| A regulatory gene of Setosphaeria turcica, named StMR, was cloned throughcandidate gene method during the earlier research period. Compared to the JGI website(http://genome.jgi-psf.org/) of S. turcica genome sequence, we found a high similarityhomologous sequence of StMR was found and named as StMR1. In order to confirm thefunction of StMR1and the regulation mechanism in DHN melanin biosynthesis, StMR1gene was cloned and analyzed by bioinformatics technique and RNAi technique. The mainresults were as follows:A regulatory gene sequence was found by searching the genome database of S. turcicapublished on JGI website (http://genome.jgi-psf.org/), which was named StMR1and clonedby PCR. The gene is located at545611-550153of the genome scaffold12negative chain. Itcontains a3282bp DNA sequence including a3021bp cDNA sequence and3introns.Bioinformatics analysis showed that the predicted protein of StMR1had conservativedomain of plant pathogenic fungi coding melanin biosynthesis regulatory transcriptionfactor. It was confirmed that the gene is the homologous gene of StMR former identified asa transcription factor modulating the melanin biosynthesis in S. turcica.According to the nucleotide sequence of StMR1and the multiple cloning site ofpSilent-1plasmid, specific primers were designed to amplify two fragments from StMR1.Then, both fragments were ligated with pSilent-1to construct the RNAi vector. The vectorwas finally transformed into protoplasts of S. turcica. Five transformants with differentStMR1-silencing efficiency were obtained by screening of hygromycin B and identifyingwith RT-PCR.Compared to the wild-type strain, the colony colour of five RNAi transformantstended to be yellow-white. The higher the StMR1-silencing efficiency was, the lighter thecolony colour became. It suggested that the lightened degree of the colony colour waspositively related with the StMR1-silencing efficiency. In addition, transformants exhibitedlong distinct septum, transparent hypha, long cells and no conidial production. StMR1-R1,StMR1-R4and StMR1-R5had transparent and long cells in which lots of vacuole-like structures were discovered, while StMR1-R3had curl hyphae. These results indicated thatStMR1was involved in DHN melanin biosynthesis, while it was also related with mycelialdevelopment and conidial formation.On the other hand, the recombination plasmid of StMR1was constructed on the baseof transformation vector pBU. It laid a foundation for the further confirmation of thefunction of StMR1. |
