球孢白僵菌高渗适应性相关基因Bbmpd的克隆与表达分析

【目的】克隆与球孢白僵菌(Beauveria bassiana)的高渗适应性相关基因,并对其功能进行分析,以揭示球孢白僵菌对高渗等逆境适应的分子机理。【方法】利用YADE法克隆T-DNA的侧翼序列并进行基因组步行,获得突变基因的全长及上游序列;利用RT-PCR技术分析突变基因的表达特性以及与Bbhog1的关系;采用同源重组技术敲除Bbmpd基因。【结果】克隆得到插入突变基因及其上、下游序列全长3037bp。该基因与编码球孢白僵菌的1-磷酸甘露醇脱氢酶基因相似性为98%。Bbmpd的表达受高渗环境(0.8mol/L NaCl)的诱导,受Bbhog1信号途径的激活调节,Bbhog1缺失导致Bbmpd表达下调。Bbmpd缺失突变体在高渗胁迫下的生长受到明显抑制。Bbmpd缺失不影响球孢白僵菌在查氏培养基上的生长和产孢。【结论】由T-DNA突变体克隆了编码球孢白僵菌1-磷酸甘露醇脱氢酶基因Bbmpd,该基因的表达受高渗环境的诱导和Bbhog1的调控,与球孢白僵菌高渗适应性相关。     

农杆菌介导的灰葡萄孢T-DNA插入突变体库构建及插入位点分析

【目的】利用农杆菌(Agrobacterium tumefaciens)介导法对灰葡萄孢(Botrytis cinerea)进行转化,构建T-DNA插入突变体库,为从分子水平上认识灰葡萄孢的致病机制打下基础。【方法】以含有pCAMBIA 1390双元载体的农杆菌对灰葡萄孢进行转化,利用潮霉素进行筛选。对抗性稳定的转化子进行生物学和形态学观察,采用离体番茄叶片进行致病性测定。利用TAIL-PCR技术对突变体中T-DNA的旁侧序列进行克隆。【结果】得到了一些突变体,表现为生长速率减缓、产孢能力下降、致病力减弱等。克隆并分析了其中一个突变体中T-DNA插入的位置和旁侧序列。【结论】本实验建立了农杆菌介导的灰葡萄孢转化体系,构建了T-DNA插入的灰葡萄孢突变体库。用TAIL-PCR进行突变体中T-DNA旁侧序列的分析是可行的。     

橡胶树胶孢炭疽菌T-DNA插入突变体库构建及其致病缺陷转化子筛选

【目的】进一步研究橡胶树胶孢炭疽菌致病分子机理。【方法】通过含ILV1基因(具氯嘧磺隆抗性)的pSULF.gfp双元载体农杆菌AGL-1介导进行橡胶树胶孢炭疽菌遗传转化,利用氯嘧磺隆抗性标记筛选转化子,对转化子PCR验证及荧光显微观察;采用离体古铜期橡胶树叶无伤接种法进行致病性缺陷转化子筛选,并对转化子进行遗传稳定性检测。【结果】获得含3 721个转化子的T-DNA插入突变体库,转化效率为150 400个转化子/106孢子,从3 721个转化子中筛选得到致病性缺陷转化子25个;随机选取20个转化子进行遗传稳定性测定,在不含氯嘧磺隆PDA平板上继代培养10次后仍保持氯嘧磺隆抗性,且表型稳定,表明插入外源基因能够稳定遗传。【结论】可以利用根癌农杆菌介导橡胶孢炭疽菌转化,构建橡胶树胶孢炭疽菌T-DNA插入突变体库,筛选致病缺陷突变菌,为进一步研究该菌致病相关基因提供材料。     

灰葡萄孢菌（Botrytis cinerea）基因组中T-DNA整合模式分析

摘要：【目的】研究灰葡萄孢菌（Botrytis cinerea）基因组中T-DNA插入位点的整合模式特征。【方法】利用农杆菌（Agrobactirium tumfacience）介导法构建灰葡萄孢菌T-DNA插入突变体库。利用热不对称交错PCR（TAIL-PCR）技术对转化子中T-DNA的旁侧序列进行扩增和克隆,对获得的旁侧序列进行比对分析。【结果】T-DNA插入在灰葡萄孢菌基因组非编码区的占69%,插入在外显子的占30%。T-DNA在插入的过程中发生了碱基缺失、增加等重组现象,其中左边界（left border,LB）整合到基因组碱基缺失较少,有的保持完整,而右边界（right border,RB）及其近邻的T-DNA区域缺失碱基较多。T-DNA的插入位点还发现有额外的序列插入。【结论】对灰葡萄孢菌中插入T-DNA的整合模式的分析为开展该菌的功能基因组学奠定了基础。     

