A predominantly expressed gene, pyruvate kinease （PK） gene, control ing oil accumulation, had been identified in previous study. To construct a PK RNAi vector, a 498-bp target PK gene sequence was amplified and tran...A predominantly expressed gene, pyruvate kinease （PK） gene, control ing oil accumulation, had been identified in previous study. To construct a PK RNAi vector, a 498-bp target PK gene sequence was amplified and transferred into the pEASY-T1vector. Subsequently, the target DNA fragments were cut off by enzymes Not I and Xho I and directional y inserted into plant RNAi platform vector pHurricane, a newly developed RNAi vector in our lab, to form the PK inverse repeats. The PK inverted repeats fragment was then cloned into a modified vector pCAMBIA1390, driven by a rapeseed seed-specific promoter napin. Restriction enzyme digestion verified the successful construction of RNA interference vector. The PK RNAi con-struction wil lay a foundation for study in the function of PK in oil accumulation and metabolism in rapeseeds.展开更多
目的:构建针对信号转导子和转录激活子6(Singnal transducers and activators of transcription 6,STAT6)基因的干扰质粒,并观察其对STAT6蛋白表达的抑制效果。方法:将构建的短发夹干扰质粒(Short hairpin RNA,shRNA)表达载体,与合成的p...目的:构建针对信号转导子和转录激活子6(Singnal transducers and activators of transcription 6,STAT6)基因的干扰质粒,并观察其对STAT6蛋白表达的抑制效果。方法:将构建的短发夹干扰质粒(Short hairpin RNA,shRNA)表达载体,与合成的pIRES2-EGFP-STAT6基因表达质粒共同转染COS7细胞,荧光显微镜下观察干扰效果,并利用Western blot观察蛋白表达的变化。结果:将pIRES2-EGFP-STAT6重组载体转染COS7细胞后,结果显示STAT6蛋白可以在细胞中高水平表达;与阴性对照组相比,STAT6 shRNA-1干扰组和STAT6 shRNA-2干扰组STAT6蛋白表达水平分别是23.1%和9.6%。结论:成功构建并筛选到具有显著干扰效率的STAT6干扰质粒,为哮喘的基因治疗研究奠定了基础。展开更多
Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ische- mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy- gen-glucose deprivatio...Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ische- mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy- gen-glucose deprivation to simulate cerebral ischemia/reperfusion injury. Chemically synthesized small interfedng RNA （siRNA）-1280, -1724 and -418 specific to toll-like receptor 3 were transfected into oxygen-glucose deprived cortical neurons to suppress the upregulation of toll-like receptor 3 protein expression. Western blotting demonstrated that after transfection with siRNA, toll-like re- ceptor 3 protein expression reduced, especially in the toll-like receptor 3-1724 group. These results suggested that siRNA-1724 is an optimal sequence for inhibiting toll-like receptor 3 expression in cortical neurons following oxygen-glucose deprivation.展开更多
OBJECTIVE In this study, RNA interference was used to evaluate the effects of HMGB1 expression on cell cycle and proliferation of the human cervical cancer cell line HeLa.METHODS We had previously constructed and scre...OBJECTIVE In this study, RNA interference was used to evaluate the effects of HMGB1 expression on cell cycle and proliferation of the human cervical cancer cell line HeLa.METHODS We had previously constructed and screened effective eukaryotic expression vectors carrying PGCsi3.0-1/ HMGB1 siRNA and PGCsi3.0-3/HMGB1 siRNA, then the vectors were transfected into HeLa cells. The expression of HMGB1 before and after transfection in HeLa cells were detected by RT-PCR and Western blot. The cell viability and proliferating activity was tested by Trypan blue dye test and MTT, and the cell cycle was determined bv flow cvtometry.RESULTS The introduction of PGCsi3.0-1/HMGB1 siRNA and PGCsi3.0-3/HMGB1 siRNA inhibited the expression of HMGB1 mRNA and protein efficiently and specifically, there was a significant difference between the siRNA groups and the control groups （P 〈 0.05）. The proliferation speed of PGCsi3.0-1 group and PGC si3.0-3 group were obviously slower than those of PGCsi3.0- Neg group and non-transfected group. Flow cytometry showed that the content of DNA in G2 phase in PGCsi3.0-1 group and PGCsi3.0-3 group were obviously more than those in PGCsi3.0- Neg group and non-transfected group, but the content in S phase was less （P 〈 0.01）. The progression of cell cycle was arrested from G2 to S phase.CONCLUSION PGCsi3.0-1/HMGB1 siRNA and PGCsi3.0-3 /HMGB1 siRNA could specially suppress the expression of HMGB1 gene, inhibit the proliferation speed of HeLa cells effectively, and arrest the progression of cell cycle from G2 to S phase. RNAi provides a new approach to the bio-therapy of cervical cancer.展开更多
