the patients.Results CR was found in 126 patients(20.5%).There was CYP2C19 681G】A polymorphism in the study population.The frequencies of the three kinds of genotypes(GG,GA,A A) in CR group and non-CR (NCR)group were 32.5%,47.6%,19.8%and 48.0%, 45.0%,7.0%,respectively.The frequency of AA genotype was significantly higher in NCR group than that in CR group (OR =3.03,95%CI:1.889～5.784,P=0.003).The A allele carriers were more likely to develop clopidogrel resistance compared with that of G allele carriers(OR=1.85,95%CI: 1.392～2.459,P=0.002).Conclusions CYP2C19 681G/A polymorphism is associated with the risk of CR,and the A allele carriers may be a possible genetic susceptibility factor for patients with CR.

HNF-4α determines hepatic differentiation of human mesenchymal stem cells from bone marrow

AIM: To investigate the differentiation status and key factors to facilitate hepatic differentiation of human bone-marrow-derived mesenchymal stem cells (MSCs). METHODS: Human MSCs derived from bone marrow were induced into hepatocyte-like cells following a previously published protocol. The differentiation status of the hepatocyte-like cells was compared with various human hepatoma cell lines. Overexpression of hepatocyte nuclear factor (HNF)-4α was mediated by adenovirus infection of these hepatocyte-like cells. The expression of interesting genes was then examined by either re-verse transcription-polymerase chain reaction (RT-PCR) or real-time RT-PCR methods. RESULTS: Our results demonstrated that the differentiation status of hepatocyte-like cells induced from human MSCs was relatively similar to poorly differentiated human hepatoma cell lines. Interestingly, the HNF-4 isoform in induced MSCs and poorly differentiated human hepatoma cell lines was identified as HNF4γ instead of HNF-4α. Overexpression of HNF-4α in induced MSCs significantly enhanced the expression level of hepatic-specific genes, liver-enriched transcription factors, and cytochrome P450 (P450) genes. CONCLUSION: Overexpression of HNF-4α improves the hepatic differentiation of human MSCs from bone marrow and is a simple way of providing better cell sources for clinical applications.

HMGR, SQS, β-AS, and Cytochrome P450 Monooxygenase Genes in Glycyrrhiza uralensis

Glycyrrhiza uralensis is frequently used in traditional Chinese medicine. This plant contains a large amount of effective constituents, including triterpenoids and flavonoids. Among them, glycyrrhizin is believed to be the marker compound to evaluate the quality of G. uralensis based on Chinese Pharmacopoeia. Many studies showed that glycyrrhizin possesses various pharmacological activities, such as antibacterial, antiviral, antitumor, anti-inflammatory, and immune-stimulating activities. In this paper, we summarized the cloning, characterization, expression, and polymorphism analysis of several functional genes involved in glycyrrhizin biosynthesis in G. uralensis.

Fine Mapping and Cloning of Leafy Head Mutant Gene pla1-5 in Rice

We identified a leafy head mutant plal-5 （plastochron 1-5） from the progeny of japonica rice cultivar Taipei 309 treated with 60Co-γ ray irradiation. The plal-5 mutant has a dwarf phenotype and small leaves. Compared with its wild type, plal-5 has more leaves and fewer tillers, and it fails to produce normal panicles at the maturity stage. Genetic analysis showed that the plal-5 phenotype is controlled by a single recessive nuclear gene. Using the map-based cloning strategy, we narrowed down the location of the target gene to a 58-kb region between simple sequence repeat markers CHR1027 and CHR1030 on the long arm of chromosome 10. The target gene cosegregated with molecular markers CHR1028 and CHR1029. There were five predicted genes in the mapped region. The results from sequencing analysis revealed that there was one base deletion in the first exon of LOC_Os10g26340 encoding cytochrome P450 CYP78A11 in the plal-5 mutant, which might result in a downstream frame shift and premature termination. These results suggest that the P450 CYP78A11 gene is the candidate gene of PLA1-5.

Current susceptibilities of brown planthopper Nilaparvata lugens to triflumezopyrim and other frequently used insecticides in China

The brown planthopper is a notorious rice pest in many areas of Asia.The evolution of insecticide resistance in Nilaparvata lugens has become a serious problem in the effective control of this pest in the paddy field.In this article,the current susceptibility of N.lugens field populations to novel mesoionic insecticide triflumezopyrim and major classes of chemical insecticides was determined and compared.The monitoring results indicated that field populations of N.lugens had developed low resistance to triflumezopyrim(resistance ratio,RR:1.3-7.3-fold)during 2015-2018 in China,and the median lethal concentration values varied from 0.05 to 0.29 mg/L.Additionally,during 2017 to 2018,field populations of N.lugens showed high resistance levels to thiamethoxam(RR:456.1-1025.6-fold),imidacloprid(RR:2195.3-6899.0-fold)and buprofezin(RR:1241.57521.7-fold),moderate to high resistance levels to dinotefuran(RR:97.6-320.1-fold),clothianidin(RR:69.4-230.1-fold)and isoprocarb(RR:44.1-108.0-fold),and low to moderate levels of resistance to chlorpyrifos(RR:12.0-29.7-fold)and nitenpyram(RR:6.9-24.1-fold).In contrast,N.lugens just showed low resistance to sulfoxaflor(RR:3.3-8.5-fold)and etofenprox(RR:5.0-9.1-fold)in the field.Additionally,the P450 gene CYP6ER1 was found to be significantly overexpressed in all five field populations of N.lugens collected in 2018 when compared with a laboratory susceptible strain.Our findings will provide useful information to delay the evolution of insecticide resistance in N.lugens.

Aldosterone biosynthesis in extraadrenal tissues

Objective To determine whether extraadrenal tissues synthesize aldoster one in addition to vascular tissue and brain. Methods Ex vivo kidney perfusion was performed in normal Wistar rats, A CEI pretreated and adrenalectomized rats prior to the perfusion experiment. Afte r equilibration for 30 minutes, 120 ml of perfusate was collected and subjected to rever se phase HPLC and then aldosterone was measured by RIA. By RT PCR and Southern blot the expression of aldosterone synthase gene CYP11B2 mRNA was studied i n both kidney tissue and cultured renal tubular epithelial cell, lung and l iver tissues. In situ hybridization was used to identify the cell types of liver and lung expressing CYP11B2 mRNA.Results Production of aldosterone in the kidney perfusate was not chang ed in adrenalectomized rats although it was decreased in the group pretreated wi th ACEI perindopril. By RT PCR and Southern blot the expression of CYP11B2 mRNA was demonstrated in both kidney tissue and cultured renal tubular epithelial cell. We have also identified CYP11B2 mRNA expression in liver and lung of rats. In si tu hybridization showed that CYP11B2 mRNA was localized in the endoplasm of live r fat storing cell (Ito cells) and type Ⅱ alveolar cells of lung.Conclusions These studies prove that kidney, liver and lung are able to produce aldosterone.