小麦种质抗纹枯病性的鉴定和遗传分析

对2300余份创新种质和引进种质抗纹枯病性进行了自然病地初步鉴定,在此基础上选择一批抗性好的种质进行了人工病圃鉴定,共评选出高抗纹枯病的创新种质14份,引进种质21份。这些种质都兼抗1－3种其它病害,且综合性状较好,其中创新种质最好,为抗纹枯病育种提供了良好的种质材料。还选用7个抗纹枯病性不同的亲本组配成半双列杂交组合。采用Hayman法进行了基因效应分析,结果表明,抗纹枯病性的遗传不符合加性－显性模型。     

Expression of a radish defensin in transgenic wheat confers increased resistance to Fusarium graminearum and Rhizoctonia cerealis

Fusarium head blight (scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat (Triticum aestivum L.) worldwide. Wheat sharp eyespot, mainly caused by Rhizoctonia cerealis, is one of the major diseases of wheat in China. The defensin RsAFP2, a small cyteine-rich antifungal protein from radish (Raphanus sativus), was shown to inhibit growth in vitro of agronomically important fungal pathogens, such as F. graminearum and R. cerealis. The RsAFP2 gene was transformed into Chinese wheat variety Yangmai 12 via biolistic bombardment to assess the effectiveness of the defensin in protecting wheat from the fungal pathogens in multiple locations and years. The genomic PCR and Southern blot analyses indicated that RsAFP2 was integrated into the genomes of the transgenic wheat lines and heritable. RT-PCR and Western blot proved that the RsAFP2 was expressed in these transgenic wheat lines. Disease tests showed that four RsAFP2 transgenic lines (RA1–RA4) displayed enhanced resistance to F. graminearum compared to the untransformed Yangmai 12 and the null-segregated plants. Assays on Q-RT-PCR and disease severity showed that the express level of RsAFP2 was associated with the enhanced resistance degree. Two of these transgenic lines (RA1 and RA2) also exhibited enhanced resistance to R. cerealis. These results indicated that the expression of RsAFP2 conferred increased resistance to F. graminearum and R. cerealis in transgenic wheat.     

28份人工合成小麦对禾谷孢囊线虫、纹枯病、条锈病和叶锈病的抗性

为了发现具有兼抗多种病害的小麦种质,本研究采用田间病圃法和人工接种法,对28份人工合成小麦的禾谷孢囊线虫、纹枯病、条锈病和叶锈病进行了抗性鉴定。人工合成小麦对这些病害表现不同程度的抗性反应。C2和C20对鉴定的4种病害都具有抗性,C5、C10和C25对这些病害都表现感病。8份材料对Heterodera avenae和H. filipjevi两种病原线虫都表现抗性反应型,也有的材料只抗一种线虫。供试材料对纹枯病的抗性表现较好,其中19份材料表现抗性反应型。9份材料对接种的条锈菌小种CY30、CY31、CY32和CY33均表现抗性反应型,5份材料对叶锈菌小种THT和PHT都具抗性。     

抗茎腐病转基因小麦新种质的筛选

为拓宽小麦茎腐病(又称茎基腐病)抗源种类,筛选抗茎腐病小麦新种质,对43份转TaPIMP1、AtNPR1和Gastrodianin基因小麦纯合株系,进行目的基因表达分析,以及茎腐病、纹枯病和赤霉病抗性鉴定。结果表明,转基因株系的目的基因均能正常表达;转基因株系间茎腐病抗性差异明显,24份转基因株系茎腐病抗性,比受体对照扬麦12显著提高;转基因株系茎腐病抗性与纹枯病抗性相关性显著,与赤霉病相关性不显著。结合农艺性状鉴定,筛选出5份抗茎腐病转基因株系,其中2份兼抗纹枯病和赤霉病,1份兼抗纹枯病,可作为长江中下游麦区茎腐病备用抗源。     

转PvPGIP2基因小麦的获得与纹枯病抗性鉴定

多聚半乳糖醛酸酶抑制蛋白(PGIP)是一种植物防卫蛋白,可阻止一些病原真菌的侵害。本研究克隆出扁豆PvP-GIP2基因编码序列,构建了受玉米泛素(ubiquitin)启动子控制的PvPGIP2基因表达载体pA25-PvPGIP2;采用基因枪法将pA25-PvPGIP2转化小麦推广品种扬麦18幼胚愈伤组织4000块,获得了203株再生植株。PCR检测出阳性植株65株,转化率为1.625%。对转PvPGIP2基因小麦T1～T2植株,进行外源基因的PCR、RT-PCR、荧光定量RT-PCR(Q-RT-PCR)分析和小麦纹枯病抗性鉴定。结果表明,转入的PvPGIP2能够在转基因小麦中遗传、转录与表达;PvPGIP2基因的表达提高了转基因植株对小麦纹枯病的抗性。     

