以潮霉素B抗性为选择标记的深黄被孢霉原生质体转化

利用亚硝基胍(MNNG)诱变方法筛选了一株深黄被孢霉潮霉素B敏感型菌株M6-22-4。采用PEG介导的方法,将含有E.coli潮霉素B抗性标记的PD4质粒转入敏感株M6-22-4原生质体,并在潮霉素B浓度为400μg/mL的选择培养基上筛选转化子,获得了1.6~2.8个转化子/μg质粒DNA的转化频率。稳定性实验表明,质粒线性化后所获得的转化子在PDA培养基上传代10代以后,转接到选择平板上有31.6%仍具有HmB抗性;随机挑选了3个转化子,通过PCR方法检测到潮霉素抗性基因的存在,Southern杂交发现,潮霉素抗性基因已经以1~2拷贝数整合到深黄被孢霉M6-22-4染色体上,这是深黄被孢霉转化系统的首次报道。     

以潮霉素B抗性为选择标记的深黄被孢霉原生质体转化

利用亚硝基胍（MNNG）诱变方法筛选了一株深黄被孢霉潮霉素B敏感型菌株M6-22-4。采用PEG介导的方法,将含有E.coli潮霉素B抗性标记的PD4质粒转入敏感株M6-22-4原生质体,并在潮霉素B浓度为400μg/mL的选择培养基上筛选转化子,获得了1.6～2.8个转化子/μg质粒DNA的转化频率。稳定性实验表明,质粒线性化后所获得的转化子在PDA培养基上传代10代以后,转接到选择平板上有31.6%仍具有HmB抗性;随机挑选了3个转化子,通过PCR方法检测到潮霉素抗性基因的存在,Southern杂交发现,潮霉素抗性基因已经以1～2拷贝数整合到深黄被孢霉M6-22-4染色体上,这是深黄被孢霉转化系统的首次报道。     

粟长蠕孢菌的原生质体转化

本文报道了利用具有潮霉素抗性标记的质粒(pAN7-1)对粟长蠕孢菌原生质体进行转化的结果。经pAN7-1质粒DNA转化处理的粟长蠕孢菌原生质体在含潮霉素(200μg/ml)的选择性培养基上出现两类转化子。一类是正常转化子,其转化率为2个转化子/μg DNA;另一类是流产转化子,其产生频率为500—600个转化子/μg DNA。DNA杂交分析结果表明,在正常转化子中质粒DNA以首尾相接、重复排列的形式整合入受体菌染色体DNA。初筛获得的转化子多数以异核状态存在,经单孢分离纯化后可通过有丝分裂稳定传代。     

携带潮霉素抗性基因的质粒pDH25转化灰霉菌研究

由丝状真菌葡萄孢属(Botrytis)引起的灰霉病在多种植物生长期和贮存期的重要病害。洋葱灰霉病菌(B. squamosa)因在致病性和生活史方面的独特表现已为研究者所注意。建立这一病菌的转化系统将为进一步研究致病性分子遗传创造条件。 丝伏真菌转化研究始于1979年,Case等首次证实外源DNA同粗糙脉孢菌(Neurospora carassa)染色体基因组整合。     

赤霉菌原生质体DNA转化

DNA转化是基因表达和其它分子生物学研究的一项重要内容。原核生物（如细菌）的DNA转化技术已相当成熟,作为真核生物的丝状真菌其DNA转化过程要相对复杂一些,且转化效率一般也不如细菌的高。但丝状真菌DNA转化的研究对于深入探讨真菌基因表达调控的机理、尤其是研究外源基因在丝状真菌中的表达有着重要意义（唐国敏,1992）。赤霉菌（Gibberellajirjtheroi）在实验室条件下易培养、生长快,是研究赤霉素储类生物合成途径的必需材料,在作为表达体系表达外源基因尤其是储类合成的关键基因方面亦有着潜在的用途。但作为赤霉菌基础研究…     

