[ Objective ] The aim of this study is to clone and analyze the actin gene from Rehmannia glutinosa. [ Method ] Degenerate primers were designed according to the conserved regions of actin sequences of Rehmannia gluti...[ Objective ] The aim of this study is to clone and analyze the actin gene from Rehmannia glutinosa. [ Method ] Degenerate primers were designed according to the conserved regions of actin sequences of Rehmannia glutinosa and its similar species, RT-PCR was next conducted to amplify the actin gene from Rehmannia glutinosa. [ Result] The amplified fragment is 724 bp and correspondingly 240 amino acids. The BLAST results indicate that the homology between the amplified fragment and other higher plants for aetin gene sequences and amino acid are more than 80% and 90%, respectively, suggesting that the amplified fragment is the actin gene of Rehmannia glutinosa. [ Conclusion] Phylogenetic analysis shows that the actin gene of Rehmannia glutinosa has an intimate genetic relationship with actin7 gene of Nicotiana tabacum.展开更多
Reverse_transcription Polymerase Chain Reaction (RT_PCR) was performed using cDNAs as templates from wheat_ Haynaldia villosa 6VS/6AL translocation line and 'Yangmai 5' induced with fungus Erysiphe gramin...Reverse_transcription Polymerase Chain Reaction (RT_PCR) was performed using cDNAs as templates from wheat_ Haynaldia villosa 6VS/6AL translocation line and 'Yangmai 5' induced with fungus Erysiphe graminis , and degenerate primers designed based on the conserved amino acid sequences of known plant disease_resistance genes. The cDNA sequences encoding cyclophilin_like and H +_ATPase_like genes were first isolated and characterized in wheat. The putative amino acid sequences of the two clones showed that they were highly homologous to those of cyclophilin proteins and H +_ATPases isolated from other plants. Thus they were designated as Ta_Cyp and Ta_MAH . The obvious expression differences could be observed between wheat_ H. villosa 6VS/6AL translocation line and susceptible wheat cultivar 'Yangmai 5', implying that the two genes may be related with the resistance of wheat_ H. villosa 6VS/6AL translocation line to disease. Southern blot indicated that the wheat genome contained 2-3 copies of Ta_Cyp gene and one copy of the Ta_MAH gene. Chinese Spring nulli_tetrasomic line analysis located the Ta_Cyp homologous genes on wheat chromosome 6A, 6B and 6D. Southern blot using Ta_Cyp clone as a probe showed that the polymorphic bands existed among the H. villosa , amphiploid of Triticum durum _ H. villosa , wheat_ H. villosa 6VS/6AL translocation line and 'Yangmai 5', suggesting that Ta_Cyp homologies exist in wheat genome as well as on the short arm of chromosome 6V in H. villosa .展开更多
The degenerate primers were designed based on the conserved NBS-LRR motifs among the known disease-resistance genes. A fragment of about 500 bp was amplified from genomic DNA of sweet potato using the specifically des...The degenerate primers were designed based on the conserved NBS-LRR motifs among the known disease-resistance genes. A fragment of about 500 bp was amplified from genomic DNA of sweet potato using the specifically designed degenerate primers. After cloning and sequencing, 20 NBS-LRR type of disease-resistance gene analogue (RGAs) in sweet potato were observed. The deduced amino acid sequence of DNA fragment contains the conserved motifs of NBS-LRR type RGAs, such as P-loop, Kinase-2α, Kinase-3α and GLPL domain. The 20 RGAs could be sorted into two subclasses, namely TIR- NBS-LRR type and non-TIR-NBS-LRR type. Compared with the known resistance genes including N, L6 and M, the percentages of homologous amino acid sequence in 10 TIR-NBS-LRR range between 21% -44%. While other 10 non-TIR-NBS-LRR assume 15% -46% homology with the known resistance genes (Prf, RPM1, RPS2, etc. ). Consequently the RGAs may further be used as molecular marker for screening the candidate disease-resistance genes in sweet potato.展开更多
[Objective] The aim was to clone the conserved sequences of cry gene and express them in Rosetta (DE3). [Method] Specific primers were designed according to NCBI database information and the conserved sequences of c...[Objective] The aim was to clone the conserved sequences of cry gene and express them in Rosetta (DE3). [Method] Specific primers were designed according to NCBI database information and the conserved sequences of cry gene were amplified by PCR from Bt transgenic cotton. Then recombinant plasmids were constructed and expressed in E. coil strain Rosetta (DE3). Finally, the effects of different concentrations and inducing time of IPTG on the expression level of protein were investigated. [Result] Two conserved sequences (304 and 853 bp respectively) of cry gene were amplified. The result of SDS-PAGE confirmed that the recombinant plasmids pGEX-4t-I-304 and pGEX-4t-1-853 could express fusion proteins by IPTG induction and the molecular weight of protein products was 39 and 62.4 kDa respectively, which was in accordance with predicted result. The optimal protein ex- pression conditions were confirmed as induction with 0.15 mmol/L IPTG for 7 h. [Conclusion] This study prepared the ground for the further detection of Bt transgenic crops.展开更多
基金National Natural Science Foundation of China (No 30472155)Beijing Natural Science Foundation (No 5062035)~~
文摘[ Objective ] The aim of this study is to clone and analyze the actin gene from Rehmannia glutinosa. [ Method ] Degenerate primers were designed according to the conserved regions of actin sequences of Rehmannia glutinosa and its similar species, RT-PCR was next conducted to amplify the actin gene from Rehmannia glutinosa. [ Result] The amplified fragment is 724 bp and correspondingly 240 amino acids. The BLAST results indicate that the homology between the amplified fragment and other higher plants for aetin gene sequences and amino acid are more than 80% and 90%, respectively, suggesting that the amplified fragment is the actin gene of Rehmannia glutinosa. [ Conclusion] Phylogenetic analysis shows that the actin gene of Rehmannia glutinosa has an intimate genetic relationship with actin7 gene of Nicotiana tabacum.
