【作者】 宫晓艳；

【导师】 姜道宏；

【作者基本信息】 华中农业大学 ， 植物病理学， 2008， 博士

【摘要】 盾壳霉(Coniothyrium minitans)是植物病原真菌核盘菌(Sclerotiniasclerotiorum)的重要生防真菌。本研究以ZS-1菌株为出发菌株,建立了农杆菌介导高效转化盾壳霉的技术体系及克隆相关基因的技术平台,构建了含30500个转化子的T-DNA标记插入转化子库,克隆到2个与产孢相关的基因,现报道如下:对农杆菌转化条件进行了优化,建立了高效转化盾壳霉的技术体系。发现ZS-1菌株的培养时间、用于转化的分生孢子浓度、分生孢子与农杆菌共培养的时间等与转化效率有关。当使用在PDA斜面上培养21d所获得的分生孢子(浓度为10~8个孢子/ml)与细菌共培养96h后获得的转化效率最高,每皿可以获得500个转化子。建立了以热不对称交错PCR(TAIL-PCR)、反向PCR(iPCR)、逆转录PCR(RT-PCR)、cDNA文库等为基础的克隆基因的技术平台,并利用此技术平台克隆了产孢相关的基因。利用上述转化方法构建了盾壳霉ZS-1菌株的T-DNA插入体库,获得了30500个转化子。自盾壳霉的插入突变体库中共筛选得到了127株产孢缺陷型突变体,其中65株突变体在PDA平皿上不能产生分生孢子,而另外的62株则可以产生少量的孢子。这些产孢缺陷型突变体在菌丝生长速度、产抗生物质、寄生菌核能力和菌落形态等方面存在差异显著性,大多数突变体的T-DNA为单位点插入,且插入位点不同。这即表明盾壳霉的分生孢子形成复杂,受多种基因调控。依据菌落形态,65株完全不产孢的突变体大致可分为7种类型,即Ⅰ类:突变体生长缓慢,菌落不能均匀扩展,共26株;Ⅱ类:突变体生长较缓慢,气生菌丝茂盛,共28株;Ⅲ类:突变体生长正常,但菌落中央的菌丝塌陷,溃解,仅1株;Ⅳ类:突变体生长正常,但与寄主(核盘菌)接触后可以恢复产孢能力,共3株;Ⅴ类:菌丝生长块,甚至超过出发菌株ZS-1,共5株;Ⅵ类:生长缓慢,菌丝稀疏,菌落中有菌丝集结,呈铁锈色,仅1株;Ⅶ类:突变体只能产生分生孢子器而不产生分生孢子,仅1株;另外还有11株菌落形态上没有明显的区别,没有具体的归为哪一类。对产孢缺陷型突变体ZS-1T2029进行了详细的研究。突变体ZS-1T2029在菌丝生长和分泌抗生物质等方面与出发菌株ZS-1相比没有显著差异;此突变体仍具寄生核盘菌的能力,在菌核上可以产生分生孢子,但其寄生腐烂菌核的能力显著下降,显微观察发现ZS-1T2029的分生孢子形成停滞于分生孢子器原基阶段。Southern杂交证实T-DNA标记在ZS-1T2029中为单位点插入。证实T-DNA插入破坏了精氨酸特异性氨甲酰磷酸合成酶基因(CMCMPS1),CMCPS1的cDNA全长为3900bp,编码含1170个氨基酸的蛋白。Southern杂交分析表明CMCPS1在盾壳霉ZS-1菌株中为单拷贝,Northern杂交显示在ZS-1T2029中没有检测到CMCPS1的mRNA,采用RNAi技术进一步证实CMCPS1与盾壳霉孢子形成相关。在PDA培养基中添加精氨酸或瓜氨酸可以恢复ZS-1T2029产孢试验表明T-DNA插入破坏CMCPS1导致不能合成精氨酸是ZS-1T2029不能产孢的原因。因此,精氨酸与盾壳霉分生孢子形成相关。在PDA培养基中添加一氧化氮供体硝普钠(SNP)可恢复ZS-1T2029产孢,而在PDA中添加一氧化氮合成酶(NOS)抑制剂L-硝基精氨酸甲酯(L-NAME)可以抑制ZS-1菌株产生分生孢子,试验结果表明精氨酸通过一氧化氮调控盾壳霉的分生孢子形成。进一步研究发现:一氧化氮在分生孢子器原基及分生孢子器中有大量的积累,而在菌丝中累积不明显;在PDB培养液中振荡培养,在ZS-1和ZS1-T2029中均可以产生NO,其释放量在第3d达到最高,但后者NO的产量显著下降。对产孢缺陷型突变体ZS-1T21882进行研究。ZS-1T21882在菌丝生长、寄生能力和分泌抗生物质等方面与出发菌株ZS-1相比没有显著差异。突变体与核盘菌菌落接触的地方及其菌核上均能够产生分生孢子,但在液体振荡培养时仅能形成分生孢子器原基。Southern杂交证实T-DNA标记在ZS-1T21882中为单拷贝插入。对ZS-1T21882中T-DNA插入破坏的基因进行了全长cDNA克隆,证实T-DNA插入破坏了谷氨酰胺磷酸核糖焦磷酸转酰胺酶基因(CMAMPRS1);CMAMPRS1的全长cDNA为1749 bp,含一个1560 bp的完整阅读框架(ORF),该ORF编码含583个氨基酸的蛋白质。CMAMPRS1在盾壳霉基因组中为单拷贝,RT-PCR结果显示此基因在突变体ZS-1T21882中不表达,而在出发菌株ZS-1中有表达。谷氨酰胺磷酸核糖焦磷酸转酰胺酶是生物体内从头合成次黄嘌呤中的第二个关键酶,研究发现在PDA培养基中添加次黄嘌呤可以恢复ZS-1T21882产孢,表明T-DNA插入破坏CMAMPRS1导致不能合成次黄嘌呤是ZS-1T21882不能产孢的原因。进一步研究发现,在PDA培养基中添加ATP、AMP和cAMP等均可以促使ZS-1T21882产孢,而添加GTP、GMP和cGMP等则不能恢复ZS-1T21882产孢。因此,推定盾壳霉孢子形成与腺嘌呤有关。更多还原

【Abstract】 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.TypeⅠ,mutants grew on PDA plates slowly,and colony could not develop radially,26 mutants were grouped into TypeⅠ.TypeⅡ,mutants grew on PDA plates slowly,but aerial hyphae were extremely developed,28 mutants could be belonded to TypeⅡ.TypeⅢ,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.TypeⅣand TypeⅤ,mutants grew on PDA plate even faster than original strain ZS-1,but mutants in TypeⅣcould produce conidia when contacted the colony or sclerotia of S.sclerotiorum,3 mutants in TypeⅣand 5 mutants in TypeⅤ. TypeⅥ,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.TypeⅦ,mutant could form empty pycnidia,but no conidium produced,only one mutants in TypeⅦ: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.更多还原