Cytochrome b5(Cyt-b5)is a small heme protein and known to be involved in a wide range of biochemical transformations,in eluding cytochrome P450 monooxyge nase(CYP)-mediated metabolism of endoge nous and exogenous comp...Cytochrome b5(Cyt-b5)is a small heme protein and known to be involved in a wide range of biochemical transformations,in eluding cytochrome P450 monooxyge nase(CYP)-mediated metabolism of endoge nous and exogenous compo un ds.Studies on Cyt-b5 are more con centrated in mammals,but are relatively rare in in sects.The characteristics and functi on of Cyt-b5 from Locusta migratoria have not been described yet.We sequeneed the full-length cDNA sequenee of Cyt-b5 from L.migratoria(LmCyt-b5)by reverse transcription-PCR(RT-PCR)based on locust transcriptome database.The phylogenetic analysis showed that LmCyt-b5 was closely related to the Cyt-b5 from Blattodea.LmCyt-b5 was highly expressed in ovary,Malpighian tubules,midgut,gastric caeca,and fat bodies.Silencing of LmCyt-b5 had no effect on the susceptibility of L.migratoria to four different insecticides.Suppression of LmCyt-b5 or silencing of both LmCyt-b5 and LmCPR did not significantly change the total CYP activity toward the substrate 7-ethoxycoumarin(7-EC).However,coexpression of LmCYP6FD1 with LmCPR and LmCyt-b5 together in Sf9 cells by using Bac-to-Bac baculovirus expression system significantly increased the catalytic activity of LmCYP6FD1 toward 7-EC as compared with the coexpression of L.mCYP6FD1 with cytochrome P450 reductase(LmCPR)or LmCyt-b5 separately.These results suggest that LmCyt-b5 plays an important role in the catalytic reaction of LmCYP6FD1 toward 7-EC in our in vitro experiments.Further study is needed to clarify the role of LmCyt-b5 in CYP-mediated catalytic reactions in L.migratoria.展开更多
The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects.In the construction of CRISPR/Cas9-mediated dsLmRNase2^(–/–)mutant locusts,we...The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects.In the construction of CRISPR/Cas9-mediated dsLmRNase2^(–/–)mutant locusts,we found that the tanned eggs which experienced a 20-min contact with the oocyst exhibited a higher success rate compared to fresh newly-laid eggs that were less tanned.However,the heritable efficiency of the dsLmRNase2 deletion to the next generation G_(1 )progeny was similar between adults derived from the tanned or less tanned engineered eggs.Further,the similar effective mutant ratios in the normally developed eggs and G_(0) adults of tanned and less tanned eggs also indicated that tanning did not reduce the absolute mutation efficiency induced by CRISPR/Cas9.Moreover,we found that the syncytial division period,which was longer than the time for tanning,conferred a window period for microinjection treatment with efficient mutation in both tanned and less tanned eggs.We further found that tanned eggs exhibited a higher hatching rate due to a reduced infection rate following microinjection.Both the anti-pressure and ultrastructure analyses indicated that the tanned eggs contained compressed eggshells to withstand increased external pressure.In summary,tanned eggs possess stronger defense responses and higher efficiency of genome editing,providing an improved model for developing Cas9-mediated gene editing procedures in locusts.展开更多
A cDNA encoding a sigma-class glutathione S-transferase of the locust, Locusta migratoria manilensis （LmGSTs1）, was cloned by reverse transcriptase-polymerase chain reaction. The 830 bp-long cDNA encoded a 615 bp op...A cDNA encoding a sigma-class glutathione S-transferase of the locust, Locusta migratoria manilensis （LmGSTs1）, was cloned by reverse transcriptase-polymerase chain reaction. The 830 bp-long cDNA encoded a 615 bp open reading frame （204 amino acid polypeptide）, which exhibited the structural motif and domain organization characteristic of GST sigma-class. It revealed 59, 57, 57, and 56% identities to sigma-class GSTs from Blattella germanica, Gryllotalpa orientalis, Nasonia vitripennis, and Pediculus humanus corporis, respectively. A recombinant protein （LmGSTs1） was functionally expressed in Escherichia coli cells in a soluble form and purified to homogeneity. LmGSTs1 was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a model substrate for GSTs, as well as with p-nitro-benzyl chloride. Its optimal activity was observed at pH 8.0 and at 30℃. Incubation for 30 min at temperatures below 50℃ scarcely affected the activity. The I50 of reactive blue （RB） was 18.5 μmol L-1. In the presence of 0.05 mmol L-1 ethacrynic acid （ECA）, LmGSTs1 showed （81±3）% of the original activities.展开更多
基金This research was supported by the National Natural Science Foundation of China(31320103921 and 31872010)and the Graduate Outstanding Irinovation Project of Shanxi Province,China(2017 BY011).
