Establishment Of Transformation System Of Geotrichum Citri-autrantii In Citrus

Sour rot is one of the most important postharvest diseases of citrus fruits except green and blue molds,and there was few studies recently on its pathogenesis in the word.The genetic transformation system was established by Agrobacterium-tumefaciens method in order to explore the pathogenic mechanism of citrus sour rot molecularly in this study.The T-DNA fragment containing the neomycin phosphotransferase gene was inserted into the genome of the recipient strain by A.tumefaciens-mediated transformation of Geotrichum citri-aurantii strain AY-1 isolated previously by our laboratory.The establishment of genetic transformation system of citrus sour rot agent by ATMT method was achieved via preliminary optimization of various conditions affecting the transformation efficiency,including the spore concentration of wild strain,co-culture time and temperature,filter papers and so on.Foreign gene was successfully inserted into the fungal genome verified by screening of antibiotics G418 and PCR amplification.The main findings were as follows:(1)The vector pTFCM-G418 was constructed,which contained neo gene,in order to establish a genetic transformation system of pathogen.(2)The result of sensitivity of G.citri-aurantii strain AY-1 to G418 showed that the growth of wild strain AY-1 was completely inhibited in media with G418 up to300?g / mL.And 1000?g / mL G418 was ultimately used as selection concentration to improve the probability of obtaining positive transformants.(3)By exploring the conditions of affecting transformation efficiency preliminarily,the optimal transformation conditions were as follows: the conidial concentration of AY-1 up to 4 × 106 conidia per mL;OD600 of A.tumefaciens AGL-1strain up to 0.4;without induction of AS in pre-culture;co-incubation at 25? for 48 h on cellophane.And the transformation efficiency was up to 60 mutants per 106 conidia.(4)The target gene of mutants,which could grew normally on PDA medium containing 1000?g/mL G418,was obtained by PCR analysis.And all of the transformants continued to be resistant to antibiotic after five successive subcultures,indicating that the foreign gene still existed in obtained mutant strain genome andcould be genetically stable.