| Mango(Mangifera indica L.)is one of the five important fruits in the world,and is also the second tropical fruit.Mango industry is one of the important characteristic agriculture support industries.Anthracnose caused by C.gloeosporioides is the most serious disease in all mango growing regions of the world.Few studies focused on its pathogenicity and related genes on mango anthracnose.C.gloeosporioides infects into the host mainly by direct penetration mediated by the a ppressorium,which are formed on the germ tube tip and pigmented with melanin.In this study,glutamate transporter Cggltl gene and transcription factor CgCMR1 gene related to appressium formation and melanin synthesis were cloned from C.gloeosporioides.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 Cggltl gene and CgCMR1 gene were obtained by protoplast transformation,hygB plate screening and PCR verification.Their some phenotypes were measured and pathogenic functions were analyzed.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.The main results were as follows:1.Bioinformatics analysis of glutamate transporter gene Cggltl in C.gloeosporioides from mango According to the genomic data of C.gloeosporioides strains HBCg01 from rubber trees(1Hevea brasiliensis),DNA sequences of Cggltl gene were obtained using PCR by homologous cloning strategy.The full-length DNA sequence of Cggltl gene was 3114 bp in size,with 68 genetic different loci from the corresponding gene in strains HBCg01 and 2162 bp from ATG to TGA.5’upstream sequence and 3’ downstream sequence were respectively 428 bp and 464 bp.Cggltl was highly similar(99%)with mRNA of glutamate transporter gene of C.gloeosporioides from strawberry in GenBank,and with three conservative domains of typical GLT sequences.2.Functional analysis of glutamate transporter gene Cggltl in C.gloeosporioides from mango Using the In-FusionR HD Cloning Kit,the Cggltl gene knockout vector pCggltlGH1 was constructed.A recombined Cggltl gene fragment containing the gfp::hygB gene was amplified from pCggltlGH1,and used to transform C.gloeosporioides protoplast by PEG method.After containing hygromycin SR plate screening,PCR verification,and target strip sequencing,glutamate transporter gene Cggltl knockout mutant was confirmed,and namedΔCgglt1.The phenotypes of Cggltl gene knockout mutant ΔCgglt1 were analysed.Compare with the blackish-brown wild-type,the colony of mutant ΔCggltl became light in color and was always white,and grew slower in term of radial growth rate.The diameter of mycelium of mutant ΔCgglt1 was not changed,while branch and septa of hyphae reduced.Conidial production and sporulation ratio of mutantΔCgglt1 decreased obviously,and no appressorial formation on the tip of germ tube.The death temperature of mycelia was 55 ℃ 20min.The optimum pH range for vegetative growth of mutant ΔCgglt1 was 6-8,which was more suitable acid environment than the wild type(pH7-9).The ability of mutant ΔCgglt1 for utilization carbon was changed.Mutant ΔCgglt1 lost the effect of regulating the alkaloid pH of PD solution.The content of the reactive oxgen species(ROS)of the mutant ΔCgglt1 was more higher and the resistance of ultraviolet radiation was more lower than that of wild type.The cellulase activity decreased slightly.Real-time fluorescence quantitative analysis showed that the expression quantity of PEL and Ecg in ΔCgglt1 were respectively 2.03 and 1.25 times of wild type,and the expression quantity of the rest genes decreased about 12%-24%,among them,LAC1 and PKS respectively decreased 44%and 36%.Mutant ΔCgglt1 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.There was little difference in the optimum temperature for mycilial growth,the utilization of nitrogen source,the resistance to osmotic stress(except sucrose and NaCl)and H2O2 with the wild type.The above findings demonstrated that the glutamate transporter gene Cggltl might be play a vital regulatory role in mycelial growth,pigmentation,conidial and appressorial formation,sensitivity to pH,carbon source utilization,ROS,UV resistance,cell wall hydrolase activity as well as virulence of C.gloeosporioides on mango.3.Bioinformatics analysis of transcription factor gene CgMR1 in C.gloeosporioides from mango Cloning and functional analysis of transcriptional activator gene CgMR1 were studied using the same method.The full-length DNA sequence of CgMRl gene was 3943 bp in size,with 42 genetic different loci from the corresponding gene in strains HBCg01,and 3117 bp from ATG to TGA.5’upstream sequence and 3’ downstream sequence were respectively 425 bp and 395 bp.CgMR1 was homologous to CMR of C.gloeosporioides from strawberry(XM 007283538.1)and C.lagenarium(AB024516.1)with a similarity of 81%～99%。.4.Functional analysis of transcription factor gene CgMR1 in C.gloeosporioides from mango The CgMRl gene knockout vector pCgMRIGH1 was constructed and transcriptional activator gene CgMR1 knockout mutant was confirmed,and named ΔCgMR1.Its phenotype was also analysed.Compare with the blackish-brown wild-type,the colony of mutant ΔCgMR1 became white,and grew slower in term of radial growth rate.The diameter of mycelium of mutant ΔCgMR1 was not changed,while branch and septa of hyphae reduced.Appressorial formation of mutant ΔCgMR1 decreased obviously and no accumulation of melanin in appressorium.The death temperature of mycelia was 50℃ 20min.The ability of utilization carbon and nitrogen was changed,in particular,the utilization of mannitol was significantly higher than that of wild type,and the utilization of pectin was slightly higher.The content of the reactive oxgen species(ROS)of the mutantΔCgMR1 was higher.The sensitivty of mutant ΔCgMR1 to osmotic stress was obviously different from the wild type,and the resistance of mutant ΔCgMR1 to H2O2 was obviously declined,and UV resistance was decreased.The cellulase activity decreased obviously.Real-time fluorescence quantitative analysis showed that the expression quantity of CgMR1 was close to 0,and the expression of PKS,THN,SCD and LAC1 related to melanin synthesis decreased about 67%-75%.The expression of PEL and ECG decreased about by 54%.Mutant ΔCgMR1 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.Compare with the wild-type,there was little difference in conidial production and sporulation ratio,the optimum temperature and pH for mycilial growth,as well as the utilization of nitrogen source.These findings shown that the transcription factor gene CgMRl might be play a vital regulatory role in mycelial growth,pigmentation,appressorial formation,carbon and nitrogen source utilization,osmotic stress,ROS,the resistance to H2O2 and UV,cell wall hydrolase activity as well as virulence of C.gloeosporioides on mango. |
PDF Full Text Request