菌寄生真菌纤细齿梗孢一丝氨酸-苏氨酸蛋白激酶基因OPK1的克隆及序列分析

 Using degenerate PCR and TAIL-PCR, a protein kinase gene OPK1 (Genebank accession No.:EU417815) was cloned from mycoparasite fungi Olpitrichum tenellum. OPK1 has an open reading frame of 2 031 bp interrupted by two introns (108 bp, 84 bp) and putatively encodes a protein of 676 aa. Phylogenetic analysis indicated that OPK1 was most similar to other serine-threonine protein kinase in fungi. Southern blot analysis indicated that OPK1 is present as a single copy in the genome. RT-PCR showed it could be transcribed both in the phase of spore germination and hyphal growth.     

梨抗黑星病AFLP标记筛选

 Pear scab caused by Venturia nashicola is one of the most destructive diseases of pears. Molecular markers linked to scab resistance gene is expected to be useful for improving pear. In this study, the F₁ population derived from the cross of ‘Huangguan’ and ‘Yali’ was analyzed genetically. The resistance of pear to scab was proved to be controlled by a single gene in a dominant manner. Bulked segregant analysis (BSA) was conducted to screen 64 fluorescent AFLP primer pairs. A marker designated as D3-365 was found to be linked to the resistant locus. Selective genotype linkage analysis showed that the genetic distance between the marker and the resistant locus was 14.9 cM.     

小麦抗白粉病基因Pm6的微卫星标记鉴定

 Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici, is a prevalent disease worldwide. Breeding and planting resistance cultivars have been proved effective and environmental friendly for control of the disease. To develop easily used PCR-based markers in marker assisted selection (MAS) for Pm6, a dominant powdery mildew resistance gene in wheat, 25 microsatellite markers on chromosome 2BL in wheat were screened between susceptible parent Yumai13 and resistance parent Timgalen carrying Pm6. F₂ population derived from Yumai13 and Timgalen was further analyzed by the marker Xgwm501. The results indicated that Xgwm501 was a co-dominant marker linked to Pm6 gene at a distance of 14.8 cM. 29 Pm-carrying varieties were tested by the marker Xgwm501 and only those carrying Pm6 showed 117 bp resistance specific band. This marker is proved to have high practicability and can be used in MAS of Pm6 gene in wheat breeding programs.     

水稻条纹病毒NS2基因原核表达产物多克隆抗体制备及受侵染水稻和灰飞虱体内NS2检测

 The NS2 gene of Rice stripe virus (RSV) was amplified by RT-PCR, cloned into pGEM-T vector and sequenced. The NS2 gene was inserted into prokaryotic expression vector pET32a to produce recombinant plasmid pET32a-NS2. The recombinant plasmid was introduced into Escherichia coli strain BL21 (DE3) pLysS. SDS-PAGE and Western blot analysis confirmed that NS2 fusion protein was expressed after induction by IPTG. The recombinant NS2 protein was purified with Ni²⁺-NTA agarose affinity chromatography and the polyclonal antibody against NS2 protein was raised in rabbit. NS2 protein was successfully detected in small brown planthopper (Laodelphax striatellus) at 1:1 600 dilution of the total protein of single planthopper and in infected rice (Oryza sativa) at 1:800 dilution of 10 mg leave by dot immunobinding assay using the polyclonal antibody.     

小麦-滨麦易位系M8657-1抗条锈病基因遗传分析和分子标记

 M8657-1, one of the wheat translocation lines derived from Leymus mollis Trin. Hara, is possessed of effective resistance at all stages to Su-ll and other dominant races of Puccinia striiformis f. sp. tritici in China. Seedlings of the parents, F₁, and F₂ progeny derived from the cross of M8657-1 (resistant) Mingxian169 (susceptible) were inoculated with Su-ll in greenhouse to identify and map the probable new stripe rust resistance gene. The results suggested that the stripe rust resistance in M8657-1 was conferred by a pair of recessive genes. Simple sequence repeat (SSR) technique was used to detect molecular marker associated with the resistance gene:208 pairs of wheat SSR primers were used to screen the two parents, as well as resistant and susceptible bulks and then three SSR markers were selected for genotyping the F₂ population. The geue, temporarily designated as YrLml, was found to be located on the chromosome 7DL and flanked by three SSR markers GDM67, WMC150 and WMC671, with the genetic distance of 5.0, 9.7 and 11.8cM, respectively.     

