两个紧密连锁的小麦苯丙氨酸解氨酶基因的分离与鉴定

利用一个小麦苯丙氨酸解氨酶基因PCR片段为探针,从小麦核DNA基因库中筛选出一个阳性噬菌体克隆,该克隆含有两个高度同源、紧密连锁、转录方向相同的小麦苯丙氨酸解氨酶基因PAL1与PAL2,它们之间的核酸序列同源性。为93％,相距约7kb,利用PAL1特异片段进行Southern分析,表明该基因在小麦基因组中具有多个拷贝。Northern杂交表明,经秆锈菌接种诱导,苯丙氨酸解氨酶基因在一对小麦抗-感近等基因系中差异表达：抗病等基因系中国春-Sr11携带与接种菌无毒性基因P11相对应的抗病基因Sr11,在接种4d后开始诱导表达,8d后表达量更高;而缺少抗病基因的感病系中国春-sr11接种6d后才开始表达,8d后的表达量与抗病系中6d时相当。用秆锈菌诱导物和几丁质寡聚物处理小麦悬浮细胞,均可在2h内激活苯丙氨酸解氨酶基因表达,但真菌诱导物在早期的诱导活性显著高于几丁质寡聚物。从转录水平证实了小麦苯丙氨酸解氨酶基因在秆锈菌诱导的抗性反应中具有重要作用。     

小麦几丁质酶基因的异种表达及其功能鉴定

几丁质酶参与植物的发育及防卫反应,并与人类疾病发生有关.文章研究了小麦几丁质酶基因Wch2经根癌农杆菌介导的烟草瞬间表达和转基因拟南芥的稳定表达,Western杂交及酶活测定证实,瞬间表达的小麦几丁质酶分子量约30 kD,具有降解几丁质多聚物的功能;Wch2在转入拟南芥后表达量高,尖孢镰刀菌接种的鉴定表明,表达Wch2的转基因植株的抗病性显著高于表达绿色荧光蛋白的对照植株.这些结果说明Wch2的异种表达,可用于植物抗病基因工程,以增强植物的抗病性.     

条锈菌诱导的小麦抗病与感病近等基因系SSH文库构建及分析

为分析条锈菌诱导下的小麦抗病与感病近等基因系之间差异表达的基因,以接种小麦条锈菌CY26小种的抗病近等基因系Yr4/6×Taichung 29幼苗叶片cDNA作为实验方,接种CY26的感病亲本Taichung 29幼苗叶片cDNA为驱动方,利用抑制消减杂交(SSH)技术构建了一个包含1 300余克隆的消减文库。对文库中600个克隆进行了反向Northern点杂交筛选,对获得的阳性克隆进一步进行了Northern杂交验证,获得显著差异的克隆12个。经测序和BlastX分析,其中6个差异表达序列的推测产物分别为亮氨酸重复序列蛋白、过氧化氢酶、硫氧还蛋白、RNA结合蛋白、抗坏血酸过氧化物酶和热激蛋白。除亮氨酸重复序列为信号传导类蛋白外、其他几个均为抗病防御类蛋白。     

小麦条锈菌PsCdc2基因的克隆及在条锈菌侵染小麦后的转录表达分析

【目的】克隆小麦条锈菌细胞分裂基因PsCdc2,分析该基因在条锈菌接种小麦后不同时间点的表达特征。【方法】利用PCR和RT-PCR技术克隆PsCdc2的cDNA序列和基因组序列,采用生物信息学技术预测分析该基因编码蛋白的保守结构域及基本特性,对该蛋白进行系统发育分析,构建进化树;运用实时荧光定量RT-PCR技术,以PsCdc2在夏孢子时期的表达情况为对照,分析该基因在亲和及非亲和互作中不同时间点的表达特征。【结果】PsCdc2基因组序列长2279 bp,由11个外显子和10个内含子构成,开放阅读框为885 bp,编码294个氨基酸,分子量为33.14 kDa,等电点为6.26。编码蛋白含两个保守的激酶特征位点,一个跨膜螺旋区域。PsCdc2基因编码蛋白与小麦秆锈菌、新型隐球菌、玉米瘤黑粉菌等多种真菌的Cdc2高度相似,其中与小麦秆锈菌的Cdc2亲缘关系最近,序列相似性达73.1%。实时荧光定量RT-PCR结果表明,在亲和组合中,该基因在条锈菌接种小麦的前期上调表达,其中接种后12 h时表达量最高,约为夏孢子中表达量的1.62倍,接种后24-268 h,基因表达基本呈下调趋势,其中96 h基因表达量最低,仅为夏孢子时期的0.07倍。在非亲和组合中,该基因表达基本呈下调趋势,在接种后各个时间点的表达量均低于在夏孢子中的表达量,其中接种后12 h时表达量最高,但仅为夏孢子中表达量的0.34倍;接种后96 h表达量最低,为夏孢子中表达量的0.02倍。【结论】PsCdc2可能通过调控条锈菌的细胞周期循环参与了侵染前期初生菌丝生长和吸器母细胞的形成,与条锈菌的致病性相关。本文首次报道了小麦条锈菌的Cdc2基因,为进一步揭示条锈菌细胞周期调控的本质及研究开发靶向Cdc2的新型农药,以及实现对小麦条锈病的新型药剂防治提供了理论基础。     

