Firmness is one of the most important fruit quality traits in strawberries.The postharvest shelf life of this soft fruit is highly limited by the loss of firmness,where cell wall disassembly plays an important role.Pr...Firmness is one of the most important fruit quality traits in strawberries.The postharvest shelf life of this soft fruit is highly limited by the loss of firmness,where cell wall disassembly plays an important role.Previous studies demonstrated that the polygalacturonase FaPG1 has a key role in remodelling pectins during strawberry softening.In this study,FaPG1 knockout strawberry plants have been generated using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens.Ten independent lines,cv.“Chandler”,were obtained,and all of them were successfully edited as determined by PCR amplification and T7 endonuclease assay.The targeted mutagenesis insertion and deletion rates were analyzed using targeted deep sequencing.The percentage of edited sequences varied from 47%up to almost 100%,being higher than 95%for seven of the selected lines.Phenotypic analyses showed that 7 out of the eight lines analyzed produced fruits significantly firmer than the control,ranging from 33 to 70%increase in firmness.There was a positive relationship between the degree of FaPG1 editing and the rise in fruit firmness.Minor changes were observed in other fruit quality traits,such as colour,soluble solids,titratable acidity or anthocyanin content.Edited fruits showed a reduced softening rate during postharvest,displayed a reduced transpirational water loss,and were less damaged by Botrytis cinerea inoculation.The analysis of four potential off-target sites revealed no mutation events.In conclusion,editing the FaPG1 gene using the CRISPR/Cas9 system is an efficient method for improving strawberry fruit firmness and shelf life.展开更多
In this study, 1 500 bp PG gene promoter was amplified from leaves of tomato cultivar ＇ Zhongshu No. 4＇. The bioinformatic analysis of PG promoter sequences was conducted. PG promoter elements were predicted and ana...In this study, 1 500 bp PG gene promoter was amplified from leaves of tomato cultivar ＇ Zhongshu No. 4＇. The bioinformatic analysis of PG promoter sequences was conducted. PG promoter elements were predicted and analyzed by PLANTCARE and PLACE. The results showed that tomato PG promoter contained multiple c/s-acting regulatory elements such as typical basic elements TATA-Box and CAAT-Box, light responsive elements 3-AF1 binding site, ATl-motif, ATCT- motif, Box 4, Box I, GA-motif, GTl-motif, Spl and MRE, heat stress-responsive element HSE, ethylene-responsive element ERE, meristem-specifie regulatory element CCGTCC-box, endosperm expression-related regulatory elements GCN4_motif and Skn-l_motif, defense and stress responsiveness element TC-rich repeats, and circadian control-related element circadian, indicating that the expression of tomato PG gene is related to light, temperature, hormone, stress and other factors. This study laid the foundation for subsequent research about regulation of PG gene expression in plants.展开更多
Three endo-polygalacturonases(endoPGs) from a newly isolated Penicillum oxalicum(CGMCC 0907) capable of utilizing waste biomass as growth substrate were separated and purified to homogeneity by ultra-filtration,affini...Three endo-polygalacturonases(endoPGs) from a newly isolated Penicillum oxalicum(CGMCC 0907) capable of utilizing waste biomass as growth substrate were separated and purified to homogeneity by ultra-filtration,affinity adsorption chromatography,CM-cellulose column chromatography,and Sephadex G-100 gel filtration chromatography with the overall yield of 64.5% from the crude enzyme.The specific activities and recovery rates of endoPG-1,endoPG-2 and endoPG-3 were 1120 U/mg and 21.6%,1350 U/mg and 25.9%,and 1560 U/mg and 17.0%,respectively.The three purified endoPGs had a close molecular weight to 41 kDa as estimated by SDS-PAGE.The optimum temperature and pH for the function of them were 65℃ and 5.0,55℃ and 5.0,50℃ and 5.5,respectively.Their pI and Km values were 5.9 and 0.78 mg/mL,6.0 and 1.2 mg/mL,and 6.1 and 2.0 mg/mL,respectively.展开更多