农杆菌介导抗储粮害虫转基因小麦(Triticum aestivum L.)的获得及分析

以本实验室选育的小麦优良品系的胚性愈伤组织为材料,采用农杆菌介导将抗虫基因豇豆胰蛋白酶抑制剂基因CpTI转入小麦培养细胞,经筛选获得抗卡那霉素的愈伤组织并再生植株。经PCR和实时PCR检测、PCR-Southern和Southernblot验证,确定了3株独立再生植株为含有CpTI的转基因植株。农杆菌菌浓度、侵染时间及转化处理方式对小麦转化率均有明显影响。3株转基因植株正常可育并结籽,形成转基因株系。外源基因在转基因植株T1代中的分离呈多样性,部分株系(转基因株系T-Ⅰ、T-Ⅲ)表现出孟德尔遗传规律。抗虫试验表明,3株转基因植株T2代籽粒对储粮害虫麦蛾具有一定的抗性,转基因株系T-Ⅰ、T-Ⅱ、T-Ⅲ及非转基因植株的T2代籽粒虫蛀率分别为19·8%、21·9%、32·9%和58·3%。转基因植株T1代群体农艺性状调查显示,3个株系具有良好的农艺性状,为小麦的遗传改良提供了新的种质抗虫材料。     

基因枪法转基因水稻中hpt基因稳定遗传

基因枪转化将潮霉素磷酸转移酶基因（ｈｐｔ）导入粳稻品种７７１７０,获得可育的转基因植株,研究外源基因遗传的稳定性。自交后代（Ｔ１和Ｔ２）经潮霉素筛选获得抗性植株和敏感植株,分子鉴定结果表明抗性植株带有ｈｐｔ基因,而敏感植株中没有ｈｐｔ基因存在。Ｔ１和Ｔ２代中潮霉素抗性表现为显性单基因位点的遗传方式,符合孟德尔分离规律,并得到分子鉴定结果的证实。Ｓｏｕｔｈｅｒｎ杂交结果显示,ｈｐｔ基因多拷贝整合在水     

小麦抗纹枯病种质资源筛选

近年来,随着耕作制度的改变和气候的变化,纹枯病在我国黄淮冬麦区和长江中下游冬麦区广泛发生,并呈逐年加重的趋势。为了筛选出可在生产或育种中利用的抗病材料,本试验对从国内外收集到的79份小麦材料及1份小麦近缘材料荆州黑麦,同时在温室和大田进行纹枯病抗性鉴定,结果表明,品种间存在纹枯病抗性差异,在小麦品种中进行小麦纹枯病抗源选择是有效的,温室和大田同时抗性表现较好的有14份材料:CI 12633、丽麦16、ARz、Cooker 983、FHB143、Italy-2、GUADA-LYPE、Ovontun、白火麦、山红麦、山农12号、小偃22、紫秆子和荆州黑麦,为纹枯病抗病育种提供稳定可靠的抗源。     

Introgression and characterization of barley yellow dwarf virus resistance from Thinopyrum intermedium into wheat.

Wheatgrasses (species of Agropyron complex) have previously been reported to be resistant to barley yellow dwarf virus (BYDV). To introgress this resistance into wheat, Triticum aestivum x Thinopyrum (Agropyron) intermedium hybrids were advanced through a backcrossing program and reaction to BYDV, as determined by enzyme-linked immunosorbent assay (ELISA), is reported for the first time in backcross populations of wide hybrids between wheat and wheatgrasses. ELISA values revealed highly resistant to highly susceptible segregants in backcrosses. BYDV resistance was expressed in some backcross derivatives. Continued selection, based on cytology and ELISA in each generation, eliminated most of the unwanted wheatgrass chromosomes and produced self-fertile BYDV resistant wheat lines. The BYDV resistant lines with 2n = 42 had normal chromosome pairing similar to wheat, and their F1 hybrids with wheat had two univalents. DNA analyses showed that the source of alien chromatin in these BYDV resistant wheat lines is distinguishable from that in other Th. intermedium derived BYDV resistant wheat lines. Chromosome pairing and restriction fragment length polymorphism analyses indicated that the 42 chromosome resistant Purdue wheat lines are substitution lines in which chromosome 7D was replaced by a chromosome from Th. intermedium that was carrying gene(s) for BYDV resistance.     

