Halophila (thellungiella Halophila) Genomic Library Construction And Important Stress Tolerance-related Gene Screening

The construction of a genomic library is essential to research on genome structure and function.A comprehensive genomic library makes it possible to screen and isolate any DNA fragments from it.With the emergence of various vector systems in recent years,genomic libraries have also developed.A method for the preparation of Thellungiella halophila genomic library was described here.This has paved a way for the cloning of other genes and other molecular biology studies.A genomic library of the Thellungiella halophila was constructed by using the broad-host-range vector Lambda FIX IIλ-phage DNA and DASH IIλ-phage DNA,The Lambda FIX II vector and the Lambda DASH II vector are replacement vectors used for cloning large fragments of genomic DNA.The Lambda FIX II vector system and the Lambda DASH II vector system are take advantage of spi (sensitve to P2 inhibition) selection. Lambda phages containing active red and gam genes are unable to grow on host strains that contain P2 phage lysogens. Lambda phages without these genes are able to grow on strains lysogenic for P2 such as XLl-Blue MRA (P2), a P2 lysogen of XLl-Blue MRA. The red and gam genes in the Lambda FIX II DNA and Lambda DASH II DNA are located on the stuffer fragment; therefore, the wild-type Lambda FIX II phage and Lambda DASH II phage cannot grow on XLl-Blue MRA (P2). When the stuffer fragment is replaced by an insert, the recombinant Lambda FIX II vector and Lambda DASH II vector becomes red-/gam-, and the phage are able to grow on the P2 lysogenic strain. Therefore, by plating the library on the XLl-Blue MRA(P2) strain, only recombinant phages are allowed to grow. Lambda FIX II vector and Lambda DASH II vector DNA containing the multiple restriction enzyme coloning sites were digested with restriction enzyme XhoI, BamHI and XhoI.Total DNA of Thellungiella halophila was extracted by CTAB method,and Dellaporta, Wood, Hicks method, and was partly digested with restriction enzyme Sau3AI, BamH I and Bgl II, and 15 to 23 kb fragments and the whole fragment were isolated and collected from Low-point-melting gel 0.7% agarosel by electrophoresis.The fragments of DNA were ligated with vectors under the role of T4 DNA ligase.The recombinant DNA moleculars were packed by theλ-phage shell protein in vitro to form active phages After infected by the packed phage with diferent dilution,grown on the NZY agar plate containing XLl-Blue MRA formedλ-phage plaques. About 1×106 plaques were obtained. It can covered Thellungiella halophila genome 14 times. So it's a good genomic library. Choosen some single clones randomly, and got the phage DNA, digested with restriction enzyme, by electrophoresis can got the inserted DNA fragments.We used fragments of cloned genes(ThP5CS1,ThP5CS12,ThP5CDH,ThPDH, ThERD5) of the metabolic pathway of Proline, the SOD family members CSD1,CSD3,ZnSOD,MSD,FSD and gene CAT as the probes to filtered the Thellungiella halophila genomic library. The first time , first filtration, we got 18 positive singnals, after the second filtration, we got 4 positive single clones. The second time ,we only used Proline series genes as the probes. First filtration, we got 9 positive singnals, the second filtration are still undergoing. No we are try our best to check out the positive single clones we have got.