Construction A Bacterial Artificial Chromosome (BAC) Genomic Library Of Heilong River Carps And It's Applying On The Study On The Genomics

The replicator of bacterial artificial chromosome (BAC) is origin from single copy plasmid F factor,therefore BAC is only few copy numbers in host bacterium and can be stable inherit without loss,recombine and table. The host of BAC is E.coli and the efficiency of transform is rather higher. The BAC can be separated with normal alkali-split method. The objective gene selection can use these methods they are blueness-blaze selection, antibiotic,dot original hybridize etc. BAC also can be directly sequencing on the insert DNA. So,the construction of BAC genomic library offers great contributions to the study of model organism genomics such as the human people, zebrafish,rat and so on. Common carps as the biggest genomics of fish have importance research value on evolution. At present there doesn't develop to study on the common carp genomics inside and outside the country. For these merits of BAC, we construct a BAC library of common carp to make preparations for study on the genomics, functional genomics,proteinome and more research on the evolution,the variation of chromosome and so on.The Heilong River carps are coming from Heilong River and are famous as Yellow River carps and Yangtze River carps. The characteristics of Heilong River carps are broad body,deep back,fire wings, and gold squamas. The Heilong River carps also have strong resistance (cold resistance and disease resistance),genetic diversity,easily grow on everywhere and therefore often use to cross breeding and select breeding. Many fine carps such as aquama carps and songpu carps are the products of the cross breeding with Heilong River carps.On this experiment, we obtained HMW DNA of Heilong River carps by isolate blood cells and embed them in low-melting-point (LMP) agarose in the form of plugs followed by cell lysis and DNA purification in the LMP agarose plug. Generation of large DNA fragments is an essential step for large DNA fragment cloning. Large DNA fragments can be generated by either physical shearing, or partial digestion of HMW DNA with a restriction enzyme that cuts relatively frequently within a genome. Firstly we determined the optimal partial digestion conditions for BAC cloning by varying the concentration of the restriction enzyme, the time of digestion with the same amount of the restriction enzyme. The partial digestion of HMW DNA is to generate equality size DNA fragments followed twice size selection. It will impact the quality of the BAC library if the fragments are over big or small. The fragments are too small to satisfy the library capability and the fragments are too big to link to vector and transform valid. Finally we performed size-selection for BamHⅠrestricted DNA Fragments from 100- 300 kb with PFGE and eluted DNA with electroelution from the agarose gel slice and then purified them with dialysis and condensed with PEG8000.The purification of restricted DNA fragment has a certainly impact on the effect of ligation. The large DNA fragments can not be ligated to the BAC vector and transformed valid. Otherwise,the valid ligation is mostly lie on the molecular weight ratio of vector: DNA. In this experiment, the BAC vector is pEZ BAC which size is 7.2 kb. The ligation condition is the molecular weight ratio of vector:DNA is 10:1,the temperature is 16℃and the time is overnight (about 16 hours). The production of ligation is also being purified and condensed as former.For BAC DNA, electrotransformation is a prevailing method. The electro- transformation efficiency is against to the size of insert DNA fragment. Furthermore, the transformation condition such as the voltage, time can impact the transformation efficiency, the insert DNA size and the content of the fake positive clone. We modulated the condition such as the preparation of the E. coli Strain DH10B electrocompetent cell,the ratio of cell: ligated DNA. Finally we transform of the Ligated DNA into E. coli DH10B by Electroporatio, the condition is 1.82kv,5.7ms. And we found that if the time of electrotransformation is under 3ms, the efficiency of transformation is deeply deduced. Subsequently we selected the positive clones with hands and performed restricted digest identification.By upwards a set of process, we constructed the BAC library of Heilong River carps for the first time. The carp BAC library consists of 46,656 BAC clones in total, and the insert DNA size is about 50~300kb, the average of size is about 100kb. The genome coverage of the BAC library is 2.45 and the possibility to find a single-copy gene in the library is about 90 percent. All these BAC clones are preserved in 378 pieces of 96-well plates and 27 pieces of 384-well plates.We used 8 gene primers to analysis the BAC library of Heilong River carps by 2 step-3D PCR. We constructed 27 superpools and directly to amplify the BAC fluid stored which it tests is not worked. So we changed method to amplify the bacterial clones which come from the BAC fluid by lineation. Here we constructed a rapid method to select the carp BAC library effectively and stable.Now in our country there has first constructed the genetic linkage map of common carp (Xiaowen Sun et.al. 2000). The genetic linkage map consists of 56 RAPD markers of common carp,26 SSLP markers of common carp,19 SSLP markers of crucian carp,70 SSLP markers of zebrafish and 91 gene markers of common carps, the total number of markers is 262 and protract 50 gene linkage group map. According to these linkage maps they ensure the genome size of the common carp is about 5789cM. The genomics of common carp maybe not the biggest genomics on the means of physics,but on the means of genetics it is the biggest genomics in those genomics which have constructed genetics linkage maps. That is, it needs more markers to construct a genetic linkage map with the same density for common carp which increases the difficulties to research include to orientation the quantity characters and to clone single genes. If we want to study the common carp's genomics further,we still utilize other model organism genomics such as human, rat and zebrafish on the economic character related gene,disease-occur related gene and so on. The genetic linkage of common carp need to increase the markers density to 1000~1500 at least. That is,each linkage group should consist of 20~30 markers on the average and the average genetic distance between markers should be 5~10cM.In order to add the density of the genetic linkage map of common carp presently and to obtain useful characteristic related genes and to perform the study on gene orientation,this experiment is to select the carp BAC library for characteristic related gene and construct big BAC contigs to catch the characteristic related functional genes and use them to add the density of genetic linkage map. We also want to orient functional genes on chromosomes to identify the table rate of the carp BAC library with BAC-FISH and chromosome walking methods and to make preparation for further research the differentiation of chromosomes and genomics. In our lab,a partial work is to add the density of the carp genetic linkage map. Now our lab has constructed the carps system for mapping and used several thousands microsatellite marker cloned by ourselves to select the characteristic related markers integrated with measure the characters. Up to date,we have obtained several molecular markers on body length and body weight. During waiting to select the markers, we worked on scrabble and perfect some technology such as FISH and chromosome microdissection. Chromosome microdissection is a bridge technology which conjoint cytogenetics and molecular genetics. To analysis the complete sequence of human genomic is need to construct high distinguish genetic map and physical map. But how to rapid and valid obtain the STS and EST of chromosome and construct clone linkage group and orderly and grading sequence the special section of chromosomes,the microdissection shows unexampled advantage.Chromosome microdissection technology has taken great achievements on chromosome disease research and diagnose,library construction,gene orientation and cloning, evolutionary genetics and tumor genetics and so on by combined with many molecular biological technologies such as PCR , microcloning , FISH , DNA sequencing,library selection,comparable genomic hybridize since it comes out at 1980s'and has well application foreground therewith its special directness and practicability on the field of molecular biology.In the experiment we scrabble and perfect the process of FISH. Because FISH need chromosome prepared from cell cultured,so we first prepared several hundred chromosome slices with perfect divide appearance by culture the blood cells of common carp. In the meanwhile, we dissected the telomere of carp chromosome with microdissection and amplified the fragment twice by PCR. Finally we gained a clear band of telomere. In the experiment,we build blood cell culture and prepare chromosomes method,and FISH and chromosome microdissection of carps to take an importance preparation to research carps gene orientation and chromosome walking and BAC-FISH.