The Identification And Construction Of Genomic DNA Library Of Ceratocystis Fimbriata

Black Rot is one of most important diseases which has caused heavy losses the sweet potato production in the world. Use of resistant culticars has been considered as an effective and important measure for preventing production from the damage caused by the disease.The separation and identification of C. fimbriata was completed and the complete genomic library was successfully constructed using lambda bacteriophage vector for screening avirulence gene and construction of expression vectors.Identification of resistance to sweet potato black rot in germplasm was conducted by using inecting-inoculation method. Among 76 accessions, 14 varieties are high resistant, 23 varieties are resistant and 39 varieties are susceptible .Genomic DNA library is a collection of recombinant DNA clones which contain the complete gene's random fragments. It is called genomic library because it have complete DNA fragments of the genome, liking a data bank of all genomic sequence.The genomic DNA was extracted from a special partial cleavaged with a four-base cutter restriction endonuclease (BamH I) to get suitable size DNA fragments. We isolated the fragments between 9～23kb using QIAGENE kit and got recombinant phages of various DNA fragments by ligating the DNA fragments with the vector DNA(lambda DASHII), packaging ligation product in vitro and transformating competent E.coli cells. Titering paekaged phage on LB plates, we calculated the packaging efficiency was 4.12×10~5pfu/μg DNA and demonstrated the genomic library's integrity. Then we selected several plaques, extracted the phage DNA and examinated the size of the inserted fragments were between 9~23kb by restriction digestion. The results indicated that DNA fragments were inserted in vectors efficiently and in full compliance with the requirements of the genome library construction.