Isolation And Genetic Analysis Of Mutants Resistant To Cadmium In Chlamydomonas Reinhardtii

Facing the worsening heavy metal pollution,researchers are gradually focusing on bioremediation methods.Compared with traditional physicochemical methods,the utilization of Chlamydomonas reinhardtii for heavy metal pollution has many advantages,such as avoiding secondary pollution and low cost.Firstly by electroporation method,we obtained a number of Chlamydomonas transformants.After screening and identification with 0.6mM and 0.4mM cadmium chloride,we discovered cadmium resistant mutants and cadmium sensitive mutants.The flanking sequence of inserting genes can be obtained by RESDA-PCR technology.By sequence alignment,we identify the mutation gene of chlamydomonas.Lastly a series of studies have been carried out through mutants and mutant genes.1.In the process of experiment,we obtained two Chlamydomonas reinhardtii cadmium resistant mutants(TC4 and CB14)and two Chlamydomonas reinhardtii cadmium sensitive mutants(CB27 and CB30).By sequence alignment,we found that the mutant gene of TC4 was SF56-ABT gene(SF56-ATP-BINDING TRANSPORT PROTEIN-RELATED).2.AccoRding to the sequence of SF56-ABT gene,we knew the mutant protein has a typical AAA domain.The complementary algae strain(TC4-6)was screened out by introducing mutant genes.The result of Western blot showed that the expression of SF56-ABT protein increased gradually under 0.1mM cadmium chloride stress.The data of determination of cadmium contend indicated that TC4 has greater adsorption capacity of cadmium than TC4-6 and 21 gr.3.Through sequence alignment,we found the mutant gene of CB14 is SRrG(small Rab-related GTPase),and the mutant gene of CB27 is FAP(Flagellar Associated Protein),and the mutant gene of CB30 is the MhABC gene.Insertion sites of three mutant genes were identified and respective protein domainswere analyzed by the relevant website.We observed the growth state of Chlamydomonas reinhardtii under the stress of cadmium at different concentrations.Then we obtained cadmium resistant mutants and cadmium sensitive mutants by screening and identification.Those indicate this subject successfully established mutant screening system related cadmium in Chlamydomonas reinhardtii.Based on result of the study about TC4,we come to a conclusion that the SF56-ABT protein may have great effect on cadmium metabolism of Chlamydomonas reinhardtii.This project discovered the other three new genes related to cadmium regulation and spcificailly analyzed these genes.Overall,four Chlamydomonas mutants related cadmium metabolism was obtained and relevant research on genes was studied in this thesis.