基因枪转化的bar基因表达盒在水稻中遗传行为复杂

基因枪介导基因表达盒(仅包括启动子、编码区和终止子)转化是基因枪转化植物的新趋势,它能消除质粒载体主干序列对转基因植物的不利影响。本文研究了基因枪转化的bar基因表达盒在转基因水稻T1～T3世代中的遗传行为。结果发现:作为筛选标记的bar基因表达盒在水稻基因组中多拷贝整合,遗传分离行为复杂,还出现了Basta抗感分离比在35:1～144:1之间的"假纯合体",但50%转基因株系中(5/10)bar基因可作为一个显性基因按孟德尔方式稳定遗传至自交T2代。虽然bar基因为多拷贝整合,30%的转基因株系(3/10)在自交低世代(T1)能获得纯合体。Southern杂交分析发现,多拷贝的bar基因表达盒倾向于连接成转基因串联子整合在水稻基因组内。我们发现在Basta抗性正常分离的株系后代中bar基因表达盒Southern杂交模式能稳定遗传,但异常分离的株系后代中bar基因表达盒的一些拷贝发生了丢失。我们推测,bar基因表达盒在水稻中遗传分离行为的复杂原因可能是bar基因表达盒多拷贝整合、基因丢失和基因表达互作。     

Resistance against Fusarium Head Blight in Transgenic Wheat Plants Expressing the ScNPR1 gene

Fusarium head blight (FHB) is a severe global wheat disease that may cause severe yield losses, especially during epidemic years. Transforming the regulatory genes in the metabolic pathways of disease resistance into wheat via transgenic methods is one way to improve resistance to FHB. ScNPR1 (Secale cereale‐NPR1), a regulatory gene for systemic acquired resistance (SAR), was isolated from S. cereale cv Jingzhouheimai and transformed into the moderately FHB‐susceptible wheat variety Ningmai 13. RT‐PCR analysis indicated that the ScNPR1 gene was stably expressed in transgenic plants. An evaluation of the resistance to FHB revealed that six ScNPR1 transgenic lines (NP1, NP2, NP3, NP4, NP5 and NP6) exhibited significantly higher FHB resistance than the wild‐type wheat Ningmai 13 and the null‐segregated plants. The expression of pathogenesis‐related (PR) genes after Fusarium graminearum inoculation was earlier or higher than those in the wild‐type variety Ningmai 13. The high expression in the early stages of PR genes should account for the enhanced FHB resistance in the transgenic lines. Our results suggest that overexpression of ScNPR1 could be used to improve FHB resistance in wheat.     

水稻谷蛋白GluA-2基因在小麦胚乳中的表达(英文)

水稻(Oryza sativa L.)谷蛋白(Glutelin)约占水稻储藏蛋白总量的80％,谷蛋白赖氨酸含量较高并易于被人体消化吸收。为了提高小麦(Triticum aestivum L. )的营养品质,将水稻谷蛋白GluA-2基因的cDNA序列导入小麦栽培品种Bobwhite(T. aestivum cv. Bobwhite)。共轰击了600个小麦幼胚,经PCR和Southern杂交鉴定,共获得4棵转GluA-2基因小麦;SDS-PAGE分析表明,GluA-2基因在3棵转基因植株及其后代中表达,在1棵转基因植株中未表达,但其内源的高分子量麦谷蛋白亚基Bx7和By9含量显著降低,并且可遗传至T_代。     

