Construction Of Actinomycetes And Petunia BAC Library And Related Gene Screening

Bacterial artificial chromosome libraries are important resources for the study of biogenomics.They have been successfully applied to map-based cloning,physical mapping and whole genome sequencing.The research work consists of two parts: the first part is the modification of the BAC vector for Actinomycetes BAC library construction and transformation,and BAC library construction of the Actinomycete 0026 strain with the modified vector;the second part is the construction and screening of Petunia hybrida BAC library.The secondary metabolites produced by Actinomycetes are of great significance in both medical and academic research.The commonly used vector for the construction of Actinomycetes BAC(bacterial artificial chromosome)library is pMSBBACs.However,this vector has a disadvantage in insert analysis of the Actinomycetes BAC libraries.In this study,we introduced the sites for endogenous enzyme I-SceI into the Actinomycetes BAC vector pMSBBACs.The modified vector,called pHZAUBACFXJ1,is more suitable for large genomic DNA cloning and heterologous expression of Actinomycetes.It is not only simple and practical in construction of Actinomycetes BAC libraries,but also favorable in insert estimation and quality evaluation of BAC libraries by I-SceI analysis.A BAC library of Actinomycete 0026 strain was successfully constructed using the pHZAUBACFXJ1 vector.The library consists of 3,456 clones and stored on 9X 384 plates.Forty randomly picked clones were analyzed by I-SceI digestion.The average insert size was estimated to be about 130 kb and the empty vector rate was about 10%.According to the genome size of 8 Mbp for the Actinomycete 0026 strain,the library covers about 40 times of the Actinomycete 0026 genome.Pools were made for rows and columns,and pool DNAs were extracted.The library was successfully used to isolate clones by collaborators.Petunia hybrida is widely used in street flowerbed and garden landscape decoration because of its long flowering period,prosperous blooming,and large flowers.It also has important significance in the study of plant flower development.In order to study the gene regulatory sequences of Petunia hybrida,we constructed a Petunia hybrida flower BAC library using the pHZAUBAC1 cloning vector.The library contains 78,336 clonesand stored in 204 X 384 plates.A total of 440 randomly picked petunia BAC cloneswere analyzed.The average insert size was approximately 120 kb and the empty vector rate was less than 2%.According to the genome size of 900 Mbp for petunia,the library covers approximately 10 times of the petunia genome.A high-efficiency secondary pool PCR screening method was used to screen the Petunia hybrida BAC library with 6 pairs of primers(SCF1-9FR,SCF2-2FR,SCF43 FR,SCF4-1FR,SCF1-12 FR,SCF101FR).A total of 9 positive BAC clones were selected and used to study the regulation mechanism of Petunia hybrida.