利用分子标记辅助选择改良珍汕97的稻瘟病抗性

利用回交育种中产生的回交群体，结合前人的研究结果构建了Pi1基因区域的局部分子标记连锁图，通过BC1F2家系的接种结果判断其基因型。将Pi1定位在RFLP标记RZ536与SSR标记RM144之间，图距分别为9．7cM、6．8cM，从而建立了一套完整的以PCR为基础的分子标记辅助选择体系。通过分子标记和抗性验证两种选择方式相结合，经过三代回交将Pi1区段快速导入受体亲本珍汕97B中。在BC3F1中利用15条ISSR引物扩增的167条随机分布在基因组中的多态性带筛选背景，得到4个背景较好的单株。经过纯合筛选及抗性验证后共得到17个带有抗性基因Pi1的改良珍汕97株系。试验表明微卫星标记在正向选择、负向选择及背景选择中都起到极大的作用。     

云南疣粒野生稻稻瘟病抗性

野生稻(Oryza rufipogo)保存有许多栽培稻(O. sativa)不具备或已经消失的优异基因资源, 是扩大栽培稻遗传背景、改良产量与品质、提高抗病虫害及抗逆境能力的重要基因库。疣粒野生稻(O. meyeriana)是中国3种野生稻资源之一, 主要分布在云南。为进一步了解其稻瘟病抗性, 首先利用来自不同稻作区的稻瘟病菌株, 通过注射接种法对疣粒野生稻进行系统的稻瘟病抗性鉴定, 发现疣粒野生稻对接种的所有稻瘟病菌株都感病。进一步采用3'/5' RACE方法, 从疣粒野生稻中克隆了水稻同源基因Pid2和Pid3, 并构建过表达转基因株系对基因功能进行了研究。结果表明, Pid2和Pid3与疣粒野生稻中同源基因间在DNA和氨基酸水平上有较大的序列差异, 过表达转基因的日本晴植株对稻瘟病菌的敏感性与对照相似。推测疣粒野生稻在自然接种条件下, 表现出的抗稻瘟病表型很可能是其旱生叶片结构特征形成了对稻瘟病菌侵染的天然屏障。对控制疣粒野生稻这一类性状基因资源的挖掘和利用, 有利于优良抗性水稻品种的培育。研究结果为疣粒野生稻的研究利用提供了新信息和新思路。     

小麦多小穗品系10-A单株籽粒产量染色体效应的研究

本文以不分枝普通多小穗品系10-A为被测材料,中国春和阿勃两套单体系列为测验系,对单株籽粒产量进行了单体分析。结果表明,被测系10-A单株籽粒产量受5A、7A、1B、2B、6B、2D和7D等染色体上的基因所控制。其中,5A、7A、2D和7D染色体上的基因表现为增效,1B、2B和6B染色体上的基因表现为减效。就作用强度而言,7A、1B、2D染色体上的基因表现为强效,5A、2B、6B和7D染色体上的基因表现为弱效。Abstract:Monosomic analysis of grain yield per plant was carried out in the common wheat (Triticum aestivam L.) multispikelet line 10-A, by using the monosomic series of the two regular lines, “Chinese Spring ” and “Abbondanza”. The grain yield per plant of the tested line 10-A was found to be controlled by seven pairs of genes, located on the chromosomes 5A、7A、2D、 7D (with increase effect) and 1B、2B、6B (with decrease effect). Of these, the chromosomes 7A、1B and 2D might carry major effect genes, and the chromosomes 5A、2B、6B and 7D might carry minor effect genes.According to analysis of the results in the past studies and the present, I was speculated that the new gene governing grain yield per plant was probably carried on the chromosome 1B of the tested line 10-A.     

稻瘟病菌致病性的分子遗传学研究进展

由稻瘟病菌引起的稻瘟病是水稻生产上危害最为严重的真菌病害,对世界粮食生产造成巨大损失。稻瘟病菌成功侵染寄主包括分生孢子萌发、附着胞形成、侵染钉分化和侵染性菌丝扩展等一系列错综复杂的过程,其中每一环节都是由特定基因控制的。稻瘟病菌与水稻的互作符合经典的基因对基因学说,二者的不亲和互作是无毒基因与抗病基因相互作用的结果。近几十年来,世界各国的科学家对稻瘟病菌致病性的生物学及其遗传的分子机制进行了深入的研究。文章就稻瘟病菌致病性的分子遗传学及其遗传变异机制的研究进行了综述,同时对功能基因的研究方法进行了总结。     