几丁质酶基因表达载体pET-chiB的构建及其在大肠杆菌中的诱导表达

几丁质酶在降解几丁质的过程中起着重要作用,目前人们已从不同微生物体中分离并克隆出了多种几丁质酶基因。实验以pET-22b( )为载体,利用从粘质沙雷氏菌(Serratia marcescens)克隆出的chiB基因,构建出原核生物表达载体pET-chiB。通过表达载体pET-chiB的诱导表达,实验结果显示该基因表达的蛋白为可溶性蛋白,其分子量约为52 kD。利用不同参数包括时间、IPTG浓度和温度诱导表达载体pET-chiB表达并对表达产物进行SDS-PAGE分析,结果显示其诱导表达的最佳参数分别为4 h,0.5 mmol/L和25℃。这些结果为几丁质酶基因的进一步研究和几丁质酶工程菌生产奠定了良好的工作基础。     

一株拮抗香蕉枯萎病的内生细菌的分离及其几丁质酶基因信号肽的分泌活性分析

目的：旨在分离并选择一株香蕉内生细菌作为内生基因工程生防菌,并克隆其几丁质酶基因的信号肽序列。方法：从香蕉植株杆下部分离并选择了一株拮抗香蕉枯萎病且具有分泌几丁质酶能力的内生细菌,对该菌株进行了形态观察、生理生化测定和16S rDNA序列分析,克隆了其几丁质酶基因的编码序列并预测了其信号肽,构建了含有信号肽和不含信号肽的几丁质酶的表达菌株BL-chi1和BL-chi2。结果：结合形态观察、生理生化特征和16S rDNA序列比对分析确定该菌株为Klebsiella属,将该菌株命名为KKWB 5;BL-chi1和BL-chi2经IPTG诱导后,均表达了与预期蛋白大小一致的蛋白,同时BL-chi1诱导后的培养基上清中出现一条约45kDa的条带,而BL-chi2和空载体的BL-pET22b诱导后的培养基上清中均无此条带;几丁质水解试验发现,BL-chi1诱导后的培养基上清中的蛋白经浓缩和纯化后都能在几丁质平板上形成透明水解圈。结论：该几丁质酶的信号肽能被BL21（DE3）所识别,将几丁质酶分泌到培养基中,并且分泌的几丁质酶具有水解几丁质的生物学活性。内生菌KKWB-5的分离及其几丁质酶分泌信号肽序列的克隆为进一步构建内生工程菌来防治香蕉枯萎病打下了基础。     

荒漠甲虫小胸鳖甲几丁质酶基因MpCht8c的克隆、鉴定与低温表达

为了挖掘荒漠昆虫小胸鳖甲Microdera punctipennis的耐寒性相关基因,从其4℃转录组数据库筛选出差异表达的几丁质酶基因片段394seq2,检测其编码蛋白的几丁质酶活性,研究该基因的表达对低温胁迫的响应情况。采用RACE技术扩增394seq2的5′端和3′端,克隆全长序列,将其ORF构建至原核表达载体pET28a,导入Transetta(DE3)感受态细胞,诱导表达融合蛋白,Western blot法检测表达蛋白的正确性;二硝基水杨酸法测定几丁质酶活性,qRT-PCR技术探究低温表达谱。结果表明,394seq2的5′-UTR为39 bp,3′-UTR为181 bp;ORF为1 140 bp。系统进化树显示该序列与赤拟谷盗几丁质酶8(TcCHT8)聚为一支,命名为MpCht8c。MpCht8c编码379个氨基酸,分子量为41.2 kDa,理论等电点pI为4.67。MpCHT8c含有完整的几丁质酶结构基序,其N端含有几丁质酶催化域,内有一个类18家族几丁质酶的保守基序KXXXXXGGW,中部是一段富PEST的连接区,C端是几丁质酶结合域,属于IV型昆虫几丁质酶。Western blot结果表明His-MpCHT8c在大肠杆菌中正确表达;融合蛋白粗酶液的几丁质酶活性为1.89 U/mL。在4℃冷胁迫0.5 h和5-9 h时Mpcht8c出现两个上调表达峰值,约为对照的2倍。研究表明荒漠昆虫小胸鳖甲的几丁质酶MpCHT8c具有几丁质酶活性,MpCht8c基因的表达可快速响应4℃低温胁迫。研究结果有助于深入研究几丁质酶在小胸鳖甲耐寒性方面的作用机理。     