Polygalacturonase-inhibiting proteins （PGIPs） are extracellular proteins that belong to the leucine-rich repeat （LRR） protein superfamily. PGIPs inhibit fungal polygalacturonases （PGs） and promote accumulation o...Polygalacturonase-inhibiting proteins （PGIPs） are extracellular proteins that belong to the leucine-rich repeat （LRR） protein superfamily. PGIPs inhibit fungal polygalacturonases （PGs） and promote accumulation of oligogalacturonides, which activate plant defense responses. PGIPs play important roles in resistance to infection of pathogens. In this study, reverse transcriptase-polymerase chain reaction （RT-PCR） and RNA ligase-mediated rapid amplification of cDNA ends （RLM-RACE） were used to isolate the full-length PGIP cDNA from Populus deltoides （GenBank accession no. of PdPGIP2 and PdPGIP4：EF684913 and EF684912）. Domain analysis revealed that the deduced amino acid sequences of PdPGIP2 and PdPGIP4 had a typical PGIP topology. Phylogenetic analysis of known PGIPs indicated that the two PdPGIPs were clustered to the defense-related PGIP clade. Using real-time RT-PCR, the expression patterns of the two PdPGIPs following treatment with a fungal pathogen and defense-related signaling molecules were studied. The expression levels of PdPGIP2 and PdPGIP4 were both up-regulated when inoculated with the phytopathogenic fungus Marssonina brunnea. Therefore, it was proposed that the two PGIPs might be involved in the resistance to Marssonina brunnea in P. deltoides.展开更多
Fruit ripening is a complex process and is regulated by many factors.Ethylene and polygalacturonase(PG),lipoxygenase(LOX),expansin(EXP) are all critical regulating factors in fruit ripening and softening process.With ...Fruit ripening is a complex process and is regulated by many factors.Ethylene and polygalacturonase(PG),lipoxygenase(LOX),expansin(EXP) are all critical regulating factors in fruit ripening and softening process.With antisense ACS tomato,Nr mutant tomato and cultivated tomato as materials,Northern blot hybridization showed that PG,LeEXP1 and LOX expressed differently in different parts of cultivated tomato fruit during ripening,which was related to fruit ripening.The ripening process of columella and radial pericarp was faster than pericarp.In both Nr mutant and antisense ACS transgenic tomato fruit,expression levels of PG,LeEXP1 and LOX were generally lower than those in cultivated fruit but still related to fruit ripening.The expression levels of PG,LeEXP1 and LOX increased in the mature green tomato fruits after 0.5 h treatment with ethylene(100 μL/L).These results indicate that gene expression of PG,LeEXP1 and LOX were positively regulated by ethylene.The time and cumulative effect of the concentration exists in the expression of PG regulated by ethylene.The regulation of LOX expression mainly depended on the fruit development after great amount of ethylene was produced.PG played a major role in ripening and softening of tomato fruit,and cooperated with the regulation of EXP and LOX.展开更多
The present study aimed to purify and characterize one polygalacturonase from L. gongylophorus (PGaseLg), the symbiotic fungus of Atta sexdens. The enzyme was isolated by salting out of crude extract followed by two c...The present study aimed to purify and characterize one polygalacturonase from L. gongylophorus (PGaseLg), the symbiotic fungus of Atta sexdens. The enzyme was isolated by salting out of crude extract followed by two chromatographic steps. PGaseLG was identified with MS analysis and molecular exclusion chromatography revealed the monomeric nature of a protein with an estimated molecular weight of about 39 kDa. PGaseLg has an optimum temperature of 60°C and optimum pH activity at 5.0. Using polygalacturonate as a substrate, the calculations of K<sub>M</sub>, V<sub>max</sub> and k<sub>cat</sub> were 0.65 mg·mL<sup>-1</sup>, 1800 μmol·min<sup>-1</sup>·mg<sup>-1</sup> and 35.97 s<sup>-1</sup>, respectively. The enzyme was stable for more than 3 h at 50°C at pH 5.0;otherwise, at lower or higher pH values, the PGaseLg was less stable. The influence of several metals, EDTA and β-mercaptoethanol on enzyme activity was also determined. Thin layer chromatography (TLC) analyses indicated that PGaseLg is an exopolygalacturonase.展开更多
