Phytoplasmas are phloem-limited bacterial plant pathogens associated with approximately1000 plant species worldwide,of which over 100 occur in *** the study,four phytoplasma strains of chinaberry witches'-broom(...
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Phytoplasmas are phloem-limited bacterial plant pathogens associated with approximately1000 plant species worldwide,of which over 100 occur in *** the study,four phytoplasma strains of chinaberry witches'-broom(CWB) from Hainan,Guangdong and Fujian provinces(CWB-hnsyl,CWB-hnsy2,CWB-fjfz,CWB-gdgz),two strains of mulberry dwarf(MD) from Zhejiang and Anhui provinces(MD-ahhf and MD-zjca),two strains of lettuce yellow(LY) from Yongan,Fujian province(LY-fjsm1 and LY-fjsm2) and a strain of paulownia witches'-broom(PaWB) from Shandong province,which were all identified as the member of 16 SrI group phytoplasmas based on 16 S rRNA gene sequences,were performed extensive sequence and phylogenetic analysis based on sequences of 16S-23 S rRNA intergenic spacer region,partial 23 S rRNA,ribosomal protein including 3' end of S19 and complete L22 and S3(rp),tuf,secA,secY,ipt,dnaK,fusA,gyrB,pyrG and rpoB gene sequence data from these nine phytoplasma strains,as well as onion yellows phytoplasma(OY-M),aster yellows witches'-broom phytoplasma(AYWB) and Acholeplasma laidlawii PG-8A strains,which were employed as *** found abundant genetic variation in these 16 SrI group phytoplasma *** phytoplasma strains from CWB,MD and LY shared 99.7%~ 100%16S rRNA gene sequences similarity,as well as 99.4%~ 100%16SrRNA,16S-23 S intergenic and partial 23 SrRNA,99.6%~ 100.0%rp,99.3%00.0%secA,98.8%00.0%secY,99.0%00.0%ipt,99.8%00.0%tuf,99.1%00%dnak,99.0%~ 100.0%fiisA,99.1%~ 100%gyrB,98.8%~ 100%pyrG and98.9%~ 100%rpoB gene sequences similarity *** phylogenetic lineage among members of these 16 Sri group phytoplasma strains were constructed based on concatenated gene of rp,tuf,secA,secY,ipt,dnaK,fusA,gyrB,pyrG and rpoB gene sequences using method of multilocus sequence analysis(MLS A).These eight strains shared 99.2%~ 100%concatenated gene similarity for one another and formed a distinct clade with 99%bootstrap support by neighbor-joining using MEGA *** the cl