Acting as one of the pattern recognition receptors （PRRs）, C-type lectin is believed to mediate pathogen recognition and plays an important role in the clearance of pathogens as part of the innate immune system. In ...Acting as one of the pattern recognition receptors （PRRs）, C-type lectin is believed to mediate pathogen recognition and plays an important role in the clearance of pathogens as part of the innate immune system. In this work, a novel C-type lectin gene （named LvLecl） was cloned from the shrimp Litopenaeus vannamei, The ORF of LvLecl is 510 bp, encoding 169 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids at the N-terminal and a carbohydrate recognition domain （CRD） at the C-terminal. LvLecl was mainly expressed in the hepatopancreas. Real-time PCR analysis indicated that the level of LvLecl transcripts significantly changed in the hepatopancreas after the shrimp were artificially challenged with LPS, Micrococcus lysodeikticus and white spot syndrome virus （WSSV）. RNAi-based silencing of LvLecl resulted in increases in mortality when the shrimp were challenged with WSSV, and the median lethal time was reduced compared with controls. Although there was no characteristic ＂EPN＂ （Glu-Pro-Ser） or ＂QPD＂ （Gin-Pro-Asp） motif, the recombinant LvLecl, expressed in Escherichia coli BL21 （DE3）, could also agglutinate M. lysodeikticus and Vibrio anguillarum. The agglutinating activities were calcium-dependent and could be inhibited by D-mannose, D-glucose, D-galactose and N-Acetyl-D-mannose. These results suggest that LvLecl might be involved in the immune response against WSSV and bacterial infections and contribute to non-self recognition as a pattern recognition receptor in the innate immune system of the shrimp L. vannamei.展开更多
During the pupal-adult eclosion process of holometabolous insects,the old cuticle is shed and replaced by a completely different new cuticle that requires tanning and expansion,along with extensive extracellular matri...During the pupal-adult eclosion process of holometabolous insects,the old cuticle is shed and replaced by a completely different new cuticle that requires tanning and expansion,along with extensive extracellular matrix(ECM)remodeling.In vertebrates,matrix metalloproteinases(MMPs),a class of zinc-dependent endopeptidases,play key roles in regulating the ECM that surrounds cells.However,little is known about these extracellular proteinases available in insects.The small hive beetle(SHB),Aethina tumida,is a widespread invasive parasite of honey bees.In this study,6 MMP homologs were identified in the SHB genome.RNA interference experiments showed that all 6 AtMmps are not required for the larval-pupal transition,only AtMmp2 was essential for pupal-adult eclosion in SHB.Knockdown of AtMmp2 resulted in eclosion defects and wing expansion failure,as well as mortality within 3 d of adult eclosion.Transcriptomic analysis revealed that knockdown of AtMmp2 significantly increased expression of the Toll and Imd pathways,chitin metabolism,and cross-linking(such as the pro-phenoloxidase activating cascade pathway and the tyrosine-mediated cuticle sclerotization and pigmentation pathway).These data revealed evolutionarily conserved functions of Mmp2 in controlling adult eclosion and wing expansion,also provided a preliminary exploration of the novel function of regulating Toll and Imd pathways,as well as new insights into how MMPs regulate insect development and defense barriers.展开更多
