Coloning And Functional Identification Of Scytalone Dehydratase Gene (SCD1) And Tetrahydroxynaphthalene Reductase Gene (THR1) From Colletotrichum Gloeosporioid Es-the Pathogen Of Mango Anthracnose Disease

Mango (Mangifera indica L.) is one of the importantly tropical and subtropical fruit only to banana. Anthracnose caused by C. gloeosporioides is the most serious disease in all mango growing regions of the world. Most studies on mango anthracnose are related to disease symptoms, pathogenic characteristics, disease occur-rence rules, control methods, few report focused on its pathogenicity and related genes. In this study,we used C.gloeosporioides as the research object and cloned scytalone dehydratase SCD1 gene and tetrahydroxynaphthalene reductase THR1 gene related to melanin synthesis. After obtaining the full-length sequence, the knockout vector was designed by inserting the green fluorescent protein gene-hygromycin B phosphotransferase gene (gfp:: hygB) into the target gene. Two knockout mutants of SCD1 gene and THR1 gene were obtained by protoplast transformation, hygB plate screening and PCR verification and preliminarily analyzed their phenotypes. It would be of great academic value in terms of revealing the pathogenisis of C.gloeosporioides.Furthermore,such studies may be useful for finding new target sites to improve control of mango anthracnose.1. Cloning the complete DNA and cDNA sequences of scytalone dehydratase SCD1 gene from C. gloeosporioides According to the genomic data of C.gloeosporioides strains HBCg01 from rubber trees (Hevea brasiliensis), DNA and cDNA sequences of SCD1 gene were obtained using PCR and RT-PCR by homologous cloning strategy. The full-length DNA and cDNA sequence of SCD1 gene respectively were 1 580 bp and 564 bp in size, with initiator codon ATG being within the 360-362 bp and terminator codon TAA being within the 1153-1155 bp.DNA sequences from ATG to TAA was 796 bp, which contains one open reading frame of 564 bp in size, encoding a putative protein of 187-amino acid with a molecular weight of 21.526 kDa and isoelectric point of 5.90. Phylogenetic analysis indicated that putative protein of SCD1 was homologous to C. musae and C. caricae scytalone dehydratase (GQ266389.1, GQ266386.1) with a similarity of 98% and 97%,and contains a conserved NTF2_like domain. Therefore, the SCD1 gene from C.gloeosporioides in this study was a scytalone dehydratase gene. The sequence had been submitted to GenBank (KX198009.1).2. Cloning the complete DNA and cDNA sequences of tetrahydroxynaphthalene reductase THR1 gene from C. gloeosporioides DNA and cDNA sequences of THR1 gene were obtained using the same methodes. The full-length DNA and cDNA sequence of THR1 gene respectively were 1 682 bp and 837 bp in size, with initiator codon ATG being within the 483-485 bp and terminator codon TAA being within the 1287-1347 bp. DNA sequences from ATG to TAA was 898 bp, which contains one open reading frame of 837 bp in size, encoding a putative protein of 278-amino acid with a molecular weight of 29.09kDa and isoelectric point of 7.01. Phylogenetic analysis indicated that putative protein of THR1 was homologous to C.tofieldiae and C.orbiculare (KZL72291.1,BAK57420.1) with a similarity of 92% and 90%, and contains a conserved NADB_Rossmann domain.Therefore, the THR1 gene from C. gloeosporioides in this study was a tetrahydroxynaphthalene reductase gene.3. Obtaining knockout mutants of SCD1 gene and THR1 gene Using the In-FusionR HD Cloning Kit , the SCD1 gene and THR1 gene knockout vector pA2SCD 1GH1 and pA2THR1GH1 were constructed. A recombined SCD1 gene fragment from pA2 SCD1GH1 or THR1 gene fragment from pA2THR1GH1 containing the gfp::hygB gene were amplified, and used to transform C.gloeosporioides protoplast by PEG method. After containing hygromycin SR plate screening, PCR verification, and target strip sequencing, three scytalone dehydratase SCD1 gene knockout mutants and three tetrahydroxynaphthalene reductase THR1 gene knockout mutants were confirmed, and named △A2SCD1-1 - △A2SCD1-3 and△A2THR1-1 - △A2THR1-3, respectively.4. Analysis of the phenotypes of SCD1 gene knockout mutant AA2SCD1-1 and THR1 gene knockout mutant △A2THR1-1 Compare with the wild-type,mutant △A2SCD1-1 displayed a close to white colony, no appressorial formation on the tip of germ tube. Aerial mycelial mass with less branches , mycelial growth rate sporulation quantity, activity of cell wall degrading enzymes, and content of intracellular melanin significantly decreased. The mycelium was more sensitive to osmotic stress response of a variety of salts and more resistant to H2O2. Mutant△A2SCD1-1 could not infect the unwounded fruits and leaves, and could infect the wounded mango fruits and leaves, but the virulence was significantly lower than that of the wild type. The phenotypes of mutant △A2THR1-1 were similar to that of mutant △A2SCD1-1 except that the mycelial branches and sensitivity to osmotic stress conditions, which were no significant difference with that of the wild type. The above findings demonstrated that the scytalone dehydratase SCD1 gene and tetrahydroxynaphthalene reductase THR1 gene might be play a vital regulatory role in mycelial growth, pigmentation, conidial and appressorial formation, as well as virulence of C. gloeosporioides on mango. In addition, SCD1 gene also regulates the adaptability of C. gloeosporioides to the osmosis stress.