绿僵菌对小麦纹枯病菌的抑制作用研究

在实验室条件下,研究了金龟子绿僵菌（Metarhizium anisopliae）对小麦纹枯病菌（Rhizoctonia cerealis）的拮抗作用及其机理。结果表明,金龟子绿僵菌与小麦纹枯病菌对峙培养以及在培养基中加入金龟子绿僵菌孢子悬浮液,对小麦纹枯病菌菌丝生长均有较好的抑制作用。测定了培养不同天数的金龟子绿僵菌Ma55发酵液对小麦纹枯病菌菌丝生长、菌核产生量及菌核萌发率的影响。结果表明,液体振荡培养25 d的金龟子绿僵菌Ma55发酵液对小麦纹枯病菌的菌丝生长、菌核产生量及菌核萌发具有显著的抑制作用,且Ma55发酵液中的抑菌活性物质具有较好的热稳定性。在光学显微镜下,未观察到Ma55对小麦纹枯病菌的重寄生现象,但发现金龟子绿僵菌与小麦纹枯病菌对峙培养处小麦纹枯病菌营养菌丝的细胞质变稀薄、菌丝部分消解或断裂。上述结果显示,金龟子绿僵菌对小麦纹枯病菌的拮抗机制主要是营养竞争、空间竞争及抗生作用等。     

水稻白叶枯病广谱抗性基因Xa21导入两用不育系培矮64S

以克隆的Xa21基因为外源基因,成熟胚愈伤组织为转化受体,应用农杆菌介导法对水稻两用型核不育系培矮64S进行转化,获46株转基因植株。PCR和Southern分析结果表明,Xa21已整合到受体基因组。用稻白叶枯病病原菌(Xanthomonasoryzaepv.oryzae)菲律宾小种6号接种鉴定,结果表明大多数转基因植株获得了抗病性。已整合的Xa21基因能够稳定地遗传,在所检测转基因株系的T1代中,Xa21基因显示3:1的分离。     

Overexpression of wheat lipid transfer protein gene TaLTP5 increases resistances to Cochliobolus sativus and Fusarium graminearum in transgenic wheat

The fungus Cochliobolus sativus is the main pathogen of common root rot, a serious soil-borne disease of wheat (Triticum aestivum L.). The fungus Fusarium graminearum is the primary pathogen of Fusarium head blight, a devastating disease of wheat worldwide. In this study, the wheat lipid transfer protein gene, TaLTP5, was cloned and evaluated for its ability to suppress disease development in transgenic wheat. TaLTP5 expression was induced after C. sativus infection. The TaLTP5 expression vector, pA25-TaLTP5, was constructed and bombarded into Chinese wheat variety Yangmai 18. Six TaLTP5 transgenic wheat lines were established and characterized. PCR and Southern blot analyses indicated that the introduced TaLTP5 gene was integrated into the genomes of six transgenic wheat lines by distinct patterns, and heritable. RT-PCR and real-time quantitative RT-PCR revealed that the TaLTP5 gene was over-expressed in the transgenic wheat lines compared to segregants lacking the transgene and wild-type wheat plants. Following challenge with C. sativus or F. graminearum, all six transgenic lines overexpressing TaLTP5 exhibited significantly enhanced resistance to both common root rot and Fusarium head blight compared to the untransformed wheat Yangmai 18.     

Mapping of QTL conferring resistance to sharp eyespot (Rhizoctonia cerealis) in bread wheat at the adult plant growth stage

Key message

Seven sharp eyespot resistance QTL were detected consistently across five environments and delimited to seven DNA marker intervals, respectively, six of which were independent of plant height and heading time.

Abstract

Sharp eyespot, caused mainly by the soil-borne fungus Rhizoctonia cerealis, is one of the important diseases of bread wheat (Triticum aestivum L.). This disease has escalated into a major threat to wheat production in some regions of the world. Wheat resistance to sharp eyespot can be a potential means to reduce the needs for application of fungicides and agricultural inputs. In the present study, the winter wheat lines, Luke and AQ24788-83, both of which possess quantitative resistance to sharp eyespot, were crossed and a population consisting 241 recombinant-inbred lines (RILs) was constructed. These RILs were assessed for sharp eyespot resistance by conducting five field and greenhouse trials during the period from 2008 to 2012, and they were genotyped with 549 simple-sequence repeat DNA markers. Seven quantitative trait loci (QTL) were detected consistently across the five trial environments to be associated with the sharp eyespot resistance. They were mapped on chromosomes 1A, 2B, 3B, 4A, 5D, 6B, and 7B. Four of these QTL are unequivocally novel, while it is possible that the other three might also be novel. Plant height and heading date of the 241 RILs were recorded in the four field trials. All of the seven disease resistance QTL were independent of plant height and heading time except one that was significantly associated with plant heading time. This association might be attributed genetically to a single QTL, or to different but closely linked QTL. In the case of single QTL, pleiotropism might be involved or the sharp eyespot resistance might be conferred in a physical instead of physiological nature.