Auxin has been regarded as the main signal molecule coordinating the growth and ripening of fruits in strawberry, the reference genomic system for Rosaceae. The mechanisms regulating auxin biosynthesis in strawberry a...Auxin has been regarded as the main signal molecule coordinating the growth and ripening of fruits in strawberry, the reference genomic system for Rosaceae. The mechanisms regulating auxin biosynthesis in strawberry are largely elusive. Recently, we demonstrated that two YUCCA genes are involved in flower and fruit development in cultivated strawberry. Here, we show that the woodland strawberry （Fragaria vesca L.） genome harbors nine loci for YUCCA genes and eight of them encode functional proteins. Transcription pattern in different plant organs was different for all eight FvYUCs. Functionality of the FvYUC6 gene was studied in transgenic strawberry over- expressing FvYUC6, which showed typical high-auxin pheno- types. Overexpression of FvYUC6 also delayed flowering and led to complete male sterility in F. vesta. Additionally, specific repression of FvYUC6 expression by RNA interference signifi- cantly inhibited vegetative growth and reduced plant fertility. The development of leaves, roots, flowers, and fruits was greatly affected in FvYUC6-repressed plants. Expression of a subset of auxin-responsive genes was well correlated with the changes of FvYUC6 transcript levels and free indole-3-acetic acid levels in transgenic strawberry. These observations are consistent with an important role of FvYUC6 in auxin synthesis, and support a main role of the gene product in vegetative and reproductive development in woodland strawberry.展开更多
Olfaction is primarily mediated by highly specified olfactory receptors （ORs）. Here, we cloned and identified an olfactory receptor, named SlituOR1 （Genbank no. JN835269）, from Spodoptera litura and found evidence...Olfaction is primarily mediated by highly specified olfactory receptors （ORs）. Here, we cloned and identified an olfactory receptor, named SlituOR1 （Genbank no. JN835269）, from Spodoptera litura and found evidence that it is a candidate pheromone receptor. It exhibited male-biased expression in the antennae, where it was localized at the base of sensilla trichoidea, the antennal sensilla mainly responsive to pheromones in moths. Conserved orthologues of this receptor, found among known pheromone receptors within the Lepidoptera, and SlituOR1 were placed among a clade of candidate pheromone re- ceptors in a phylogeny tree of insect OR gene sequences. SlituOR1 showed differential expression in S. litura populations attracted to traps baited with different ratios of the two sex pheromone components （9Z,11E）-tetradecadienyl acetate （Z9E11-14：OAc） and （9Z,12E）-tetradecadienyl acetate （Z9E12-14：OAc）. Knocking down of SlituOR1 by RNA interference reduced the electroantennogram （EAG） response to Z9E11-14：OAc, and this result is consistent with the field trapping experiment. We infer that variation in transcrip- tion levels of olfactory receptors may modulate sex pheromone perception in male moths and could provide some of the flexibility required to maintain the functionality of com- munication with females when a population is adapting to a new niche and reproductive isolation becomes an advantage.展开更多
Mutations in the photorespiration pathway dis- play a lethal phenotype in atmospheric air, which can be fully recovered by elevated C02. An exception is that mutants of peroxisomal hydroxypyruvate reductase （HPR1） d...Mutations in the photorespiration pathway dis- play a lethal phenotype in atmospheric air, which can be fully recovered by elevated C02. An exception is that mutants of peroxisomal hydroxypyruvate reductase （HPR1） do not have this phenotype, indicating the presence of cytosolic bypass in the photorespiration pathway. In this study, we constructed overexpression of the OsHPR1 gene and RNA interference plants of OsHPR1 and OsHPR2 genes in rice （Oryza sativo L. cv. Zhonghua 11）. Results from reverse transcription-polymerase chain reaction （RT-PCR）, Western blot, and enzyme assays showed that HPR1 activity changed significantly in corresponding transgenic lines without any effect on HPR2 activity, which is the same for HPR2. However, metabolite analysis and the serine glyoxylate aminotransferase （SGAT） activity assay showed that the metabolite flux of photorespiration was disturbed in RNAi lines of both HPR genes. Furthermore, HPR1 and HPR2 proteins were located to the peroxisome and cytosol, respectively, by transient expression experiment. Double mutant hprl x hpr2 was generated by crossing individual mutant of hprl and hpr2. The phenotypes of all transgenic lines were determined in ambient air and C02-elevated air. The phenotype typical of photorespiration mutants was observed only where activity of both HPRI and HPR2 were downregulated in the same line. These findings demonstrate that two hydroxypyruvate reductases encoded by OsHPRI and OsHPR2 are involved in photorespiratory metabolism in rice.展开更多