The pectin is a backbone of the plant cell wall, its network structure will systemicly resolve when the plant cell splits up and forms. The pectinase produced by Rhizoctonia mainly acts on the pectin of cell wall, and...The pectin is a backbone of the plant cell wall, its network structure will systemicly resolve when the plant cell splits up and forms. The pectinase produced by Rhizoctonia mainly acts on the pectin of cell wall, and causes the maceration of tissue and the death of protoplast. Polygalacturonase (PG) can decompose the galacturonic acid of disease tissue. The research defined the PG activities of extracellular metabolite of the different virulence Rhizoctonia isolates, and testifid the effect of Trichoderma viride to PG activities, and clarified the mechanisms of biocontrol by Trichoderma. The test methods as following: Firstly, to select the isolates of different virulence: WK-47, WK-141 and WK-160 strain of Rhizoctonia AG-D and YW-2 strain of Rhizoctonia AG1-IA and TCS-1 strain of Trichoderma viride. Secondly, to culture TCS-1 on PD, and draw a group of fermented liquid in every 24 hours, and draw 7 times. Thirdly, to culture quietly Rhizoctonia isolates with Czapek-Dox at 25℃ for 15 days, filter and centrifuge (2350 g×30 min), fetch the clear liquid, put it into the ammonium sulfate according to 60% saturation degree, put it quietly for 30 min at 4℃, centrifuge (21000 g×30 min) at 4℃, remove the clear liquid, dissolve the deposit with sodium acetate buffer (25 mmol/L, pH5.5), dialysis for 48 h in the same buffer,and change the buffer every 12 h, Fourthly, to put TCS-1 fermented broth of different times in the tubes, one mL per a tube, add 0.5 mL PG to every tube, react for 4 h in 30 ℃ water, the same time fetch the test tube filled with the same treated liquid that was not dealed in 30℃ water.Finally,to testify PG activities with DNS’s test. In all, PG of Rhizoctonia had high activities and virulence. The conrtrol efficacy of T.viride to PG activities of WK-47, WK-141, WK-160 and YW-2 were 95%,94%,95%,92% separately, fermented time had a great influence to control efficacy, the third fermented broth did the best. Through effect to PG activities T. viride can reduce the virulence of Rhizoctonia, and protect the hosts. The specific mechanism, qualitative and quantitative research of antagonistic substance in the fermented broth will be further carried out.展开更多
基金supported by the Ministerio de Ciencia,Innovación y Universidades and FEDER EU funds(grant numbers AGL2017-86531-C2-1R and PID2020-118468RB-C21)the University of Malaga(grant number B1-2020_09)+1 种基金CS-R was awarded a PhD Fellowship from the Ministerio de Ciencia,Innovación y Universidades(PRE2018-085509)PhD Program Advanced Biotechnology,University of Málaga.
文摘Firmness is one of the most important fruit quality traits in strawberries.The postharvest shelf life of this soft fruit is highly limited by the loss of firmness,where cell wall disassembly plays an important role.Previous studies demonstrated that the polygalacturonase FaPG1 has a key role in remodelling pectins during strawberry softening.In this study,FaPG1 knockout strawberry plants have been generated using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens.Ten independent lines,cv.“Chandler”,were obtained,and all of them were successfully edited as determined by PCR amplification and T7 endonuclease assay.The targeted mutagenesis insertion and deletion rates were analyzed using targeted deep sequencing.The percentage of edited sequences varied from 47%up to almost 100%,being higher than 95%for seven of the selected lines.Phenotypic analyses showed that 7 out of the eight lines analyzed produced fruits significantly firmer than the control,ranging from 33 to 70%increase in firmness.There was a positive relationship between the degree of FaPG1 editing and the rise in fruit firmness.Minor changes were observed in other fruit quality traits,such as colour,soluble solids,titratable acidity or anthocyanin content.Edited fruits showed a reduced softening rate during postharvest,displayed a reduced transpirational water loss,and were less damaged by Botrytis cinerea inoculation.The analysis of four potential off-target sites revealed no mutation events.In conclusion,editing the FaPG1 gene using the CRISPR/Cas9 system is an efficient method for improving strawberry fruit firmness and shelf life.
