Construction Of A T-DNA Insertional Library Of Coniothyrium Minitans And CDNA Cloning Of Two Conidiation Associated Genes

Coniothyrium minitans is an important sclerotial mycoparasite of Sclerotinia sclerotiorum.In this dissertation,a high efficient Agrobacterium tumefacies mediated transformation system for Coniothyrium minitans and the technical platform of cloning conidiation associated genes were established by optimization of transformation conditions,a T-DNA insertional library with 30500 transformants was constructed,and two conidiation associated genes were clonied and analysed.The high efficient transformation system for Coniothyrium minitans with Agrobacterium tumefacies was established by optimizing several transformation conditions.Our results showed that the cultivation time for strain ZS-1 on PDA slant,conidia concentration and co-cultivation time were key factors for transformation efficiency.To obtain the highest efficiency,conidia were collected from a 21-day-colony developed on PDA slant with centrifuge,and then the conidial pellet were resuspended and adjusted to 10⁸ conidia/ml with bacterial liquid from inducible medium(OD₆₀₀=0.15),subquently,one hundred microliter of mixture was spreaded on cellophane membrane laid on the solided co-culture medium,and incubated at 23℃for 96h.To clone the genes flanking the T-DNA in certain inserts,a technical platforms based on Thermal asymmetric interlaced PCR(TAIL-PCR),Inverse PCR (iPCR),Reverse transcription PCR(RT-PCR) and cDNA library screening were built, and this plateform were successfully used to clone two conidia-associated genes.A T-DNA insertional library with 30500 transformants was contructed with the transformation system described above,but the conidial concentration was adjusted to 10⁷ conidia/ml to obtain individual transformants.In the library,127 conidiation deficient mutants were screened out,and among them 65 mutants were proved not to produce any conidia on PDA plate,and 62 mutants were proved to could produce a few conidia on PDA plate.These mutants were varied in phenotypes including hyphal extension rate,colony morphology,pathogencity to S.sclerotiorum,antifungal abibility and antibacterial ability.Southern blots showed that the T-DNA marker was single copy in most tested mutants,and the insertion sites were varied.These evidence suggested that conidiation of C.minitans was likely complicated,and was controlled by lots of genes.Base on the colony morphology of mutants,the 65 mutants could be grouped into seven types.Typeâ… ,mutants grew on PDA plates slowly,and colony could not develop radially,26 mutants were grouped into Typeâ… .Typeâ…¡,mutants grew on PDA plates slowly,but aerial hyphae were extremely developed,28 mutants could be belonded to Typeâ…¡.Typeâ…¢,mutants grew on PDA plate as fast as original strain ZS-1,but hyphae in colony center were collapse,and then degraded,only one mutant in this group.Typeâ…£and Typeâ…¤,mutants grew on PDA plate even faster than original strain ZS-1,but mutants in Typeâ…£could produce conidia when contacted the colony or sclerotia of S.sclerotiorum,3 mutants in Typeâ…£and 5 mutants in Typeâ…¤. Typeâ…¥,the hyphae of this type mutant were very sparse,and in some place,the mycelia interlaped and turned to ferruginous color,only one mutant was found I this type.Typeâ…¦,mutant could form empty pycnidia,but no conidium produced,only one mutants in Typeâ…¦:other 11 mutants were not grouped,because their morphology shape has not obvious discrepancy,but these mutants couldn't produce conidiation..Conidiation-deficiency mutant ZS-1T2029 was studied in detail.There was not significant difference between ZS-1T2029 and ZS-1 on growth rate and secretion of antibiotics,the pathogenicity to S.sclerotiorum was declined significantly,however, conidiation of this mutants could restored when grew on sclertia.ZS-1T2029 could only form primordia both on PDA plate or in PDB liquid,and the primordia could not develop to pycnidia.Southern blot showed that only one copy of T-DNA was inserted in ZS-1T2029,and a gene named CMCPS1,which encodes carbamoyl-phosphate synthase(CPS),was cloned initially based on TAIL-PCR from ZS-1T2029,and combined with iPCR and RT-PCR.The full length of CMCPS1's cDNA was 3900 bp, and sequencing analysis showed that there might include an complete ORF(122-3634nt) encoding 1170 amino acids.Southern blot showed one copy of CMCPS1 in C.minitans. Northern blots showed that CMCPS1 was expressed in original strain ZS-1,but not in mutant ZS-1T2029.CMCPS1 was confirmed to be a gene associated with conidiation with gene silence strategy using RNAi technique.Amending arginine or citrulline into PDA could restore conidiation of ZS-1T2029,suggested that blocking of arginine biosynthesis by the T-DNA insertional disruption of CMCPS1 was the reason of conidiation deficiency of ZS-1T2029.Further results showed that conidiation of ZS-1T2029 could be restored with sodium nitroprussiate(SNP),a nitric oxide donor, and the conidiation of ZS-1 could be suppressed by N,G-nitro L-arginine(L-NAME), an inhibitor of inducible nitric oxide synthase.The highest amount of nitric oxide in mycelia was examined at the early stage of conidiation(the 3^rd day) with nitrate reductase when C.minitans was shaken in modified PDB;in situ probing with 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate,strong fluorescence signal could be checked in immature pycnidia,primordia and in mycelial mass where pycnidia were initially differentiated.These evidence supported that arginine via NO regulated the conidiation of C.minitans.Conidiation-deficiency mutant ZS-1T21882 was also studied.There was not any significant difference between ZS-1T21882 and ZS-1 on growth rate and secretion of antibiotics.ZS-1T21882 could form conidia when it contacted colony or sclerotia of S. sclerotiorumsclerotiorum.The pathogencity to S.sclerotiorum was not different from original strain ZS-1.ZS-1T21882 could produce primordia in shaken liquid medium, but the primordia failed to develop to pycnidia.Southern blot showed that only one copy of T-DNA was inserted in this mutant,and a gene named CMAMPRS1,which encodes amidophosphoribosyltransferase,was cloned initially based on TAIL-PCR from ZS-1T2029,and combined with RT-PCR.The full length of CMAMPRS1's cDNA was 1749 bp,and sequencing analysis showed that there might include an complete ORF(122-3634nt) encoding 583 amino acids.Southern blot showed one copy of CMAMPRS1 in C.minitans,and no mRNA of CMAMPRS1 was checked with RT-PCR in ZS-1T21882.CMAMPRS1 is the second key enzyme in the process of IMP synthesis.Amending IMP into PDA could restore the conidiation of ZS-1T21882, suggested that the insertional disruption of CMAMPRS1 was the cause for this mutant's deficiency.Further experiments showed that ATP,AMP and cAMP could restore conidiation of ZS-1T21882 when amended into PDA,but GTP,GMP and cGMP could not.These evidence suggested that C.minitans needed more adenine during conidiation,and the likely role for adenine was as substrate to form cAMP,an important transduction signal.