一种新的抗真菌几丁质酶基因的分离及其植物表达载体的构建

以西瓜尖镰孢菌诱导、提纯的豇豆抗真菌 I类几丁质酶 N端前 1 0个氨基酸序列测定的基础上 ,设计合成了引物 ,运用 PCR等分子生物学技术 ,从豇豆基因组中分离克隆了该特异几丁质酶成熟蛋白基因 ,测定分析了其全序列。该新基因全长 894bp,无内含子 ;具 Aat I、Aat II、Bgl I、Dpn I、Dpn II、Eco R II、Hae I、Hae II、Hae III、Hinf I、Hpa II、Mae II、Mae III、Nba I、Oxa I和 Sst IV酶切位点 43个 ;豇豆、Vigna unguiculata、菜豆、豌豆、烟草、小麦、水稻的同源性依次递减。扩增克隆了菜豆几丁质酶信号肽基因 ,并将其与豇豆几丁质酶成熟蛋白基因连接 ,再与 p BI1 2 1重组 ,成功构建了特异几丁质酶基因的植物表达载体 ,为进一步培育抗真菌病转基因西瓜新品种打下了坚实基础。     

BTH对小麦叶片防卫相关酶的系统诱导作用

用0.4 mmol/L的苯并噻二唑(BTH)溶液处理小麦幼苗第1叶和第2叶2 d后接种白粉菌,比色法测定第3叶接种前后过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、几丁质酶和β-1,3-葡聚糖酶的活性,结果表明BTH处理或接种均可使这4种酶活性升高。BTH诱导酶活性的系统增强与小麦对白粉病的诱导抗性密切相关。     

杆状病毒几丁质酶基因结构与功能的研究进展

杆状病毒几丁质酶基因是杆状病毒的非必需基因,是高度保守的基因。该基因在杆状病毒复制晚期表达产生几丁质酶,该酶N端具信号肽,中部是酶的活性区,C端是酶的内质网结合区。杆状病毒几丁质酶同时具有内切和外切几丁质酶活性,主要功能是水解昆虫体内的组成型几丁质。杆状病毒几丁质酶对于虫体液化是必需的,同时它还是原组织蛋白酶(pro-V-Cath)的分子伴侣,并与病毒侵染机制相关联。杆状病毒的几丁质酶基因与细菌的几丁质酶基因可能源于共同的祖先。     

Rust resistance in Triticum cylindricum Ces. (4x, CCDD) and its transfer into durum and hexaploid wheats.

In order to counteract the effects of the mutant genes in races of leaf rust (Puccinia recondita f.sp. tritici Rob. ex Desm.) and stem rust (P. graminis f.sp. tritici Eriks. &Henn.) in wheat, exploration of new resistance genes in wheat relatives is necessary. Three accessions of Triticum cylindricum Ces. (4x, CCDD), Acy1, Acy9, and Acy11, were tested with 10 races each of leaf rust and stem rust. They were resistant to all races tested. Viable F1 plants were produced from the crosses of the T. cylindricum accessions as males with susceptible MP and Chinese Spring ph1b hexaploid wheats (T. aestivum, 6x, AABBDD), but not with susceptible Kubanka durum wheat (T. turgidum var. durum, 4x, AABB), even with embryo rescue. In these crosses the D genome of hexaploid wheat may play a critical role in eliminating the barriers for species isolation during hybrid seed development. The T. cylindricum rust resistance was expressed in the F1 hybrids with hexaploid wheat. However, only the cross MP/Acy1 was successfully backcrossed to another susceptible hexaploid wheat, LMPG-6. In the BC2F2 of the cross MP/Acy1//LMPG-6/3/MP, monosomic or disomic addition lines with resistance to either leaf rust race 15 (infection types (IT) 1=, 1, or 1+; addition line 1) or stem rust race 15B-1 (IT 1 or 1+; addition line 2) were selected. Rust tests and examination of chromosome pairing of the F1 hybrids and the progeny of the disomic addition lines confirmed that the genes for rust resistance were located on the added T. cylindricum C-genome chromosomes rather than on the D-genome chromosomes. The T. cylindricum chromosome in addition line 2 was determined to be chromosome 4C through the detection of RFLPs among the genomes using a set of homoeologous group-specific wheat cDNA probes. Addition line 1 was resistant to the 10 races of leaf rust and addition line 2 was resistant to the 10 races of stem rust, as was the T. cylindricum parent. The added C-genome chromosomes occasionally paired with hexaploid wheat chromosomes. Translocation lines with rust resistance (2n = 21 II) may be obtained in the self-pollinated progeny of the addition lines through spontaneous recombination of the C-genome chromosomes and wheat chromosomes. Such translocation lines with resistance against a wide spectrum of rust races should be potentially valuable in breeding wheat for rust resistance.     