壳寡糖诱导烟草防御酶系活性变化及PR-1a基因表达研究

 The activity change of defensive enzymes and PR-1a gene expression of tobacco (Nicotiana tabacum) seedling induced by chito-oligosaccharides were studied. The results showed that high level systemic acquired resistance (SAR) was expressed in tobacco plants treated with chito-oligosaccharides solution at the concentration of 50 μg/mL. PAL activity increased greatly with 2 peaks, the activity of SOD decreased initially followed by an increase with higher increment, and the activity of POD peaked early followed by a gentle fall in chito-oligosaccharide treated plants. The PR-1a gene was strongly expressed in tobacco due to systemic acquired resistance induced by chito-oligosaccharides. At 168 h after inoculation the expression quantity (co-pies/2 μL) of PR-1a gene was increased to 2 469.6 in treated tobacco leaf, reached 392.6% than that at 0 h after inoculation, it was increased 3.05 times of that in untreated control.     

分子标记辅助选择小麦抗白粉病兼抗赤霉病聚合体

 Sumai 3, a wheat variety resistant to Fusarium head blight(FHB), was crossed with Neimai 9, a commercial wheat cultivar with the resistance to powdery mildew.The SCAR(sequence characterized amplified region) markers of powdery mildew resistance gene Pm21 and four SSR(simple sequence repeats)markers flanking the major FHB resistance QTL(Qfhs.ndsu-3BS) in Sumai 3 were used to detect the resistance loci by marker assisted selection(MAS) in the plants of the F₂ population.Identification of resistance to both powdery mildew and FHB in field showed that 12 plants resistant to both diseases were obtained.In addition, the agronomic traits of these plants were better than those of Sumai 3, and are perhaps the excellent parental materials for wheat breeding.     

香蕉线条病毒ORFⅡ基因的原核表达及抗体制备

 ORFⅡ gene of Banana streak virus GuangDong isolate (BSV-GD) was amplified from a BSV-GD recombinant plasmid by PCR, and the gene was expressed by being cloned into prokaryote expression vector pET-28b (+). The fusion protein was about 16.5 kDa in size and was soluble with SDS-PAGE analysis. The purified protein was obtained by using the histidine labeling kit of N-terminus of protein. The antiserum was obtained by immunizing healthy rabbits with the purified protein. Western blot and ELISA analysis showed that the special antiserum of BSV possessed high titer, which was tested as 1:51 200. The study was a base for further research on BSV including ORFⅡ gene function and virus detection.     

一个小麦茉莉酮酸酯诱导蛋白基因的克隆和鉴定

 用基因芯片技术结合分池法(bulked segregating analysis,BSA)对参与小麦(Triticum aestivum L.)"兰考90(6)"抗白粉病反应或与抗病基因连锁的EST进行了分析。从"中国春"中克隆了一个与Ta-JA1高度相似的新的小麦茉莉酮酸酯诱导蛋白基因(GenBank登录号:EU035635),命名为Ta-JA2。Ta-JA2和Ta-JA1的cDNA序列有99%相同,编码304个氨基酸组成的多肽。Ta-JA2具有植物病原应答诱导蛋白-dirigent-类蛋白的典型保守功能域和jacalin-类植物血球凝集素的典型保守功能域。Ta-JA2主要在叶片和茎中表达,在根和幼穗中几乎不表达;在幼叶、壮叶和旗叶中的表达水平依次增强;在"中国春"和一个二粒小麦(Triticum dicoccoides)品系幼叶中的表达受白粉菌(Blumeria graminis f.sp.tritici)的诱导而增强;在"兰考90(6)21-12"叶片中的表达保持稳定而较高的水平。根据氨基酸序列的相似性建立了Ta-JA2类似蛋白的树形图,提供了植物中此类基因的进化信息。     

小麦凝集素类受体激酶TaLecRLK1的鉴定及抗锈病功能分析

凝集素类受体激酶(lectin receptor-like kinase, LecRLKs)是一类在植物应答多种生物/非生物胁迫中发挥重要作用的类受体激酶。本研究在小麦与条锈菌互作的转录组中筛选到1个在小麦与条锈菌非亲和互作中显著上调表达的LecRLKs基因TaLecRLK1。该基因全长2 292 bp,编码蛋白含有1个胞外信号肽、B-lectin结构域、PAN＿AP结构域、跨膜域和胞内酪氨酸激酶域。qRT-PCR分析表明：TaLecRLK1在小麦与条锈菌非亲和互作早期诱导表达,在烟草及小麦原生质体中瞬时表达,TaLecRLK1-GFP定位在细胞膜上。利用大麦条纹花叶病毒介导的基因沉默技术(BSMV-VIGS)沉默TaLecRLK1,接种无毒性条锈菌小种CYR23后,沉默叶片表面产生少量夏孢子堆,组织学观察发现沉默植株中条锈菌菌丝长度增长、侵染点附近活性氧积累面积减少;TaPR1、TaPR2、TaPR5的表达受到抑制,TaCAT和TaSOD则被迅速诱导表达。综上所述,TaLecRLK1对小麦抗条锈病起到正调控作用。     