和其他生物一样,植物病原真菌也是生活在多变环境中,外界环境对其影响贯穿于整个生命过程。关于病原真菌如何感受外界环境刺激,这些刺激又是如何调控和决定病原真菌的生长、繁殖和致病已成为植物病理学研究热点之一。生物体接受和传递外界环境刺激的过程称信号传导。G蛋白偶联受体是生物体内一类重要的跨膜蛋白,与G蛋白偶联受体相结合的异三聚体G蛋白(beterotrimeric G-protein)在信号传递中起着中枢作用,通过其构象的改变将胞外信号整合到胞内,激活环腺苷酸途径和促分裂活化蛋白激酶级联信号途径,从而调控一系列基因的转录,最终引起生物对外界刺激作出应答。已知G蛋白在真核细胞生物中是保守的,能与鸟嘌呤核苷酸结合,并自身具有GTP酶水解活性的信号传导分子。G蛋白分子量大,由α、β和γ三个亚基组成。其中Gα亚基分子量最大,一般382kD,具有鸟苷酸结合位点,通过GTP结合态(激活态)和GDP结合态(失活态)间的转换行使信息传递,调控细胞对外界环境刺激的应答(McCudden et al.,2005)。根据Gα亚基氨基酸序列的相似性和下游效应元件的相关性,Gα亚基在丝状真菌中可以分为Ⅰ、Ⅱ和Ⅲ三大类(Liande et al.,2007),大多数丝状真菌具有3个不同种类的Gα亚基。第Ⅰ类Gα亚基的氨基末端具有肉豆蔻酰基化位点(Buss et al.,1987),羧基末端具有百日咳毒素结合位点(West et al.,1985)。该类Gα亚基主要参与调控真菌的营养生长、有性与无性繁殖、致病性和真菌毒素合成等多种生理过程(Liande et al.,2007;Bolker,1998)。第Ⅱ类Gα亚基没有第Ⅰ类Gα亚基和第Ⅲ类Gα亚基保守,功能也没有第Ⅰ类Gα亚基和第Ⅲ类Gα亚基明显。但有研究表明,第Ⅱ类Gα亚基可能在碳源应答途径中具有一定的作用(Li and Borkovich,2006)。第Ⅲ类Gα亚基在丝状真菌中高度保守,正调控细胞内cAMP水平(Bolker et al.,1998)。位于G蛋白信号途径下游的腺苷酸环化酶(adenylate cyclase,AC)是膜结合蛋白,分子量为150kD,氨基端和羧基端都朝向细胞质。AC在细胞内有两个膜结合区和两个催化结构域,其中每个膜结合区分别有6个跨膜的α螺旋,催化结构域中有2个保留序列Cla和C2a,这两个区域主要负责AC的活性。在Mg或Mn存在时,AC能催化三磷酸腺苷(adenosine triphosphate,ATP)形成环腺苷酸(cyclic adenosine monophosphate,cAMP)并释放出焦磷酸。在许多真菌中,AC及其产物cAMP’对菌落生长、致病性和抗逆性等方面有重要的调控作用(Choi&Dean,1997;Kronstad,1997;Loubradou et al.,1996)。已有的研究证明,Gα亚基及其下游的cAMP信号途径主要传递营养和性激素等的刺激,参与植物病原真菌的生长发育、产孢、交配、侵染相关结构的分化以及致病过程(Bolker,1998;Lengleler et al.,2000;Yu,2006)。柑橘绿霉病菌(Penicillium digitatum)是采后柑橘的最主要病害,其造成的损失通常占采后柑橘腐烂损失的90%(Kanetis et al.,2007)。尽管该病菌的营养方式为死体营养型,只能通过伤口侵染,但病菌的寄主却只局限于成熟的柑橘类果实果皮。这一独特的寄主-病原互作方式,引起我们对其信号传递途径的兴趣。以稻瘟病菌G蛋白α亚基(MagB、MagC和MagA)和腺苷酸环化酶(Mac1)为参考序列,在已测序的柑橘绿霉病菌基因组序列中(Sun et al.,2013)分别搜索到上述基因的同源基因PdgpaA、PdgpaB、PdgpaC和Pdac1。通过pfam数据库分析发现PdgpaA、PdgpaB和PdgpaC均含有保守的G蛋白α亚基结构域,其E值分别为6.5e-127、7.2e-139、5.4e-119;Pdac1含有保守的腺苷酸环化酶结构域,其E值为1.1e-26。通过运用重叠PCR和农杆菌介导的遗传重组技术,构建获得了PdgpaB、PdgpaC和Pdac1缺失突变体。表型分析研究表明,PdgpaB(第Ⅱ类Gα亚基)和PdgpaC(第Ⅲ类Gα亚基)的缺失均不影响柑橘绿霉病菌在正常条件,离子胁迫、氧化胁迫,阴离子去污剂SDS、荧光增白剂和刚果红胁迫下的菌丝生长、孢子形成和孢子萌发,对柑橘类果实的致病性没有显著的影响。然而,尽管花费了巨大的努力,始终未能敲除PdGpa2,推测该基因在柑橘绿霉菌G蛋白信号途径中具有关键作用,其缺失可能导致菌体死亡。而PdAc1的缺失使得柑橘绿霉菌的营养生长严重受阻,形成的菌落极为致密,分生孢子产生提前,并
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