In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by ...In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGsl and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5℃ for the wild-type enzyme to Tm=65.5℃ for the mutant EGsl and 67.5℃ for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher （28%） for the former or much lower （21.6%） for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.展开更多
In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was ...In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was constructed and the vector was introduced into tobacco with the agrobacterium-mediated method. PCR results showed that the RrGlu gene was integrated into the tobacco genome.展开更多
In order to reveal which role the callose played in R. rugosa pollination incompatibility, the full-length cDNA sequence of β-1,3-glucanase gene was cloned for the first time from the stylus of Rosa rugosa “Tanghong...In order to reveal which role the callose played in R. rugosa pollination incompatibility, the full-length cDNA sequence of β-1,3-glucanase gene was cloned for the first time from the stylus of Rosa rugosa “Tanghong” with RT-PCR and RACE methods and named as RrGlu. The full-length cDNA is 1380 bp with an open reading frame of 1041 bp, encoding 346 amino acids. The derived protein has a molecular weight of 37.85 kD, a calculated pI of 9.12, a pfam00332 conserved domain at position 36 - 345, and belongs to glycosyl hydrolase family 17. The derived protein is a hydrophilic protein secreted into the vacuole. There is a signal peptide cleavage site at position 34 - 35, a transmembrane domain at position 13 - 32, six Ser phosphorylation sites, three Thr phosphorylation sites, three Tyr phosphorylation sites, one N-glycosylation site, and five O-glycosylation sites. There are 31.50% α-helixes, 30.92% random coil, 25.14% extended peptide chain, and 12.43% β-corner structure. This protein and the Glu protein from eight other species, including Prunus persica, share a sequence homology of greater than 72%;all of the proteins contain a pfam00332 conserved domain and a β-1,3-glucanase active center sequence (LIVM)-X-(LIVMFYW)3-(STAG)-E-(ST)-G-W-P-(ST)-X-G. Furthermore, their phylogenetic relationships are consistent with their traditional classifications. These results were meaningful to reveal the molecular mechanism of R. rugosa pollination incompatibility and improve the theory and techniques of breeding ornamental R. rugosa.展开更多
利用SMART（switching mechanism at 5’end of the RNA transcript）技术构建了红肉猕猴桃品种‘红阳’（Actinidia chinesis cv‘Hongyang’）内果皮组织的全长cDNA文库，此文库的构建有助于克隆与次生代谢相关的基因，特别是红肉猕...利用SMART（switching mechanism at 5’end of the RNA transcript）技术构建了红肉猕猴桃品种‘红阳’（Actinidia chinesis cv‘Hongyang’）内果皮组织的全长cDNA文库，此文库的构建有助于克隆与次生代谢相关的基因，特别是红肉猕猴桃花青素特异合成代谢的基因。文库滴度为6．7×10^4cfu／mL，库容为2．72×10^8cfu／mL，文库重组率99．8％，插入片段多数分布在700～1000bp。随机挑选1014个克隆进行测序，测序成功963个，经过序列拼接去除低质量序列后获得632个unigenes，包括92个contigs和540个singletons，获得已知功能unigenes共441个。从所测克隆中得到一个花青素途径的结构基因AcF3H，其cDNA序列长1369bp（GenBank登录号：FJ542819），CDS区为1101bp，编码366个氨基酸的多肽。与拟南芥、葡萄及龙胆已知乃日氨基酸序列比对，发现该基因十分保守。通过RT—PCR技术对苍溪栽培的不同发育时期‘红阳’猕猴桃果实AcF3H基因的表达进行了分析。结果表明，果肉转色前AcF3H基因表达量较高，而转色初期表达量降低，此后随着果实着色加深表达量维持在较高水平。展开更多
基金Project supported by the National Natural Science Foundation of China (No. 20276064) and Natural Science Foundation of ZhejiangProvince (No. Z304076), China
文摘In order to improve the thermostability of β- 1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGsl and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5℃ for the wild-type enzyme to Tm=65.5℃ for the mutant EGsl and 67.5℃ for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher （28%） for the former or much lower （21.6%） for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.
文摘In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was constructed and the vector was introduced into tobacco with the agrobacterium-mediated method. PCR results showed that the RrGlu gene was integrated into the tobacco genome.
文摘In order to reveal which role the callose played in R. rugosa pollination incompatibility, the full-length cDNA sequence of β-1,3-glucanase gene was cloned for the first time from the stylus of Rosa rugosa “Tanghong” with RT-PCR and RACE methods and named as RrGlu. The full-length cDNA is 1380 bp with an open reading frame of 1041 bp, encoding 346 amino acids. The derived protein has a molecular weight of 37.85 kD, a calculated pI of 9.12, a pfam00332 conserved domain at position 36 - 345, and belongs to glycosyl hydrolase family 17. The derived protein is a hydrophilic protein secreted into the vacuole. There is a signal peptide cleavage site at position 34 - 35, a transmembrane domain at position 13 - 32, six Ser phosphorylation sites, three Thr phosphorylation sites, three Tyr phosphorylation sites, one N-glycosylation site, and five O-glycosylation sites. There are 31.50% α-helixes, 30.92% random coil, 25.14% extended peptide chain, and 12.43% β-corner structure. This protein and the Glu protein from eight other species, including Prunus persica, share a sequence homology of greater than 72%;all of the proteins contain a pfam00332 conserved domain and a β-1,3-glucanase active center sequence (LIVM)-X-(LIVMFYW)3-(STAG)-E-(ST)-G-W-P-(ST)-X-G. Furthermore, their phylogenetic relationships are consistent with their traditional classifications. These results were meaningful to reveal the molecular mechanism of R. rugosa pollination incompatibility and improve the theory and techniques of breeding ornamental R. rugosa.
文摘利用SMART（switching mechanism at 5’end of the RNA transcript）技术构建了红肉猕猴桃品种‘红阳’（Actinidia chinesis cv‘Hongyang’）内果皮组织的全长cDNA文库，此文库的构建有助于克隆与次生代谢相关的基因，特别是红肉猕猴桃花青素特异合成代谢的基因。文库滴度为6．7×10^4cfu／mL，库容为2．72×10^8cfu／mL，文库重组率99．8％，插入片段多数分布在700～1000bp。随机挑选1014个克隆进行测序，测序成功963个，经过序列拼接去除低质量序列后获得632个unigenes，包括92个contigs和540个singletons，获得已知功能unigenes共441个。从所测克隆中得到一个花青素途径的结构基因AcF3H，其cDNA序列长1369bp（GenBank登录号：FJ542819），CDS区为1101bp，编码366个氨基酸的多肽。与拟南芥、葡萄及龙胆已知乃日氨基酸序列比对，发现该基因十分保守。通过RT—PCR技术对苍溪栽培的不同发育时期‘红阳’猕猴桃果实AcF3H基因的表达进行了分析。结果表明，果肉转色前AcF3H基因表达量较高，而转色初期表达量降低，此后随着果实着色加深表达量维持在较高水平。