来自簇毛麦抗条锈病新基因的SSR标记

 用小麦条锈菌条中30号生理小种,对小麦抗病种质小麦-簇毛麦易位系V9128-1和铭贤169的杂交后代进行抗条锈性遗传分析,小麦-簇毛麦易位系V9128-1的抗病性符合1对显性抗条锈病基因控制。并根据F₂抗、感病单株分离比例组建抗感池,用SSR技术寻找与抗病基因连锁的分子标记。从121个SSR引物组合中筛选到2个与抗病基因YrV1(暂命名)紧密连锁的微卫星标记Xgwm566和Xgwm376,遗传距离分别为3.6和5.5cM;因此,该抗条锈病基因位于小麦3B染色体短臂上。这2个标记不仅能在小麦-簇毛麦易位系V9128-1中检测到,而且在抗病基因供体亲本簇毛麦中也能检测到。综合抗病基因来源和分子生物学试验结果,可以推断,YrV1很可能是1个来自簇毛麦并与已知抗条锈病基因不同的新基因。     

β-氨基丁酸诱导烟草产生PR蛋白及对TMV的抑制作用

 本文研究了β-氨基丁酸(BABA)诱导系统侵染寄主烟草NC89产生PR蛋白及其对TMV的抑制作用。用5mmol/LBABA对抗性烟和非抗性烟进行叶面喷施处理均可以抑制烟草叶片中TMV的产生,平均抑制率分别达到59%和49%。BABA和SA处理均可以诱导2种烟草叶片中PR1、PR2和PR5基因的表达,其中对PR1的诱导作用相对较强。分别用BABA和SA处理非抗性烟草,在处理后的不同时间内2种药剂对PR1基因的诱导规律基本相同。用BABA和SA分别预处理非抗性烟草2d后接种TMV,2种药剂对烟草叶片中PR1基因的影响是完全不同的,其中,BABA处理后接种TMV抑制PR1基因的表达,在接种后第72h已经检测不到PR1基因的存在,SA处理后接种TMV对PR1基因的影响呈现弱-强-弱的变化趋势,在接种后第24hPR1的表达量达到最强,以后逐渐减弱。试验结果表明:BABA能够提高非抗性烟草对TMV的抗性,但是BABA诱导烟草产生抗TMV的作用机制可能是一种不同于目前普遍公认的SA的作用机制。     

番茄抗晚疫病基因Ph-3的分子标记开发及应用

正番茄晚疫病由卵菌疫霉属的致病疫霉菌(Phytophthora infestans)侵染引起。病菌对番茄致病性强,侵染寄主后通过气流散发孢子,多次重复侵染植株,引起晚疫病爆发,对番茄生产造成严重危害[1]。利用抗晚疫病资源,培育番茄抗病品种是降低晚疫病危害的有效途径。在野生番茄中发现     

小簇麦易位系V9128-3抗条锈病基因的遗传分析和SSR分子标记

 用7个我国当前流行的条锈菌生理小种对V9128-3的抗条锈性进行了评价,表明本易位系对我国优势流行小种具有良好的抗病性。以Su-4对V9128-3与铭贤169配置的F₁、BC₁F₁、F₂及F₃代群体进行了遗传分析,并对其中1个F₂群体进行了SSR标记,再用BC₁F₁群体的部分单株和F₃家系进行连锁标记的初步验证。遗传分析表明了V9128-3对Su-4的抗病性由1对显性核基因独立控制,从219对SSR引物中筛选到2个位于2AL上的该基因YrHV(暂命名)两侧的标记Xgwm356和Xwmc658,遗传距离分别为8.5和5.6cM,所用部分BC₁F₁单株和F₃家系验证了该2个标记与YrHV连锁性。将此标记可用于小麦抗条锈病分子标记辅助育种。     