文摘Reverse_transcription Polymerase Chain Reaction (RT_PCR) was performed using cDNAs as templates from wheat_ Haynaldia villosa 6VS/6AL translocation line and 'Yangmai 5' induced with fungus Erysiphe graminis , and degenerate primers designed based on the conserved amino acid sequences of known plant disease_resistance genes. The cDNA sequences encoding cyclophilin_like and H +_ATPase_like genes were first isolated and characterized in wheat. The putative amino acid sequences of the two clones showed that they were highly homologous to those of cyclophilin proteins and H +_ATPases isolated from other plants. Thus they were designated as Ta_Cyp and Ta_MAH . The obvious expression differences could be observed between wheat_ H. villosa 6VS/6AL translocation line and susceptible wheat cultivar 'Yangmai 5', implying that the two genes may be related with the resistance of wheat_ H. villosa 6VS/6AL translocation line to disease. Southern blot indicated that the wheat genome contained 2-3 copies of Ta_Cyp gene and one copy of the Ta_MAH gene. Chinese Spring nulli_tetrasomic line analysis located the Ta_Cyp homologous genes on wheat chromosome 6A, 6B and 6D. Southern blot using Ta_Cyp clone as a probe showed that the polymorphic bands existed among the H. villosa , amphiploid of Triticum durum _ H. villosa , wheat_ H. villosa 6VS/6AL translocation line and 'Yangmai 5', suggesting that Ta_Cyp homologies exist in wheat genome as well as on the short arm of chromosome 6V in H. villosa .
文摘The degenerate primers were designed based on the conserved NBS-LRR motifs among the known disease-resistance genes. A fragment of about 500 bp was amplified from genomic DNA of sweet potato using the specifically designed degenerate primers. After cloning and sequencing, 20 NBS-LRR type of disease-resistance gene analogue (RGAs) in sweet potato were observed. The deduced amino acid sequence of DNA fragment contains the conserved motifs of NBS-LRR type RGAs, such as P-loop, Kinase-2α, Kinase-3α and GLPL domain. The 20 RGAs could be sorted into two subclasses, namely TIR- NBS-LRR type and non-TIR-NBS-LRR type. Compared with the known resistance genes including N, L6 and M, the percentages of homologous amino acid sequence in 10 TIR-NBS-LRR range between 21% -44%. While other 10 non-TIR-NBS-LRR assume 15% -46% homology with the known resistance genes (Prf, RPM1, RPS2, etc. ). Consequently the RGAs may further be used as molecular marker for screening the candidate disease-resistance genes in sweet potato.
基金Supported by Scientific Research Fund for Doctoral Program of Wuhan Polytechnic University (2006696)~~
文摘[Objective] The aim was to clone the conserved sequences of cry gene and express them in Rosetta (DE3). [Method] Specific primers were designed according to NCBI database information and the conserved sequences of cry gene were amplified by PCR from Bt transgenic cotton. Then recombinant plasmids were constructed and expressed in E. coil strain Rosetta (DE3). Finally, the effects of different concentrations and inducing time of IPTG on the expression level of protein were investigated. [Result] Two conserved sequences (304 and 853 bp respectively) of cry gene were amplified. The result of SDS-PAGE confirmed that the recombinant plasmids pGEX-4t-I-304 and pGEX-4t-1-853 could express fusion proteins by IPTG induction and the molecular weight of protein products was 39 and 62.4 kDa respectively, which was in accordance with predicted result. The optimal protein ex- pression conditions were confirmed as induction with 0.15 mmol/L IPTG for 7 h. [Conclusion] This study prepared the ground for the further detection of Bt transgenic crops.