[ Objective] The aim of this study was to provide a method for solving the problems in preparing BAC vector with High-copy plasmid pUC119- Bluelox BAC. [ Method ] With selecting a proper single restriction site, seque...[ Objective] The aim of this study was to provide a method for solving the problems in preparing BAC vector with High-copy plasmid pUC119- Bluelox BAC. [ Method ] With selecting a proper single restriction site, sequences of a single copy BAC vector plasmid were inserted into proper site of High-copy plasmid pUC119 vector. [ Result] The gene sequence of BAC vector lost control function of single copy number in new plasmid pUC119-BAC and was copied through High-copy form. The gene sequence of BAC vector basic function was completely cutted off through single enzyme digestion and the control function of single copy could be recovered by auto-connection. [ Conclusion] The High-copy pUC119-BAC plasmid was used to copy and amplify high copy of basic function gene sequence in BAC vector, besides that it could be used to construct transfer vector of molecular cloned recombinant virus or BAC library.展开更多
We have constructed a full BAC library for the superior eaxly indica variety of OryZa sativa, Guang Lu Ai4. The MAX Efficiency DHlOB with increased stabilltyof inserts was used as BAC host cells. The potent pBelo BACI...We have constructed a full BAC library for the superior eaxly indica variety of OryZa sativa, Guang Lu Ai4. The MAX Efficiency DHlOB with increased stabilltyof inserts was used as BAC host cells. The potent pBelo BACII with double selection markers was used as cloning vector. The cloning efficiency we have reached was as high as 98%, and the transformation efficiency was raised up to 1Oo transformants / pg of large fragment DNA. The BAC recombinant transformants were picked at random and analyzed for the size of inserts, which turned out to be of 120 kb in length on average. We have obtained more than 20,000 such BAC clones. According to conventional probabillty equation, they covered the entire rice genome of 420,000 kb in length. The entire length of inserts of the library obtained has the 5- to 6- fold coverage of the genome. To our knowledge, this is the first reported full BAC library for a complex genome.展开更多
Pepper （Capsicum annuum. L.） is a widely cultivated vegetable crop worldwide and has the second largest planting area and the first largest vegetable output and value in China. Pepper root-knot nematode （Meloidogyn...Pepper （Capsicum annuum. L.） is a widely cultivated vegetable crop worldwide and has the second largest planting area and the first largest vegetable output and value in China. Pepper root-knot nematode （Meloidogyne spp.） is one of the most serious pests of pepper, which caused huge losses every year. Previous studies showed that the Me3 gene is resistant to a wide range of Meloidogyne species, including M. arenaria, M. javanica, and M. incognita. HDA149, a double haploid pepper genotype, harboring the root-knot nematode resistance gene Me3, was used to construct bacterial artificial chro- mosome library （BAC） via the vector of CopyControFM pCC1 in this study. The library consists of 210 200 BAC clones and is equivalent to 5.3 pepper genomes. The average insert size is 95 kb, and most of them are 90-120 kb; but the empty clones are less than 3%. In order to screen the BAC library easily, 550 super pools with 384 BAC clones of each pool were further developed in this study. Specific primers from Me3 gene locus were used for BAC library screening, and more than 20 positive BAC clones were obtained. Then the selected positive BAC clones were analyzed by restriction enzyme digestion, BAC-end sequencing, marker development, and new positive BAC clones exploration, respectively. Finally, the contig with total length of about 300 kb linked to the Me3 locus was constructed based on chromosome walking strategy, which made a solid foundation for the cloning of the important root-knot nematode resistance gene Me3.展开更多
Based on the sequence of a resistance gene analog FZ14 derived from Zizania latifolia （Griseb.）, a pair of specific PCR primers FZ14P1/FZ14P2was designed to isolate candidate disease resistance gene. The pooled-PCR ...Based on the sequence of a resistance gene analog FZ14 derived from Zizania latifolia （Griseb.）, a pair of specific PCR primers FZ14P1/FZ14P2was designed to isolate candidate disease resistance gene. The pooled-PCR approach was adopted using the primer pair to screen a genomic transformation-competent artificial chromosome （TAC） library derived from Z. latifolia. A positive TAC clone （ZR1） was obtained and confirmed by sequence analysis. The results indicated that ZR1 consisted of conserved motifs similar to P-loop （kinase la）, kinase 2, kinase 3a and GLPL （Gly-Leu-Pro-Leu）, suggesting that it could be a portion of NBS-LRR type of resistance gene. Using Agrobacterium-mediated transformation of Nipponbare mature embryo, a total of 48 independent transgenic To plants were obtained. Among them, 36 plants were highly resistant to the virulent bacterial blight strain PXO71. The results indicate that ZR1 contains at least one functional bacterial blight resistance gene.展开更多
文摘[ Objective] The aim of this study was to provide a method for solving the problems in preparing BAC vector with High-copy plasmid pUC119- Bluelox BAC. [ Method ] With selecting a proper single restriction site, sequences of a single copy BAC vector plasmid were inserted into proper site of High-copy plasmid pUC119 vector. [ Result] The gene sequence of BAC vector lost control function of single copy number in new plasmid pUC119-BAC and was copied through High-copy form. The gene sequence of BAC vector basic function was completely cutted off through single enzyme digestion and the control function of single copy could be recovered by auto-connection. [ Conclusion] The High-copy pUC119-BAC plasmid was used to copy and amplify high copy of basic function gene sequence in BAC vector, besides that it could be used to construct transfer vector of molecular cloned recombinant virus or BAC library.