Cloning and in silico Mapping of Resistance Gene Analogues Isolated from Rice Lines Containing Known Genes for Blast Resistance

We amplified resistance gene analogues (RGAs) from the genomic DNA of 10 rice lines having varying degree of resistance to Magnaporthe grisea by using degenerate primers and various RGAs were mapped in silico on different rice chromosomes. The amplified products were grouped into 3–8 restriction fragment length polymorphic classes by using Mbo1 and Alu1 restriction enzymes. Of 98 RGAs obtained in this study, 65 RGA clones showed more than 95% homology with various RGAs sequences present in the GenBank. Phylogenetic analysis of these RGAs formed 11 groups. Using sequence homology approach, RGAs isolated in this study were physically mapped on 23 loci on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 10, 11 and 12. Twenty RGAs were mapped near to the chromosomal regions containing known genes/QTLs for rice blast, bacterial leaf blight and sheath blight resistance. Thirty‐nine RGA sequences also contained open reading frame representing signature of potential disease resistance genes.     

Identification of Two Blast Resistance Genes in a Rice Variety, Digu

Blast, caused by Magnaporthe grisea is one of most serious diseases of rice worldwide. A Chinese local rice variety, Digu, with durable blast resistance, is one of the important resources for rice breeding for resistance to blast (M. grisea) in China. The objectives of the current study were to assess the identity of the resistance genes in Digu and to determine the chromosomal location by molecular marker tagging. Two susceptible varieties to blast, Lijiangxintuanheigu (LTH) and Jiangnanxiangnuo (JNXN), a number of different varieties, each containing one blast resistance gene, Pik^s, Pia, Pik, Pi‐b, Pi‐k^p, Pi‐ta², Pi‐ta, Pi‐z, Pi‐i, Pi‐k^m, Pi‐z^t, Pi‐t and Pi‐11, and the progeny populations from the crosses between Digu and each of these varieties were analysed with Chinese blast isolates. We found that the resistance of Digu to each of the two Chinese blast isolates, ZB13 and ZB15, were controlled by two single dominant genes, separately. The two genes are different from the known blast resistance genes and, therefore, designated as Pi‐d(t)1 and Pi‐d(t)2. By using bulked segregation method and molecular marker analysis in corresponding F₂ populations, Pi‐d(t)1 was located on chromosome 2 with a distance of 1.2 and 10.6 cM to restriction fragment length polymorphism (RFLP) markers G1314A and G45, respectively. And Pi‐d(t)2 was located on chromosome 6 with a distance of 3.2 and 3.4 cM to simple sequence repeat markers RM527 and RM3, respectively. We also developed a novel strategy of resistance gene analogue (RGA) assay with uneven polymerase chain reaction (PCR) to further tag the two genes and successfully identified two RGA markers, SPO01 and SPO03, which were co‐segregated toPi‐d(t)1 and Pi‐d(t)2, respectively, in their corresponding F₂ populations. These results provide essential information for further utilization of the Digu's blast resistance genes in rice disease resistance breeding and positional cloning of these genes.     

Genetic Analysis of Resistance to Rice Blast: A Study on the Inheritance of Resistance to the Blast Disease Pathogen in an F3 Population of Rice

Blast caused by the fungus Magnaporthae grisea (Herbert) Borr. (anamorphe Pyricularia oryza Cav.) is a serious disease of rice (Oryza sativa L.). One method to overcome this disease is to develop disease resistant cultivars. Due to the genetic plasticity in the pathogen genome, there is a continuous threat to the effectiveness of the developed cultivars. Additional studies of the genetics of resistance, virulence stability and functional genomics are required to accelerate research into understanding the molecular basis of blast disease resistance. In this study, individual plants of the F₃ population derived from Pongsu Seribu 2 and Mahsuri were used for pathogenesis assays and inheritance studies of blast resistance. The study was performed with two of the most virulent Malaysian M. grisea pathotypes: P7.2 and P5.0. For blast screening, plants were scored based on the IRRI Standard Evaluation System (SES). F₃ populations showed a segregation ratio of 3R:1S for pathotype P7.2, indicating that resistance to this pathotype is likely controlled by a single nuclear gene. Chi‐square analysis showed that the F₃ families segregated in a 15R:1S ratio for pathotype P5.0. Therefore, locus interactions or epitasis of blast resistance occur against pathotype P5.0 in the F₃ population derived from Pongsu Seribu 2 and Mahsuri. This can be explained by the presence of two independent dominant genes that when present simultaneously, provide resistance to the M. gresia pathotype P5.0. These results indicated that blast resistance in rice is due to the combined effects of multiple loci with major and minor effects. The genetic data generated here will be useful in the breeding of local cultivars for resistance to field blast. The methodology reported here will facilitate the mapping of genes and quantitative trait loci (QTLs) underlying the blast resistance trait.     