Heterologous expression of genes mediating enhanced fungal resistance in transgenic wheat.

Three cDNAs encoding the antifungal protein Ag-AFP from the fungus Aspergillus giganteus, a barley class II chitinase and a barley type I RIP, all regulated by the constitutive Ubiquitin1 promoter from maize, were expressed in transgenic wheat. In 17 wheat lines, stable integration and inheritance of one of the three transgenes has been demonstrated over four generations. The formation of powdery mildew (Erysiphe graminis f. sp. tritici) or leaf rust (Puccinia recondita f. sp. tritici) colonies was significantly reduced on leaves from afp or chitinase II- but not from rip I-expressing wheat lines compared with non-transgenic controls. The increased resistance of afp and chitinase II lines was dependent on the dose of fungal spores used for inoculation. Heterologous expression of the fungal afp gene and the barley chitinase II gene in wheat demonstrated that colony formation and, thereby, spreading of two important biotrophic fungal diseases is inhibited approximately 40 to 50% at an inoculum density of 80 to 100 spores per cm2.     

Transient transformation of the rust fungus Puccinia graminis f. sp. tritici

The biotrophic rust fungus Puccinia graminis f. sp. tritici (Pgt) was transformed by particle bombardment. The promoter from the Pgt translation elongation factor 1alpha (EF-1alpha) gene was fused to the bacterial marker genes hygromycin B phosphotransferase (hpt) and beta-glucuronidase (GUS). Transformation constructs were introduced into uredospores of Pgt, an obligate pathogen of wheat, by biolistic bombardment. Uredospores transformed with the construct containing the hpt gene germinated and initiated branching on selective medium, indicating that they had acquired resistance to hygromycin B. However, transformants stopped growing 5 days after bombardment. GUS activity in uredospores and germlings was histochemically detected 4-16 h after bombardment. GUS expression was also obtained using the INF24 promoter from the bean rust fungus Uromyces appendiculatus, demonstrating that heterologous genes can be expressed in P. graminis under the control of regulatory sequences from closely related organisms.     

Inheritance and expression of stripe rust resistance in common wheat (Triticum aestivum) transferred from Aegilops tauschii and its utilization

Stripe rust is one of the most destructive diseases for wheat crops in China. Two stripe rust physiological strains, i.e. CYR30 (intern. name: 175E191) and CYR31 (intern. name: 293E175) have been the dominant and epidemic physiological strains since 1994. One Aegilops tauschii accession (SQ-214) from CIMMYT was found immune from or highly resistant to Chinese new stripe rust races CYR30 and CYR31 at adult stage. SQ-214 was crossed with a highly susceptible Ae. tauschii accession As-80. Analysis of data from F1-F2 populations of SQ-214/As-80 revealed that the resistance was controlled by a single dominant gene. To exploit the resistance for wheat breeding, SQ-214 was crossed with Chinese Spring (CS) and backcrossed by two Chinese commercial wheat varieties MY26 and SW3243. The resistance from SQ-214 was suppressed in the F1 hybrids (CS/SQ-214) and the F2 population of CS/SQ-214//MY26. However, the resistance of SQ-214 was expressed in several F2 individuals of CS/SQ-214//SW3243. Eleven advanced lines with high level of resistance to the Chinese stripe rust CYR30 and CYR31 have been developed. This result suggested that SW3243 does not suppress the expression of the Chinese stripe rust and should be used as wheat germplasm for exploiting resistance of Ae. tauschii in wheat breeding. The gliadin electrophoretic pattern of the eleven advanced lines with high stripe rust resistances was compared with their parents SQ-214, CS and SW3243 by acid polyacrylamide gel electrophoresis. The omega-gliadin bands of Gli-Dt1 in Ae. tauschii SQ-214 were transferred to some advanced lines and freely expressed in common wheat genetic background. One of advanced lines possesses a null Gli-D1 allele, where the omega-gliadin bands encoding by the Gli-D1 allele were absent. The potential utilization of this advanced line for wheat quality and stripe rust resistance breeding is also discussed in this paper.     