基金Supported by Natural Science Foundation of Yunnan Province(2011FB049)National Natural Science Foundation of China(31260481,31460516)+1 种基金Fund of Yunnan Education Department(2013Y251)Development Fund of the Department of Life Science and Technology,Kunming University(GXKM201505)
文摘In this study, 1 500 bp PG gene promoter was amplified from leaves of tomato cultivar ＇ Zhongshu No. 4＇. The bioinformatic analysis of PG promoter sequences was conducted. PG promoter elements were predicted and analyzed by PLANTCARE and PLACE. The results showed that tomato PG promoter contained multiple c/s-acting regulatory elements such as typical basic elements TATA-Box and CAAT-Box, light responsive elements 3-AF1 binding site, ATl-motif, ATCT- motif, Box 4, Box I, GA-motif, GTl-motif, Spl and MRE, heat stress-responsive element HSE, ethylene-responsive element ERE, meristem-specifie regulatory element CCGTCC-box, endosperm expression-related regulatory elements GCN4_motif and Skn-l_motif, defense and stress responsiveness element TC-rich repeats, and circadian control-related element circadian, indicating that the expression of tomato PG gene is related to light, temperature, hormone, stress and other factors. This study laid the foundation for subsequent research about regulation of PG gene expression in plants.
基金Supported by National Natural Science Foundation of China (No. 30600082)National Key Technology R&D Program of China (No. 2008BADA7B01)Beijing Municipal Commission of Education (No. KM200811417006)
文摘Three endo-polygalacturonases(endoPGs) from a newly isolated Penicillum oxalicum(CGMCC 0907) capable of utilizing waste biomass as growth substrate were separated and purified to homogeneity by ultra-filtration,affinity adsorption chromatography,CM-cellulose column chromatography,and Sephadex G-100 gel filtration chromatography with the overall yield of 64.5% from the crude enzyme.The specific activities and recovery rates of endoPG-1,endoPG-2 and endoPG-3 were 1120 U/mg and 21.6%,1350 U/mg and 25.9%,and 1560 U/mg and 17.0%,respectively.The three purified endoPGs had a close molecular weight to 41 kDa as estimated by SDS-PAGE.The optimum temperature and pH for the function of them were 65℃ and 5.0,55℃ and 5.0,50℃ and 5.5,respectively.Their pI and Km values were 5.9 and 0.78 mg/mL,6.0 and 1.2 mg/mL,and 6.1 and 2.0 mg/mL,respectively.
基金the National Basic Research Program of China (No 2007CB116307)Forestry Scientific and Technical Supporting Programs (No 2006BAD01A15-3)
文摘Polygalacturonase-inhibiting proteins （PGIPs） are extracellular proteins that belong to the leucine-rich repeat （LRR） protein superfamily. PGIPs inhibit fungal polygalacturonases （PGs） and promote accumulation of oligogalacturonides, which activate plant defense responses. PGIPs play important roles in resistance to infection of pathogens. In this study, reverse transcriptase-polymerase chain reaction （RT-PCR） and RNA ligase-mediated rapid amplification of cDNA ends （RLM-RACE） were used to isolate the full-length PGIP cDNA from Populus deltoides （GenBank accession no. of PdPGIP2 and PdPGIP4：EF684913 and EF684912）. Domain analysis revealed that the deduced amino acid sequences of PdPGIP2 and PdPGIP4 had a typical PGIP topology. Phylogenetic analysis of known PGIPs indicated that the two PdPGIPs were clustered to the defense-related PGIP clade. Using real-time RT-PCR, the expression patterns of the two PdPGIPs following treatment with a fungal pathogen and defense-related signaling molecules were studied. The expression levels of PdPGIP2 and PdPGIP4 were both up-regulated when inoculated with the phytopathogenic fungus Marssonina brunnea. Therefore, it was proposed that the two PGIPs might be involved in the resistance to Marssonina brunnea in P. deltoides.