抗赤霉病普通小麦-大赖草易位系T5AS/5LrL的分子细胞遗传学鉴定

 大赖草作为小麦野生近缘植物,对赤霉病表现较好的抗性, 将其赤霉病抗性基因转入普通小麦, 对创新小麦赤霉病抗性种质有重要意义。本研究在获得抗赤霉病普通小麦-大赖草异附加系基础上, 采用1200R ⁶⁰Co-γ射线处理小麦-大赖草二体附加系 DA5Lr花粉, 授予已去雄的普通小麦中国春,对其后代(M1)种子根尖细胞有丝分裂中期染色体进行GISH分析,获得了1株具有1条普通小麦-大赖草易位染色体的植株,让其自交,对自交后代中具有2条易位染色体植株的花粉母细胞减数分裂中期I进行观察,发现2条易位染色体形成了稳定的环状二价体,表明该植株为纯合体。利用顺序GISH-双色FISH分析,结合小麦D组专化探针Oligo-pAs1-2和B组专化探针Oligo-pSc119.2-2,进一步鉴定出该普通小麦-大赖草易位系为T5AS/5LrL,且筛选出了可追踪该易位系的3个EST-STS分子标记BE591127 、BQ168298和BE591737。赤霉病抗性鉴定结果表明,易位系T5AS/5LrL连续3年的病小穗率分别为7.69%、10.29%和8.66%,显著低于感病品种中国春和绵阳85-45。该易位系的育成为小麦赤霉病抗病性遗改良提供了新种质。     

大豆斑疹病菌胞外纤维素酶编码基因的鉴定及其调控分析

 植物病原黄单胞菌的胞外纤维素酶基因具有差异性和多样性。大豆斑疹病菌(Xanthomonas axonopodis pv. glycines,Xag)具有胞外纤维素酶活性,但其编码基因和调控特性尚不明确。为了鉴定Xag的胞外纤维素酶编码基因,本研究从NEAU001菌株基因组内筛选出6个候选的纤维素酶编码基因。通过异源表达实验发现,engXCA和egl2基因分别编码的蛋白EngXCA和Egl2具有水解羧甲基纤维素的能力。酶活性测定实验表明,engXCA和egl2基因决定Xag的胞外纤维素酶活性。另外,对已知的10个毒性调控基因突变株进行胞外纤维素酶活性测定,揭示Xag胞外纤维素酶活性受DSF(diffusible signal factor)信号通路和全局性调控子Clp(Crp-like protein)正调控。荧光定量PCR结果进一步证实,RpfF和Clp参与正调控engXCA和egl2基因的mRNA水平。这些结果暗示,DSF信号通路可能通过Clp调控engXCA和egl2基因的表达,实现对Xag胞外纤维素酶活性的调控。     

菰黑粉菌MAPK同源基因UeKss1的克隆及表达

菰黑粉菌作为茭白植株体内的内生真菌,其二型态转换与茭白孕茭密切相关,而MAPK途径在真菌二型态转换中具有重要调控作用。本研究在菰黑粉菌中克隆得到了一个MAPK基因UeKss1,通过与酵母菌中的MAPK途径蛋白进行聚类分析表明其属于Kss1的同源蛋白。进一步的系统进化分析表明,UeKss1与黑粉菌属真菌的Kss1同源性最高,达80%以上,且它的丝/苏氨酸双特异性蛋白激酶催化结构域高度保守。不同碳源诱导下菰黑粉菌的生长特征及UeKss1表达分析发现,只有蔗糖培养基能诱导菰黑粉菌菌丝形成,而在PDA、葡萄糖和麦芽糖培养基中,该菌都保持酵母型生长;UeKss1基因的表达量在PDA和葡萄糖培养基中变化不显著,但在麦芽糖培养基中,该基因的表达随着培养时间的延长持续上调表达,而在蔗糖培养基中,UeKss1的表达量虽然也呈现上升趋势,但是远小于麦芽糖的诱导表达量。对UeKss1进行的原核表达纯化,经Western blot验证得到纯度较高的UeKss1蛋白。研究结果可为UeKss1基因的功能研究,阐明其在菰黑粉菌二型态转换中的作用机制提供帮助。     

三个小麦野生近缘种抗条锈性传递的初步研究

 以当前小麦条锈病菌的优势小种条中29号、30号和31号测定了小麦近缘植物长穗偃麦草、簇毛麦和华山新麦草及其各自与小麦的杂交后代的抗条锈性。试验结果表明,3个小麦近缘植物均含有宝贵的抗条锈基因,此类基因具有较强的传递性能,可在小麦遗传背景下高度表达,表现出良好的抗条锈性能,具有广阔的应用前景。