腺苷酸核糖基化因子BbarfA介导白僵菌孢子萌发和毒力

【目的】探究腺苷酸糖基化因子ARF在球孢白僵菌(Beauveria bassiana)中存在种类及生物学功能。【方法】利用BLASTp搜索球孢白僵菌非冗余蛋白数据库,鉴定ARF并进行聚类分析,结合表达分析、反义抑制、超量表达野生型基因和GTP解离位点与结合位点突变的基因,解析其中1个ARF与白僵菌发育分化、逆境胁迫反应和毒力的关系。【结果】球孢白僵菌中存在至少6个ARF或类似蛋白,分别聚类于酵母、人类ARF及其类似蛋白的不同类群。其中BBA_01574与人类的ARF3、ARF4和ARF5聚为一类,命名为BbarfA。BbarfA在成熟的分生孢子和球形膨大时期表达明显高于芽管伸长期。反义抑制BbarfA加速了孢子萌发,提高了菌株毒力,而超量表达BbarfA和点突变GTP解离区域的BbarfA则延迟了孢子萌发速度,降低了菌株毒力。尽管BbarfA转录受高盐、髙渗、氧化和高温胁迫的诱导,但遗传修饰的转化子与野生菌株对上述胁迫反应的敏感性无明显差异。【结论】BbarfA介导分生孢子萌发和毒力。     

拟南芥ABA敏感突变体asm1的分离与鉴定

以拟南芥(Arabidopsis thaliana)为研究材料,从T-DNA突变体库中筛选分离得到1株脱落酸(ABA)敏感突变体asm1(ABA sensitive mutant 1,asm1),在含有ABA的培养基中,与野生型相比,asm1突变体的根伸长明显受到抑制,且其种子萌发结果显示asm1对ABA同样表现出敏感特性。在生长发育方面,asm1突变体抽苔时间提前,植株矮化,并且荚果长度明显小于野生型。利用远红外成像系统分析发现,在干旱胁迫下asm1突变体叶面温度高于野生型;失水率分析显示突变体失水率降低以及水分散失减少。遗传学分析表明,asm1是单基因隐性突变且与一个T-DNA插入共分离;通过图位克隆成功获得候选基因ASM1。RT-PCR结果显示,在突变体中ASM1的表达受到抑制,并且能够调控多种ABA信号通路和胁迫应答基因的表达水平。研究结果表明,ASM1可能参与调控ABA信号转导并应答干旱胁迫。     

香蕉枯萎病菌中Hog1 MAPK同源基因FoHog1敲除突变体的生物学特性

【目的】研究香蕉枯萎病菌4号生理小种中促分裂原活化蛋白激酶基因FoHog1的结构特点及其功能【方法】通过PCR和RT-PCR的方法获得了FoHog1基因序列并进行生物信息学分析,利用PEG介导的原生质体转化法得到了FoHog1基因缺失突变体,分析敲除突变体与野生型的生物学特性差异【结果】FoHog1基因编码一个含有357个氨基酸的蛋白,该蛋白在不同种镰刀菌中高度保守。通过对敲除突变体的研究发现,该基因缺失后菌丝密度下降,产孢量与菌丝干重明显降低,对乙酸钠和氯化铵的利用率下降,对温度、pH及渗透压等外源胁迫更为敏感。通过致病力实验发现,基因敲除突变体的定殖能力有所降低【结论】尖孢镰刀菌古巴专化型4号生理小种中FoHog1基因参与调控菌丝生长、分生孢子生成、乙酸钠和氯化铵代谢、渗透压胁迫反应及致病相关过程。     