Insect cytochrome P450 monooxygenases （CYPs or P450s） play an important role in detoxifying insecticides leading to resistance in insect populations. A polyphagous pest, Spodoptera litura, has developed resistance t...Insect cytochrome P450 monooxygenases （CYPs or P450s） play an important role in detoxifying insecticides leading to resistance in insect populations. A polyphagous pest, Spodoptera litura, has developed resistance to a wide range of insecticides. In the present study, a novel P450 gene, CYP321B1, was cloned from S. litura. The function of CYP321B1 was assessed using RNA interference （RNAi） and monitoring resistance levels for three commonly used insecticides, including chlorpyrifos, β-cypermethrin and methomyl. The full-length complementary DNA sequence of CYP321B1 is 1814 bp long with an open reading frame of 1 488 bp encoding 495 amino acid residues. Quantitative reverse-transcriptase polymerase chain reaction analyses during larval and pupal develop- ment indicated that CYP321B1 expression was highest in the midgut of fifth-instar larvae, followed by fat body and cuticle. The expression of CYP321B1 in the midgut was up- regulated by chlorpyrifos,β-cypermethrin and methomyl with both lethal concentration at 15% （LC15） （50, 100 and 150 μg/mL, respectively） and 50%（LC50） dosages （100,200 and 300μg/mL, respectively）. Addition of piperonyl butoxide （PBO） significantly increased the toxicity ofchlorpyrifos,β-cypermethrin and methomyl to S. litura, suggesting a marked synergism of the three insecticides with PBO and P450-mediated detoxification. RNAi- mediated silencing of CYP321B1 further increased mortality by 25.6% and 38.9% when the fifth-instar larvae were exposed to chlorpyrifos and β-cypermethrin, respectively, at the LCso dose levels. The results demonstrate that CYP321B1 might play an important role in chlorpyrifos and β-cypermethrin detoxification in S. litura.展开更多
基金Supported by Natural Science Foundation of Jiangsu Province(BK2011668)Special Fund for Independent Innovation of Agricultural Science and Technology in Jiangsu Province[CX(10)406]+1 种基金Opening Project of the Key Lab of Biology of Oil Crops of Ministry of Agriculture(201001)National 948 Project of China(2011-G23)~~
文摘A predominantly expressed gene, pyruvate kinease （PK） gene, control ing oil accumulation, had been identified in previous study. To construct a PK RNAi vector, a 498-bp target PK gene sequence was amplified and transferred into the pEASY-T1vector. Subsequently, the target DNA fragments were cut off by enzymes Not I and Xho I and directional y inserted into plant RNAi platform vector pHurricane, a newly developed RNAi vector in our lab, to form the PK inverse repeats. The PK inverted repeats fragment was then cloned into a modified vector pCAMBIA1390, driven by a rapeseed seed-specific promoter napin. Restriction enzyme digestion verified the successful construction of RNA interference vector. The PK RNAi con-struction wil lay a foundation for study in the function of PK in oil accumulation and metabolism in rapeseeds.
基金supported by the National Natural Science Foundation of China,No.30970995the Postgraduate Science Research Innovation Project of Higher Learning University of Jiangsu Province in China,No.CXLX11_0735
文摘Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ische- mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy- gen-glucose deprivation to simulate cerebral ischemia/reperfusion injury. Chemically synthesized small interfedng RNA （siRNA）-1280, -1724 and -418 specific to toll-like receptor 3 were transfected into oxygen-glucose deprived cortical neurons to suppress the upregulation of toll-like receptor 3 protein expression. Western blotting demonstrated that after transfection with siRNA, toll-like re- ceptor 3 protein expression reduced, especially in the toll-like receptor 3-1724 group. These results suggested that siRNA-1724 is an optimal sequence for inhibiting toll-like receptor 3 expression in cortical neurons following oxygen-glucose deprivation.