基金Supported by National Project of Scientific and Technical Supporting Programs Funded by Ministry of Science and Technology of China (No.2006BAD22B01)National Natural Science Foundation of China (No.30800767)Postdoctoral Fund of China (No.20080430725)
文摘Fruit ripening is a complex process and is regulated by many factors.Ethylene and polygalacturonase(PG),lipoxygenase(LOX),expansin(EXP) are all critical regulating factors in fruit ripening and softening process.With antisense ACS tomato,Nr mutant tomato and cultivated tomato as materials,Northern blot hybridization showed that PG,LeEXP1 and LOX expressed differently in different parts of cultivated tomato fruit during ripening,which was related to fruit ripening.The ripening process of columella and radial pericarp was faster than pericarp.In both Nr mutant and antisense ACS transgenic tomato fruit,expression levels of PG,LeEXP1 and LOX were generally lower than those in cultivated fruit but still related to fruit ripening.The expression levels of PG,LeEXP1 and LOX increased in the mature green tomato fruits after 0.5 h treatment with ethylene(100 μL/L).These results indicate that gene expression of PG,LeEXP1 and LOX were positively regulated by ethylene.The time and cumulative effect of the concentration exists in the expression of PG regulated by ethylene.The regulation of LOX expression mainly depended on the fruit development after great amount of ethylene was produced.PG played a major role in ripening and softening of tomato fruit,and cooperated with the regulation of EXP and LOX.
文摘The present study aimed to purify and characterize one polygalacturonase from L. gongylophorus (PGaseLg), the symbiotic fungus of Atta sexdens. The enzyme was isolated by salting out of crude extract followed by two chromatographic steps. PGaseLG was identified with MS analysis and molecular exclusion chromatography revealed the monomeric nature of a protein with an estimated molecular weight of about 39 kDa. PGaseLg has an optimum temperature of 60°C and optimum pH activity at 5.0. Using polygalacturonate as a substrate, the calculations of K<sub>M</sub>, V<sub>max</sub> and k<sub>cat</sub> were 0.65 mg·mL<sup>-1</sup>, 1800 μmol·min<sup>-1</sup>·mg<sup>-1</sup> and 35.97 s<sup>-1</sup>, respectively. The enzyme was stable for more than 3 h at 50°C at pH 5.0;otherwise, at lower or higher pH values, the PGaseLg was less stable. The influence of several metals, EDTA and β-mercaptoethanol on enzyme activity was also determined. Thin layer chromatography (TLC) analyses indicated that PGaseLg is an exopolygalacturonase.
文摘The pectin is a backbone of the plant cell wall, its network structure will systemicly resolve when the plant cell splits up and forms. The pectinase produced by Rhizoctonia mainly acts on the pectin of cell wall, and causes the maceration of tissue and the death of protoplast. Polygalacturonase (PG) can decompose the galacturonic acid of disease tissue. The research defined the PG activities of extracellular metabolite of the different virulence Rhizoctonia isolates, and testifid the effect of Trichoderma viride to PG activities, and clarified the mechanisms of biocontrol by Trichoderma. The test methods as following: Firstly, to select the isolates of different virulence: WK-47, WK-141 and WK-160 strain of Rhizoctonia AG-D and YW-2 strain of Rhizoctonia AG1-IA and TCS-1 strain of Trichoderma viride. Secondly, to culture TCS-1 on PD, and draw a group of fermented liquid in every 24 hours, and draw 7 times. Thirdly, to culture quietly Rhizoctonia isolates with Czapek-Dox at 25℃ for 15 days, filter and centrifuge (2350 g×30 min), fetch the clear liquid, put it into the ammonium sulfate according to 60% saturation degree, put it quietly for 30 min at 4℃, centrifuge (21000 g×30 min) at 4℃, remove the clear liquid, dissolve the deposit with sodium acetate buffer (25 mmol/L, pH5.5), dialysis for 48 h in the same buffer,and change the buffer every 12 h, Fourthly, to put TCS-1 fermented broth of different times in the tubes, one mL per a tube, add 0.5 mL PG to every tube, react for 4 h in 30 ℃ water, the same time fetch the test tube filled with the same treated liquid that was not dealed in 30℃ water.Finally,to testify PG activities with DNS’s test. In all, PG of Rhizoctonia had high activities and virulence. The conrtrol efficacy of T.viride to PG activities of WK-47, WK-141, WK-160 and YW-2 were 95%,94%,95%,92% separately, fermented time had a great influence to control efficacy, the third fermented broth did the best. Through effect to PG activities T. viride can reduce the virulence of Rhizoctonia, and protect the hosts. The specific mechanism, qualitative and quantitative research of antagonistic substance in the fermented broth will be further carried out.