Targeted spatio-temporal expression based characterization of state of infection and time-point of maximum defense in wheat NILs during leaf rust infection

Leaf rust, caused by the fungus Puccinia triticina, is the most devastating disease of wheat worldwide, which sometimes becomes epidemic. The pathogen evolves into new strains, making its control difficult. Though more than 60 leaf rust resistant genes are now known, only limited insight is available into the molecular mechanism involved in this host pathogen interaction. In the present study, quantitative real-time PCR based differential gene expression profiling was examined for five target genes encoding for chitinase3, β-1,3/1,4 glucanase, thaumatin-like protein, peroxidase2 and mitogen activated protein kinase1 to unravel their coordinated action during compatible and incompatible interaction, to inhibit the pathogen progression and to identify the time-period of maximum defense activity. Spatio-temporal expression profiling suggested that the maximum defense activity occurred at 12-24?hours post inoculation, whereas the state of infection and degree of resistance was predicted using coordinated unique expression signatures of target genes. The significant differences of targeted gene expression between resistant mock inoculated, resistant infected and susceptible infected plants were evaluated using t test at significance level of p?相似文献   

Identification of markers linked to the race Ug99 effective stem rust resistance gene Sr28 in wheat (Triticum aestivum L.)

Wheat stem rust caused by Puccinia graminis f. sp. tritici can cause devastating yield losses in wheat. Over the past several decades, stem rust has been controlled worldwide through the use of genetic resistance. Stem rust race TTKSK (Ug99), first detected in Uganda in 1998, threatens global wheat production because of its unique virulence combination. As the majority of the currently grown cultivars and advanced breeding lines are susceptible to race TTKSK, sources of resistance need to be identified and characterized to facilitate their use in agriculture. South Dakota breeding line SD 1691 displayed resistance to race TTKSK in the international wheat stem rust nursery in Njoro, Kenya. Seedling screening of progeny derived from SD 1691 crossed to susceptible LMPG-6 indicated that a single resistance gene was present. Allelism and race-specificity tests indicated the stem rust resistance gene in SD 1691 was Sr28. The chromosome arm location of Sr28 was previously demonstrated to be 2BL. We identified molecular markers linked to Sr28 and validated this linkage in two additional populations. Common spring wheat cultivars in the central United States displayed allelic diversity for markers flanking Sr28. These markers could be used to select for Sr28 in breeding populations and for combining Sr28 with other stem rust resistance genes.     

Proteins in intercellular washing fluid from noninoculated and rust-affected leaves of wheat and barley

Proteins in intercellular washing fluid (IWF) from wheat (Triticum aestivum) and barley (Hordeum vulgare) leaves were separated by two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue (CBB) or silver. Intracellular protein from the cut ends of leaves accounted for only a small proportion of total protein in IWF from wheat leaves. When these were heavily infected with the stem rust fungus (Puccinia graminis f. sp. tritici) and grown at 19°C, four infection-related CBB-stainable proteins were detected in IWF.

To compare IWF proteins from wheat and barley leaves infected with the same pathogen, conditions were established that permitted luxuriant growth of stem rust of wheat in barley (exposure to chloroform before inoculation and maintenance at 25°C thereafter). Under these conditions, at least 10 infection-related silver-stainable proteins were detected in IWF from infected wheat in addition to the more than 50 that were of host origin. The electrophoretic properties of 8 of the infection-related proteins were the same as those of 8 infection-related proteins in IWF from barley.

IWF from wheat and barley grown under these conditions was analyzed for Concanavalin A-binding glycoproteins immobilized on nitrocellulose membrane replicas made from gels. Of the many infection-related glycoproteins that were detected in IWF from stem rust-affected wheat, approximately 20 occupied the same positions as those from stem rust-affected barley. The glycoprotein pattern of IWF prepared from wheat leaves grown at 19°C and infected with the leaf rust fungus (P. recondita f. sp. tritici) was markedly different to that of IWF from the same host infected with the stem rust fungus. We conclude that IWF from rust-affected cereal leaves may be a useful source of surface or extracellular proteins from the parasitic mycelium.