文摘We have constructed a full BAC library for the superior eaxly indica variety of OryZa sativa, Guang Lu Ai4. The MAX Efficiency DHlOB with increased stabilltyof inserts was used as BAC host cells. The potent pBelo BACII with double selection markers was used as cloning vector. The cloning efficiency we have reached was as high as 98%, and the transformation efficiency was raised up to 1Oo transformants / pg of large fragment DNA. The BAC recombinant transformants were picked at random and analyzed for the size of inserts, which turned out to be of 120 kb in length on average. We have obtained more than 20,000 such BAC clones. According to conventional probabillty equation, they covered the entire rice genome of 420,000 kb in length. The entire length of inserts of the library obtained has the 5- to 6- fold coverage of the genome. To our knowledge, this is the first reported full BAC library for a complex genome.
基金supported by the National High-Tech R&D Program in China (2013AA102603)the Natural Science Foundation of Shandong Province,China (ZR2014YL014)+3 种基金the Youth Scientific Research Foundation of Shandong Academy of Agricultural Sciences,China (2014QNZ03)the Taishan Scholars Program of Shandong Province,China (2016-2020)the National Natural Science Foundation of China (31101425)Prof. Alain Palloxin,French National Institute for Agricultural Research (INRA),for kindly providing the pepper genotype HDA149
文摘Pepper （Capsicum annuum. L.） is a widely cultivated vegetable crop worldwide and has the second largest planting area and the first largest vegetable output and value in China. Pepper root-knot nematode （Meloidogyne spp.） is one of the most serious pests of pepper, which caused huge losses every year. Previous studies showed that the Me3 gene is resistant to a wide range of Meloidogyne species, including M. arenaria, M. javanica, and M. incognita. HDA149, a double haploid pepper genotype, harboring the root-knot nematode resistance gene Me3, was used to construct bacterial artificial chro- mosome library （BAC） via the vector of CopyControFM pCC1 in this study. The library consists of 210 200 BAC clones and is equivalent to 5.3 pepper genomes. The average insert size is 95 kb, and most of them are 90-120 kb; but the empty clones are less than 3%. In order to screen the BAC library easily, 550 super pools with 384 BAC clones of each pool were further developed in this study. Specific primers from Me3 gene locus were used for BAC library screening, and more than 20 positive BAC clones were obtained. Then the selected positive BAC clones were analyzed by restriction enzyme digestion, BAC-end sequencing, marker development, and new positive BAC clones exploration, respectively. Finally, the contig with total length of about 300 kb linked to the Me3 locus was constructed based on chromosome walking strategy, which made a solid foundation for the cloning of the important root-knot nematode resistance gene Me3.
基金supported by the National Natural Science Foundation of China (Grant No. 30760115)Transgenic Program (Grant No. 2008ZX08001-002)
文摘Based on the sequence of a resistance gene analog FZ14 derived from Zizania latifolia （Griseb.）, a pair of specific PCR primers FZ14P1/FZ14P2was designed to isolate candidate disease resistance gene. The pooled-PCR approach was adopted using the primer pair to screen a genomic transformation-competent artificial chromosome （TAC） library derived from Z. latifolia. A positive TAC clone （ZR1） was obtained and confirmed by sequence analysis. The results indicated that ZR1 consisted of conserved motifs similar to P-loop （kinase la）, kinase 2, kinase 3a and GLPL （Gly-Leu-Pro-Leu）, suggesting that it could be a portion of NBS-LRR type of resistance gene. Using Agrobacterium-mediated transformation of Nipponbare mature embryo, a total of 48 independent transgenic To plants were obtained. Among them, 36 plants were highly resistant to the virulent bacterial blight strain PXO71. The results indicate that ZR1 contains at least one functional bacterial blight resistance gene.
