Ⅰ. INTRODUCTIONRice stripe disease is one of the main diseases to rice. Its pathogeny is rice stripe virus (called RSV for short). RSV can damage 37 species of gramineal crops, such as rice, wheat and corn, and decre...Ⅰ. INTRODUCTIONRice stripe disease is one of the main diseases to rice. Its pathogeny is rice stripe virus (called RSV for short). RSV can damage 37 species of gramineal crops, such as rice, wheat and corn, and decrease the yield seriously. In 1975 Koganezawa first reported that RSV possessed the branched filamentous particle. Then a series of papers was展开更多
Complementary DNAs to turnip mosaic virus (a radish Raphanus stativus isolate) genomicRNA were synthesized using oligo (dT) as primer and cloned into the vector λ-ZAP Ⅱ. Afterhybridization with a single-stranded cDN...Complementary DNAs to turnip mosaic virus (a radish Raphanus stativus isolate) genomicRNA were synthesized using oligo (dT) as primer and cloned into the vector λ-ZAP Ⅱ. Afterhybridization with a single-stranded cDNA probe and the sequencing of inserted DNA,positive clones with poly--A tails were obtained. One clone containing 1429-base pair insertwas sequenced. The coat protein gene was identified based on the molecular weight of theTuMV coat protein and the consensus sequences of the polyprotein processing sites ofpotyviruses. The 5’ end of the coat protein gene was modified by PCR to introduce aninitiation codon, ATG, and two restriction enzyme sites. The gene was then manipulatedinto a binary vector pBIN437 which was derived from pBI121, and the plant expressionvector is being used to transform Brassica napus.展开更多
The Barley yellow dwarf virus (BYDV) GAV isolate was preserved at the Institute of Plant Protection of the Chinese Academy of Agricultural Sciences. The cDNA of BYDV GAV coat protein (CP) gene was amplified from the e...The Barley yellow dwarf virus (BYDV) GAV isolate was preserved at the Institute of Plant Protection of the Chinese Academy of Agricultural Sciences. The cDNA of BYDV GAV coat protein (CP) gene was amplified from the extracted RNA of BYDV GAV by using the polymerase chain reaction (PCR), and cloned into pGEM-7zf(+). Its complete nucleotide sequence has been determined by means of Sanger’s dideoxy-mediated chain-termination method. The result showed that BYDV GAV CP gene has 600nt. It shares 97.5% and 96.5% identity with CP gene of BYDV MAV-PS1 in terms of nucleotide and amino acid sequences respectively.展开更多
The IPTG inducible expression vector containing the BYDV GAV coat protein gene was constructed and transferred into E.coli BL21(DE3).High level expression of the specific protein was achieved by IPTG induction.The res...The IPTG inducible expression vector containing the BYDV GAV coat protein gene was constructed and transferred into E.coli BL21(DE3).High level expression of the specific protein was achieved by IPTG induction.The results of SDS PAGE and Western blottong show that the expression product which accumulates 19.5% of the total cellular proteins estimated by scanning is 24 kD BYDV GAV coat protein plus eleven amino acids of pET 5a.展开更多
Based on a full-length infectious cDNA clone, gene modifications of Tobacco necrosis virus A Chinese isolate (TNV-AC) were made by site-directed mutagenesis or nucleotide deletions for in vitro transcrip-tion of mutan...Based on a full-length infectious cDNA clone, gene modifications of Tobacco necrosis virus A Chinese isolate (TNV-AC) were made by site-directed mutagenesis or nucleotide deletions for in vitro transcrip-tion of mutant viral RNAs. Mechanical inoculations of Chenopodium amaranticolor with in vitro tran-scripts, containing a single nucleic acid substitution at the presumed transcriptional start sites for the two subgenomic (sg) RNAs, showed that the sgRNA1 and sgRNA2 of TNV-AC were initiated at G2184 or G2460, respectively. Mutagenesis of the translational initiation-codons for the open-reading frame (ORF) P8 or P6 encoded by sgRNA1 indicated that each of the two genes was essential for formation of local lesions on C. amaranticolor leaves, perhaps by blocking virus cell-to-cell movement, but were not necessary for viral RNA replication in the protoplast of tobacco cell BY-2. Results of prokaryotic expression showed that the ORF coding for coat protein on TNV-AC sgRNA2 was initiatively translated by the first AUG codon at nucleotides 2612―2614. Site-directed mutation of translational start codons, and deletion of the entire coding region, showed that the intact TNV-AC coat protein was dispensable for establishment of TNV-AC infection in C. amaranticolor, otherwise the numbers of local lesions and the viral RNA accumulation level were reduced, or the time to symptom appearance significantly delayed. These results suggested that the nucleotide sequence around the translational start codon coding for TNV-AC coat protein gene may play an important role in the local symptoms. Aspects of the involve-ment of the coat protein in the TNV-AC life cycle were discussed.展开更多