文摘OBJECTIVE In this study, RNA interference was used to evaluate the effects of HMGB1 expression on cell cycle and proliferation of the human cervical cancer cell line HeLa.METHODS We had previously constructed and screened effective eukaryotic expression vectors carrying PGCsi3.0-1/ HMGB1 siRNA and PGCsi3.0-3/HMGB1 siRNA, then the vectors were transfected into HeLa cells. The expression of HMGB1 before and after transfection in HeLa cells were detected by RT-PCR and Western blot. The cell viability and proliferating activity was tested by Trypan blue dye test and MTT, and the cell cycle was determined bv flow cvtometry.RESULTS The introduction of PGCsi3.0-1/HMGB1 siRNA and PGCsi3.0-3/HMGB1 siRNA inhibited the expression of HMGB1 mRNA and protein efficiently and specifically, there was a significant difference between the siRNA groups and the control groups （P 〈 0.05）. The proliferation speed of PGCsi3.0-1 group and PGC si3.0-3 group were obviously slower than those of PGCsi3.0- Neg group and non-transfected group. Flow cytometry showed that the content of DNA in G2 phase in PGCsi3.0-1 group and PGCsi3.0-3 group were obviously more than those in PGCsi3.0- Neg group and non-transfected group, but the content in S phase was less （P 〈 0.01）. The progression of cell cycle was arrested from G2 to S phase.CONCLUSION PGCsi3.0-1/HMGB1 siRNA and PGCsi3.0-3 /HMGB1 siRNA could specially suppress the expression of HMGB1 gene, inhibit the proliferation speed of HeLa cells effectively, and arrest the progression of cell cycle from G2 to S phase. RNAi provides a new approach to the bio-therapy of cervical cancer.
基金Supported by the National Basic Research Program of China (973 Program) (No. 2006CB101804)the National Natural Science Foundation of China (No. 30972245)Public Industry (Agriculture) Specific Research Program (No. 200803012)
文摘Acting as one of the pattern recognition receptors （PRRs）, C-type lectin is believed to mediate pathogen recognition and plays an important role in the clearance of pathogens as part of the innate immune system. In this work, a novel C-type lectin gene （named LvLecl） was cloned from the shrimp Litopenaeus vannamei, The ORF of LvLecl is 510 bp, encoding 169 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids at the N-terminal and a carbohydrate recognition domain （CRD） at the C-terminal. LvLecl was mainly expressed in the hepatopancreas. Real-time PCR analysis indicated that the level of LvLecl transcripts significantly changed in the hepatopancreas after the shrimp were artificially challenged with LPS, Micrococcus lysodeikticus and white spot syndrome virus （WSSV）. RNAi-based silencing of LvLecl resulted in increases in mortality when the shrimp were challenged with WSSV, and the median lethal time was reduced compared with controls. Although there was no characteristic ＂EPN＂ （Glu-Pro-Ser） or ＂QPD＂ （Gin-Pro-Asp） motif, the recombinant LvLecl, expressed in Escherichia coli BL21 （DE3）, could also agglutinate M. lysodeikticus and Vibrio anguillarum. The agglutinating activities were calcium-dependent and could be inhibited by D-mannose, D-glucose, D-galactose and N-Acetyl-D-mannose. These results suggest that LvLecl might be involved in the immune response against WSSV and bacterial infections and contribute to non-self recognition as a pattern recognition receptor in the innate immune system of the shrimp L. vannamei.
基金funded by GDAS Special Project of Science and Technology Development(2022GDASZH-2022010106)National Natural Science Foundation of China(Grant No.32200405),Guangzhou Basic and Applied Basic Research Foundation(202201010255)+1 种基金Guangdong Basic and Applied Basic Research Foundation(2021A1515012102,2021A1515010630)Science and Technology Planning Project of Yunfu City(2022020425).