文摘Cytochrome b5(Cyt-b5)is a small heme protein and known to be involved in a wide range of biochemical transformations,in eluding cytochrome P450 monooxyge nase(CYP)-mediated metabolism of endoge nous and exogenous compo un ds.Studies on Cyt-b5 are more con centrated in mammals,but are relatively rare in in sects.The characteristics and functi on of Cyt-b5 from Locusta migratoria have not been described yet.We sequeneed the full-length cDNA sequenee of Cyt-b5 from L.migratoria(LmCyt-b5)by reverse transcription-PCR(RT-PCR)based on locust transcriptome database.The phylogenetic analysis showed that LmCyt-b5 was closely related to the Cyt-b5 from Blattodea.LmCyt-b5 was highly expressed in ovary,Malpighian tubules,midgut,gastric caeca,and fat bodies.Silencing of LmCyt-b5 had no effect on the susceptibility of L.migratoria to four different insecticides.Suppression of LmCyt-b5 or silencing of both LmCyt-b5 and LmCPR did not significantly change the total CYP activity toward the substrate 7-ethoxycoumarin(7-EC).However,coexpression of LmCYP6FD1 with LmCPR and LmCyt-b5 together in Sf9 cells by using Bac-to-Bac baculovirus expression system significantly increased the catalytic activity of LmCYP6FD1 toward 7-EC as compared with the coexpression of L.mCYP6FD1 with cytochrome P450 reductase(LmCPR)or LmCyt-b5 separately.These results suggest that LmCyt-b5 plays an important role in the catalytic reaction of LmCYP6FD1 toward 7-EC in our in vitro experiments.Further study is needed to clarify the role of LmCyt-b5 in CYP-mediated catalytic reactions in L.migratoria.
基金This work was supported by the National Natural Science Foundation of China(32070502,31730074,32072419 and 31501905).
文摘The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects.In the construction of CRISPR/Cas9-mediated dsLmRNase2^(–/–)mutant locusts,we found that the tanned eggs which experienced a 20-min contact with the oocyst exhibited a higher success rate compared to fresh newly-laid eggs that were less tanned.However,the heritable efficiency of the dsLmRNase2 deletion to the next generation G_(1 )progeny was similar between adults derived from the tanned or less tanned engineered eggs.Further,the similar effective mutant ratios in the normally developed eggs and G_(0) adults of tanned and less tanned eggs also indicated that tanning did not reduce the absolute mutation efficiency induced by CRISPR/Cas9.Moreover,we found that the syncytial division period,which was longer than the time for tanning,conferred a window period for microinjection treatment with efficient mutation in both tanned and less tanned eggs.We further found that tanned eggs exhibited a higher hatching rate due to a reduced infection rate following microinjection.Both the anti-pressure and ultrastructure analyses indicated that the tanned eggs contained compressed eggshells to withstand increased external pressure.In summary,tanned eggs possess stronger defense responses and higher efficiency of genome editing,providing an improved model for developing Cas9-mediated gene editing procedures in locusts.
基金supported by the Funds for International Cooperation and Exchange of the National Natural Science Foundation of China (30810103907)the National Natural Science Foundation of China (30870302)+2 种基金the Public Welfare Fund for Agriculture, Ministry of Agriculture, China (200903021)the China Postdoctoral Science Foundation (Special Program, 201003656 Regular Program, 20090451359)
文摘A cDNA encoding a sigma-class glutathione S-transferase of the locust, Locusta migratoria manilensis （LmGSTs1）, was cloned by reverse transcriptase-polymerase chain reaction. The 830 bp-long cDNA encoded a 615 bp open reading frame （204 amino acid polypeptide）, which exhibited the structural motif and domain organization characteristic of GST sigma-class. It revealed 59, 57, 57, and 56% identities to sigma-class GSTs from Blattella germanica, Gryllotalpa orientalis, Nasonia vitripennis, and Pediculus humanus corporis, respectively. A recombinant protein （LmGSTs1） was functionally expressed in Escherichia coli cells in a soluble form and purified to homogeneity. LmGSTs1 was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a model substrate for GSTs, as well as with p-nitro-benzyl chloride. Its optimal activity was observed at pH 8.0 and at 30℃. Incubation for 30 min at temperatures below 50℃ scarcely affected the activity. The I50 of reactive blue （RB） was 18.5 μmol L-1. In the presence of 0.05 mmol L-1 ethacrynic acid （ECA）, LmGSTs1 showed （81±3）% of the original activities.