The plant expression vector of the coat protein(CP) gene of cucumber mosaic virus (CMV) BS strain was used to transform three kinds of pepper (Capsicum annuum) tissues (cotyledon, stem and root) by agrobacterium-m...The plant expression vector of the coat protein(CP) gene of cucumber mosaic virus (CMV) BS strain was used to transform three kinds of pepper (Capsicum annuum) tissues (cotyledon, stem and root) by agrobacterium-mediated co-cultivation. 53%-68.4% of the total tissues (639) can be induced to be calli, but only cotyledon calli can be further regenerated to form shoots (regenerated efficiency 39.7%). 70%(42/60) of the putative transformed plants were confirmed to have CP gene in their genomes by Southern blot. The mRNAs and the CP were respectively found in 80% of transgenic plants by Northern blot and DAS-ELISA. 24 of the transgenic plants expressing CP gene of BS strain showed three kinds of resistant level (severe symptom, delay of symptomatic development, no symptom) to infection of CMV-BS and of CMV-P. However, there was distinctly higher resistance to inoculation of CMV-BS than that with CMV-P in these transgenic plants.展开更多
文摘Ⅰ. INTRODUCTIONRice stripe disease is one of the main diseases to rice. Its pathogeny is rice stripe virus (called RSV for short). RSV can damage 37 species of gramineal crops, such as rice, wheat and corn, and decrease the yield seriously. In 1975 Koganezawa first reported that RSV possessed the branched filamentous particle. Then a series of papers was
文摘Complementary DNAs to turnip mosaic virus (a radish Raphanus stativus isolate) genomicRNA were synthesized using oligo (dT) as primer and cloned into the vector λ-ZAP Ⅱ. Afterhybridization with a single-stranded cDNA probe and the sequencing of inserted DNA,positive clones with poly--A tails were obtained. One clone containing 1429-base pair insertwas sequenced. The coat protein gene was identified based on the molecular weight of theTuMV coat protein and the consensus sequences of the polyprotein processing sites ofpotyviruses. The 5’ end of the coat protein gene was modified by PCR to introduce aninitiation codon, ATG, and two restriction enzyme sites. The gene was then manipulatedinto a binary vector pBIN437 which was derived from pBI121, and the plant expressionvector is being used to transform Brassica napus.
文摘The Barley yellow dwarf virus (BYDV) GAV isolate was preserved at the Institute of Plant Protection of the Chinese Academy of Agricultural Sciences. The cDNA of BYDV GAV coat protein (CP) gene was amplified from the extracted RNA of BYDV GAV by using the polymerase chain reaction (PCR), and cloned into pGEM-7zf(+). Its complete nucleotide sequence has been determined by means of Sanger’s dideoxy-mediated chain-termination method. The result showed that BYDV GAV CP gene has 600nt. It shares 97.5% and 96.5% identity with CP gene of BYDV MAV-PS1 in terms of nucleotide and amino acid sequences respectively.
文摘The IPTG inducible expression vector containing the BYDV GAV coat protein gene was constructed and transferred into E.coli BL21(DE3).High level expression of the specific protein was achieved by IPTG induction.The results of SDS PAGE and Western blottong show that the expression product which accumulates 19.5% of the total cellular proteins estimated by scanning is 24 kD BYDV GAV coat protein plus eleven amino acids of pET 5a.
基金the National Natural Science Foundation of China (Grants Nos. 30470077 and 30325001).
文摘Based on a full-length infectious cDNA clone, gene modifications of Tobacco necrosis virus A Chinese isolate (TNV-AC) were made by site-directed mutagenesis or nucleotide deletions for in vitro transcrip-tion of mutant viral RNAs. Mechanical inoculations of Chenopodium amaranticolor with in vitro tran-scripts, containing a single nucleic acid substitution at the presumed transcriptional start sites for the two subgenomic (sg) RNAs, showed that the sgRNA1 and sgRNA2 of TNV-AC were initiated at G2184 or G2460, respectively. Mutagenesis of the translational initiation-codons for the open-reading frame (ORF) P8 or P6 encoded by sgRNA1 indicated that each of the two genes was essential for formation of local lesions on C. amaranticolor leaves, perhaps by blocking virus cell-to-cell movement, but were not necessary for viral RNA replication in the protoplast of tobacco cell BY-2. Results of prokaryotic expression showed that the ORF coding for coat protein on TNV-AC sgRNA2 was initiatively translated by the first AUG codon at nucleotides 2612―2614. Site-directed mutation of translational start codons, and deletion of the entire coding region, showed that the intact TNV-AC coat protein was dispensable for establishment of TNV-AC infection in C. amaranticolor, otherwise the numbers of local lesions and the viral RNA accumulation level were reduced, or the time to symptom appearance significantly delayed. These results suggested that the nucleotide sequence around the translational start codon coding for TNV-AC coat protein gene may play an important role in the local symptoms. Aspects of the involve-ment of the coat protein in the TNV-AC life cycle were discussed.
文摘The plant expression vector of the coat protein(CP) gene of cucumber mosaic virus (CMV) BS strain was used to transform three kinds of pepper (Capsicum annuum) tissues (cotyledon, stem and root) by agrobacterium-mediated co-cultivation. 53%-68.4% of the total tissues (639) can be induced to be calli, but only cotyledon calli can be further regenerated to form shoots (regenerated efficiency 39.7%). 70%(42/60) of the putative transformed plants were confirmed to have CP gene in their genomes by Southern blot. The mRNAs and the CP were respectively found in 80% of transgenic plants by Northern blot and DAS-ELISA. 24 of the transgenic plants expressing CP gene of BS strain showed three kinds of resistant level (severe symptom, delay of symptomatic development, no symptom) to infection of CMV-BS and of CMV-P. However, there was distinctly higher resistance to inoculation of CMV-BS than that with CMV-P in these transgenic plants.