农杆菌介导的获取疏绵状嗜热丝孢菌插入突变体体系的建立

[目的]建立疏绵状嗜热丝孢菌的稳定遗传转化体系并获得插入突变体.[方法]利用农杆菌介导的方法建立疏绵状嗜热丝孢菌的遗传转化体系 ;分别通过Southern杂交、克隆转移DNA(T-DNA)侧翼序列来确定T-DNA在疏绵状嗜热丝孢菌基因组中的拷贝数和插入位点.[结果]成功建立了可靠的疏绵状嗜热丝孢菌的遗传转化体系.共培养过程中使用萌发孢子是成功建立疏绵状嗜热丝孢菌遗传转化体系的必要条件.疏绵状嗜热丝孢菌萌发的孢子与农杆菌在28℃共培养48h时,转化效率最高.乙酰丁香酮(AS)在农杆菌预培养及疏绵状嗜热丝孢菌萌发的孢子与农杆菌的共培养阶段都是必需的,且在共培养阶段当AS浓度为500 μM时转化效率最高.Southern杂交验证表明,79.2％的转化子为T-DNA单拷贝插入,且通过热不对称PCR (TAIL-PCR)分析得出T-DNA在该菌基因组中的插入位点是随机的.通过该转化系统筛选到部分表型突变体.[结论]我们首次报道了利用ATMT技术成功转化嗜热真菌-疏绵状嗜热丝孢菌,证明了该方法是一种简单有效的获得插入突变体的方法,并为该嗜热真菌进行基因定位提供了工具.     

胶孢炭疽菌热休克蛋白基因Cghsp90的RNAi突变体获得与功能分析

为揭示胶孢炭疽菌(Colletotrichum gloeosporioides)热休克蛋白基因Cghsp90在抗逆境胁迫中的功能,利用RNAi技术结合PEG介导的原生质体转化方法,获得了Cghsp90的RNAi突变体菌株,经实时荧光定量PCR (qRT-PCR)对野生型菌株和突变体菌株间Cghsp90基因差异表达分析以及NaCl、H_2O_2、SDS、Congo Red、18℃(Cold)和33℃(Hot)等非生物胁迫条件下野生型菌株和突变体菌株的胁迫耐受性分析。通过特异性引物检测鉴定、qRT-PCR分析,获得Cghsp90的RNAi突变体pSilent-1:Cghsp90-1和pSilent-1:Cghsp90-2;与野生型菌株相比,侵染过程中Cghsp90基因的表达量显著低于野生型菌株,8个致病相关基因显著下调表达;突变体菌株的产孢量下降、分生孢子畸形、萌发率下降;在非生物胁迫条件下,突变体菌株较野生型菌株更为敏感;在胶孢炭疽菌的分生孢子中研究Cghsp90基因的形态建成与萌发,对应变逆境胁迫以及调控致病相关基因等方面发挥重要的作用。     

酿酒酵母转座标签插入突变体263-H9中高盐胁迫基因的确定

突变体263-H9是利用mTn3转座标签对酿酒酵母(Saccharomyces cerevisiae) W303-1A诱变、筛选得到的。该突变体表现出对多种逆境胁迫(1.5 mol/L山梨醇高渗透压胁迫、0.65 mol/L NaCl高盐胁迫和15℃低温胁迫)敏感的表型特征, 而且与其他突变体不同其转座标签的插入位点是GIP2和YER053C-A的基因间隔区域。本文通过基因敲除、基因组文库功能互补等多种分子生物学和遗传学方法, 确定了突变体263-H9的敏感表型不是由于转座标签的插入直接引起的, 而是盐胁迫反应信号传导途经中重要的基因PBS2发生部分缺失, 造成该基因不能正常表达, 而导致的表型变化。     

铜绿假单胞菌中一个新的吩嗪合成调节基因

[目的]在次抑制浓度四环素条件下,研究铜绿假单胞菌phzAl操纵子的调节基因及调节途径.[方法]对转座突变库中phaAl操纵子表达发生变化的突变体,进行随机PCR、基因测序及比对,确定突变位点.并以发光杆菌的荧光素酶基因操纵子luxCDABE为报道基因,研究基因调节作用及调节路径.[结果]在两株突变体PAM0487和PAM0487R中phzAl操纵子的表达降低,这两株突变体的突变基因确定为假定钼元素转运蛋白调节子PA0487基因.[结论]PA0487是phzAl操纵子表达的一个新的正向调节子,并对密度感应系统相关基因的表达有凋节作用.     