文摘During the pupal-adult eclosion process of holometabolous insects,the old cuticle is shed and replaced by a completely different new cuticle that requires tanning and expansion,along with extensive extracellular matrix(ECM)remodeling.In vertebrates,matrix metalloproteinases(MMPs),a class of zinc-dependent endopeptidases,play key roles in regulating the ECM that surrounds cells.However,little is known about these extracellular proteinases available in insects.The small hive beetle(SHB),Aethina tumida,is a widespread invasive parasite of honey bees.In this study,6 MMP homologs were identified in the SHB genome.RNA interference experiments showed that all 6 AtMmps are not required for the larval-pupal transition,only AtMmp2 was essential for pupal-adult eclosion in SHB.Knockdown of AtMmp2 resulted in eclosion defects and wing expansion failure,as well as mortality within 3 d of adult eclosion.Transcriptomic analysis revealed that knockdown of AtMmp2 significantly increased expression of the Toll and Imd pathways,chitin metabolism,and cross-linking(such as the pro-phenoloxidase activating cascade pathway and the tyrosine-mediated cuticle sclerotization and pigmentation pathway).These data revealed evolutionarily conserved functions of Mmp2 in controlling adult eclosion and wing expansion,also provided a preliminary exploration of the novel function of regulating Toll and Imd pathways,as well as new insights into how MMPs regulate insect development and defense barriers.
基金the Science and Technology Commission of Shanghai Municipality (Shanghai Rising-Star Program (09QA1405300) to K. D., Key Program (12391901400) to Q. H. G.)Shanghai Academy of Agricultural Sciences (S & T Development Program (2012(13)) to K. D.). V. V. was funded by MICINN (Spain), grant no. BIO2010-15630
文摘Auxin has been regarded as the main signal molecule coordinating the growth and ripening of fruits in strawberry, the reference genomic system for Rosaceae. The mechanisms regulating auxin biosynthesis in strawberry are largely elusive. Recently, we demonstrated that two YUCCA genes are involved in flower and fruit development in cultivated strawberry. Here, we show that the woodland strawberry （Fragaria vesca L.） genome harbors nine loci for YUCCA genes and eight of them encode functional proteins. Transcription pattern in different plant organs was different for all eight FvYUCs. Functionality of the FvYUC6 gene was studied in transgenic strawberry over- expressing FvYUC6, which showed typical high-auxin pheno- types. Overexpression of FvYUC6 also delayed flowering and led to complete male sterility in F. vesta. Additionally, specific repression of FvYUC6 expression by RNA interference signifi- cantly inhibited vegetative growth and reduced plant fertility. The development of leaves, roots, flowers, and fruits was greatly affected in FvYUC6-repressed plants. Expression of a subset of auxin-responsive genes was well correlated with the changes of FvYUC6 transcript levels and free indole-3-acetic acid levels in transgenic strawberry. These observations are consistent with an important role of FvYUC6 in auxin synthesis, and support a main role of the gene product in vegetative and reproductive development in woodland strawberry.
基金This study was funded by the Special Fund for Agro- scientific Research in the Public Interest in China （Grant No. 201203036）, Natural Science Foundation of Zhejiang （Project No. D3080388） and National 973 Project （Project No. 2012CB11410005）.