Light-Enhanced Resistance to Magnaporthe grisea Infection in the Rice Sekiguchi Lesion Mutants

The rice sl mutant showed two types of responses to Magnaporthe grisea infection by light treatments. One was an sl -mutant-type response characterized by Sekiguchi lesion expression under light waves of 400–700 nm, and the other was a wild-type response characterized by blast and/or necrotic spot lesion expression in the dark or at wavelength between 290 and 330 nm. There was a large difference in the resistance to M. grisea infection between the mutant- and wild-type responses in the rice sl mutant. When the mutant-type response was induced in the rice sl mutant, the disease resistance was enhanced relative to that in the wild-type response. Enhanced resistance was demonstrated by two components: (a) the number of Sekiguchi lesions was reduced relative to that of blast or necrotic lesions; (b) sporulation of M. grisea was not induced in Sekiguchi lesions. The enhanced resistance was dependent on light of 400–700 nm.     

Transformation of Chlamydomonas reinhardtii CW-15 with the Hygromycin Phosphotransferase Gene as a Selectable Marker

To transform Chlamydomonas reinhardtiiDang. cells, plasmid pCTVHyg was constructed with the use of theEscherichia colihygromycin phosphotransferase gene (hpt) controlled by the SV40 early promoter. Cells of the CW-15 mutant strain were transformed by electroporation, with the yield reaching 10³ hygromycin-resistant (Hyg^R) clones per 10⁶ recipient cells. The exogenous DNA integrated in the Ch. reinhardtii nuclear genome showed stable transmission for approximately 350 cell generations, while hygromycin resistance was expressed as an unstable character. Codon usage was compared for the hptgene and Ch. reinhardtiinuclear genes. The results testified that codon usage bias, which is characteristic of Ch. reinhardtii, is not the major factor affecting foreign gene expression. The advantages of the selective system for studying Ch. reinhardtii transformation with heterologous genes are discussed.     

Construction of a Binary Vector for Knockout and Expression Analysis of Rice Blast Fungus Genes

We developed an efficient method to analyze gene function and expression of the rice blast fungus. We constructed a GATEWAY binary vector, which generates a gene-targeted disruptant carrying a green fluorescent protein gene under the native promoter of the target gene. Using this method, the knockout efficiency and expression patterns of two hypothetical genes were determined.     

Population Structure of Magnaporthe grisea from North-western Himalayas and its Implications for Blast Resistance Breeding of Rice

Neutral and pathogenicity markers were used to analyse the population structure of Magnaporthe grisea rice isolates from the north‐western Himalayan region of India. Random amplified polymorphic DNA (RAPD)‐based DNA fingerprinting of 48 rice isolates of M. grisea with five primers (OPA‐04, OPA‐10, OPA‐13, OPJ‐06 and OPJ‐19) showed a total of 65 RAPD bands, of which 54 were polymorphic. Cluster analysis of 48 rice isolates of M. grisea on the basis of these 65 RAPD bands revealed the presence of high genotypic diversity and continuous DNA fingerprint variation in the pathogen population. No correlation was observed between RAPD patterns and virulence characteristics of the pathogen. The observed population structure contrasted with presumed clonal reproductive behaviour of the pathogen and indicated the possibility of ongoing genetic recombination in the pathogen population. Analysis of the virulence organization of five RAPD groups (RG1–RG5) using 20 rice genotypes comprising at least 15 resistance genes revealed that no combination of resistance genes would confer resistance against all RAPD fingerprint groups present in the M. grisea rice population. The possible implications of the observed population structure of M. grisea for blast resistance breeding have been discussed.