Time- and Cost-Efficient Identification of T-DNA Insertion Sites through Targeted Genomic Sequencing

Forward genetic screens enable the unbiased identification of genes involved in biological processes. In Arabidopsis, several mutant collections are publicly available, which greatly facilitates such practice. Most of these collections were generated by agrotransformation of a T-DNA at random sites in the plant genome. However, precise mapping of T-DNA insertion sites in mutants isolated from such screens is a laborious and time-consuming task. Here we report a simple, low-cost and time efficient approach to precisely map T-DNA insertions simultaneously in many different mutants. By combining sequence capture, next-generation sequencing and 2D-PCR pooling, we developed a new method that allowed the rapid localization of T-DNA insertion sites in 55 out of 64 mutant plants isolated in a screen for gyrase inhibition hypersensitivity.     

灰葡萄孢分生孢子产生相关基因的克隆及功能分析

[目的]克隆灰葡萄孢分生孢子产生相关基因,并研究其功能,为进一步研究灰葡萄孢分生孢子产生机理和灰葡萄孢侵染及致病机理奠定基础.[方法]通过筛选灰葡萄孢ATMT突变体库,获得一株不能产生分生孢子的突变菌株BCt78,采用PCR和Southern Blotting技术,对突变菌株BCt78进行分子鉴定.利用TAIL-PCR技术获得T-DNA插入位点的侧翼序列;将所获得侧翼序列与灰葡萄孢基因组数据库中的已知基因序列进行BLAST分析,推测出T-DNA的插入位点;通过PCR进一步验证T-DNA的插入位点,利用RT-PCR技术确定突变基因;最后对突变菌株的菌落形态、生长速度、胞壁降解酶活力、粗毒素的生物活性、对番茄叶片的致病能力及部分致病相关基因的表达情况进行研究.[结果]TAIL-PCR结果证实T-DNA插入到灰葡萄孢BCIG 12707.1基因的ATG起始密码子区;RT-PCR结果证实突变基因为BCIG_12707.1,该基因DNA全长为135 bp,编码一个44个氨基酸的假定蛋白(Hypothetical protein).突变菌株在PDA培养基上菌落呈灰白色,生长速度减慢,不能产生分生孢子及菌核;对番茄叶片的致病性增强,且胞壁降解酶(PG、PMG和Cx)活力增强;突变菌株中参与细胞壁降解的角质酶基因cutA和多聚半乳糖醛酸酶基因Bepg1,信号转导途径基因(PKA1、PKA2、Bac、Bmp3),产毒素基因BcBOT2(Sesquiterpene synthase),漆酶基因Lac1,跨膜蛋白基因Btp1表达都增强.[结论]BC1G_ 12707.1基因在灰葡萄孢分生孢子产生、菌核形成及致病力等方面起重要作用.     

利用根癌农杆菌T-DNA 插入突变寻找参与漆酶葡萄糖阻遏的关键基因

【目的】新型隐球酵母(Cryptococcus neoformans)是人类重要致病真菌,主要毒性因子之一漆酶的表达受葡糖糖阻遏,机制未知。本文拟寻找参与葡萄糖阻遏的关键基因。【方法】建立根癌农杆菌介导的转化方法(Agrobacterium tumefanciens-mediated transformation,ATMT)建立一个容量约200000的随即插入突变文库,在高浓度葡萄糖条件下从中筛选葡萄糖去阻遏的突变株。通过Southern确定突变株中T-DNA的拷贝数,利用反向PCR获得依赖葡萄糖的漆酶阻遏基因序列。【结果】筛选到了30株葡萄糖去阻遏突变株,Southern blot发现83%的葡萄糖去抑制突变株含有单个T-DNA拷贝。初步鉴定了可能参与漆酶阻遏的10个不同生物学功能基因,如参与碳水化合物的代谢,固醇的合成,几丁质的合成,GPI脂锚钩的合成等等。【结论】ATMT突变策略可以找到一些参与漆酶葡萄糖阻遏的关键基因,为理解漆酶在致病过程中的作用机制和工业改进漆酶活性提供参考。     

Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana.