文摘Olfaction is primarily mediated by highly specified olfactory receptors （ORs）. Here, we cloned and identified an olfactory receptor, named SlituOR1 （Genbank no. JN835269）, from Spodoptera litura and found evidence that it is a candidate pheromone receptor. It exhibited male-biased expression in the antennae, where it was localized at the base of sensilla trichoidea, the antennal sensilla mainly responsive to pheromones in moths. Conserved orthologues of this receptor, found among known pheromone receptors within the Lepidoptera, and SlituOR1 were placed among a clade of candidate pheromone re- ceptors in a phylogeny tree of insect OR gene sequences. SlituOR1 showed differential expression in S. litura populations attracted to traps baited with different ratios of the two sex pheromone components （9Z,11E）-tetradecadienyl acetate （Z9E11-14：OAc） and （9Z,12E）-tetradecadienyl acetate （Z9E12-14：OAc）. Knocking down of SlituOR1 by RNA interference reduced the electroantennogram （EAG） response to Z9E11-14：OAc, and this result is consistent with the field trapping experiment. We infer that variation in transcrip- tion levels of olfactory receptors may modulate sex pheromone perception in male moths and could provide some of the flexibility required to maintain the functionality of com- munication with females when a population is adapting to a new niche and reproductive isolation becomes an advantage.
基金supported by the National Natural Science Foundation of China (U1201212 31170222)+1 种基金the Shenzhen Overseas Talents Innovation and Entrepreneurship Funding Scheme (The Peacock Scheme)China Postdoctoral Science Foundation (2013M530374)
文摘Mutations in the photorespiration pathway dis- play a lethal phenotype in atmospheric air, which can be fully recovered by elevated C02. An exception is that mutants of peroxisomal hydroxypyruvate reductase （HPR1） do not have this phenotype, indicating the presence of cytosolic bypass in the photorespiration pathway. In this study, we constructed overexpression of the OsHPR1 gene and RNA interference plants of OsHPR1 and OsHPR2 genes in rice （Oryza sativo L. cv. Zhonghua 11）. Results from reverse transcription-polymerase chain reaction （RT-PCR）, Western blot, and enzyme assays showed that HPR1 activity changed significantly in corresponding transgenic lines without any effect on HPR2 activity, which is the same for HPR2. However, metabolite analysis and the serine glyoxylate aminotransferase （SGAT） activity assay showed that the metabolite flux of photorespiration was disturbed in RNAi lines of both HPR genes. Furthermore, HPR1 and HPR2 proteins were located to the peroxisome and cytosol, respectively, by transient expression experiment. Double mutant hprl x hpr2 was generated by crossing individual mutant of hprl and hpr2. The phenotypes of all transgenic lines were determined in ambient air and C02-elevated air. The phenotype typical of photorespiration mutants was observed only where activity of both HPRI and HPR2 were downregulated in the same line. These findings demonstrate that two hydroxypyruvate reductases encoded by OsHPRI and OsHPR2 are involved in photorespiratory metabolism in rice.
文摘Insect cytochrome P450 monooxygenases （CYPs or P450s） play an important role in detoxifying insecticides leading to resistance in insect populations. A polyphagous pest, Spodoptera litura, has developed resistance to a wide range of insecticides. In the present study, a novel P450 gene, CYP321B1, was cloned from S. litura. The function of CYP321B1 was assessed using RNA interference （RNAi） and monitoring resistance levels for three commonly used insecticides, including chlorpyrifos, β-cypermethrin and methomyl. The full-length complementary DNA sequence of CYP321B1 is 1814 bp long with an open reading frame of 1 488 bp encoding 495 amino acid residues. Quantitative reverse-transcriptase polymerase chain reaction analyses during larval and pupal develop- ment indicated that CYP321B1 expression was highest in the midgut of fifth-instar larvae, followed by fat body and cuticle. The expression of CYP321B1 in the midgut was up- regulated by chlorpyrifos,β-cypermethrin and methomyl with both lethal concentration at 15% （LC15） （50, 100 and 150 μg/mL, respectively） and 50%（LC50） dosages （100,200 and 300μg/mL, respectively）. Addition of piperonyl butoxide （PBO） significantly increased the toxicity ofchlorpyrifos,β-cypermethrin and methomyl to S. litura, suggesting a marked synergism of the three insecticides with PBO and P450-mediated detoxification. RNAi- mediated silencing of CYP321B1 further increased mortality by 25.6% and 38.9% when the fifth-instar larvae were exposed to chlorpyrifos and β-cypermethrin, respectively, at the LCso dose levels. The results demonstrate that CYP321B1 might play an important role in chlorpyrifos and β-cypermethrin detoxification in S. litura.