The purpose of this project was to identify large numbers of Arabidopsis genes with essential functions during seed development. More than 120,000 T-DNA insertion lines were generated following Agrobacterium-mediated transformation. Transgenic plants were screened for defective seeds and putative mutants were subjected to detailed analysis in subsequent generations. Plasmid rescue and TAIL-PCR were used to recover plant sequences flanking insertion sites in tagged mutants. More than 4200 mutants with a wide range of seed phenotypes were identified. Over 1700 of these mutants were analyzed in detail. The 350 tagged embryo-defective (emb) mutants identified to date represent a significant advance toward saturation mutagenesis of EMB genes in Arabidopsis. Plant sequences adjacent to T-DNA borders in mutants with confirmed insertion sites were used to map genome locations and establish tentative identities for 167 EMB genes with diverse biological functions. The frequency of duplicate mutant alleles recovered is consistent with a relatively small number of essential (EMB) genes with nonredundant functions during seed development. Other functions critical to seed development in Arabidopsis may be protected from deleterious mutations by extensive genome duplications.     

Characterization of a rice chlorophyll-deficient mutant using the T-DNA gene-trap system

We have previously generated a large pool of T-DNA insertional lines in rice. In this study, we screened those T-DNA pools for rice mutants that had defective chlorophylls. Among the 1,995 lines examined in the T2 generation, 189 showed a chlorophyll-deficient phenotype that segregated as a single recessive locus. Among the mutants, 10 lines were beta-glucuronidase (GUS)-positive in the leaves. Line 9-07117 has a T-DNA insertion into the gene that is highly homologous to XANTHA-F in barley and CHLH in ARABIDOPSIS: This OsCHLH gene encodes the largest subunit of the rice Mg-chelatase, a key enzyme in the chlorophyll branch of the tetrapyrrole biosynthetic pathway. In the T2 and T3 generations, the chlorina mutant phenotypes are co-segregated with the T-DNA. We have identified two additional chlorina mutants that have a Tos17 insertion in the OsCHLH gene. Those phenotypes were co-segregated with Tos17 in the progeny. GUS assays and RNA blot analysis showed that expression of the OsCHLH gene is light inducible, while TEM analysis revealed that the thylakoid membrane of the mutant chloroplasts is underdeveloped. The chlorophyll content was very low in the OschlH mutants. This is the first report that T-DNA insertional mutagenesis can be used for functional analysis of rice genes.     

快速筛选拟南芥受精和早期胚胎发生相关基因的方法

阐明拟南芥受精和早期胚胎发生过程对理解被子植物生殖发育有着重要的指导意义,而利用正向遗传学方法研究拟南芥突变体的表型及其分子机理是探究植物基因功能最常用的一种方法。基于常规的插入突变(包括T-DNA和转座子)、化学诱变(如ethylmethane sulfonate,EMS)和高能射线方法构建的突变体库中假阳性突变体多,难以高效筛选到受精和早期胚胎发生相关基因的突变体。为解决这一难题,本研究建立了一种构建T-DNA插入突变体文库的新方法。即在载体p CAMBIA1302的T-DNA元件上增加花粉特异荧光标记基因(p LAT52∷EGFP),并遗传转化具有四分体花粉的Columbia野生型拟南芥突变体qrt1-2;对获得的突变体库可利用花粉荧光快速排除假阳性突变体,并采用反向PCR(inverse-PCR)扩增技术确定突变位点。此方法在筛选拟南芥受精和早期胚胎发生相关基因突变体上的成功应用表明,其是一种效率高、针对性强、操作相对快捷方便的拟南芥突变体筛选方法。     

Cloning of rel from Listeria monocytogenes as an osmotolerance involvement gene

Transposon insertional mutants of Listeria monocytogenes were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained. The cloned gene from the transposon insertion site of the mutant, named rel, was 2,214 bp in length and had very high homology to relA of Bacillus subtilis, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively designated (p)ppGpp] synthetase during stringent response. The mutant showed a deficiency in (p)ppGpp accumulation. In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift. The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included rel of L. monocytogenes was introduced into the mutant. After exposure to methyl glucoside, the rel mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth. The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl. Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in L. monocytogenes and that its mechanism is different from that for the accumulation of compatible solutes.