无选择标记和载体骨干序列的Xa21转基因水稻的获得

利用双右边界T-DNA载体通过根癌农杆菌介导法将水稻白叶枯病广谱抗性基因Xa21导入杂交稻重要恢复系C418中。T0代共获得27个独立转基因株系,通过田间抗性鉴定与PCR分析,有17个株系的Xa21基因分子鉴定为阳性,且对白叶枯病原菌P6生理小种具有抗性。通过对17个株系的后代植株进行田间抗性鉴定,分子标记辅助选择及Southern杂交分析,结果显示4个株系的T1代植株中能分离出无潮霉素标记基因的Xa21转基因植株。无选择标记Xa21转基因株系的获得率为15%。PCR检测还表明,这些无选择标记的Xa21转基因植株不带有载体骨架序列。通过对转基因后代进一步的抗性鉴定与PCR辅助选择,获得了无选择标记和载体骨架序列的转基因Xa21纯合的抗白叶枯病水稻。     

由农杆菌介导将白叶枯病抗性基因Xa21转入我国的5个水稻品种

利用农杆菌介导的转化系统将已克隆的Xa21基因转入我国5个水稻主栽品种, 获得了110个独立的转基因系. 转基因植株的PCR和Southern分析揭示Xa21基因已整合到受体基因组. 已整合的Xa21基因能稳定遗传, 单拷贝整合的转化体在自交T1代呈现抗感3:1的分离. 接种实验表明转基因T0植株和Xa21-PCR阳性T1植株对白叶枯病的高度抗性. 经过筛选的Xa21纯合的具有优良品质的抗性转基因系可以作为品种直接种植, 或者用于杂交稻育种.     

来源于疣粒野生稻的白叶枯病新抗源的鉴定

疣粒野生稻对白叶枯病高抗甚至免疫。粳稻品种8411与疣粒野生稻体细胞杂交获得了2个高抗所有栽培稻白叶枯病抗性基因均不能抵御的国际广致病菌系P6的新种质SH5和SH76,遗传分析鉴定出新种质含有1个抗P6小种的显性基因。本研究以高感白叶枯病水稻品种IR24及携带不同抗性基因的16个材料为参照,对分蘖期、孕穗期的SH5和SH76分别接种11个白叶枯病小种,抗性分析表明SH5和SH76的抗谱广,与IRBB21(Xa21)抗谱一致,与IRBB5(xa5)、IRBB7(Xa7)和Asominori(Xa17)的较相近。用Xa21的分子标记pTA248和XA21检测,确定SH5和SH76不携带Xa21基因,前期研究结果证实新种质中不含xa5和Xa7;与Asominori的杂交试验表明其抗性基因与Xa17基因不等位。这些结果表明SH5和SH76中存在1个抗P6小种的新基因。     

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。     

云南野生稻抗白叶枯病类Xa21基因的鉴定

水稻白叶枯病是水稻生产上的主要细菌病害之一。从野生稻中发掘优异的水稻白叶枯病抗性材料,可以拓宽栽培稻抗白叶枯病遗传基础。经过温室接菌鉴定和PCR标记分析,对云南野生稻进行Xa21基因的检测鉴定。温室接菌鉴定表明,云南野生稻对广谱致病小种PX099及云南强致病菌Y8具有较好的抗性能力,特别是疣粒野生稻对致病菌株达到免疫程度;PCR标记分析表明,云南野生稻不含有Xa21基因,但含有与Xa21基因某些区域同源的片段。本研究结果为寻找新的抗源材料及快速发掘利用云南野生稻中的抗白叶枯病基因提供理论依据。     

云南野生稻中的抗白叶枯病基因Xa21的鉴定

水稻白叶枯病是水稻生产上的主要细菌病害之一。从野生稻中发掘优异的水稻白叶枯病抗性材料,可以拓宽栽培稻抗白叶枯病遗传基础。经过温室接菌鉴定和PCR标记分析,对云南野生稻进行Xa21基因的检测鉴定。温室接菌鉴定表明,云南野生稻对广谱致病小种PX099及云南强致病菌Y8具有较好的抗性能力,特别是疣粒野生稻对致病菌株达到免疫程度;PCR标记分析表明,云南野生稻不含有Xa21基因,但含有与Xa21基因某些区域同源的片段。本研究结果为寻找新的抗源材料及快速发掘利用云南野生稻中的抗白叶枯病基因提供理论依据。     

水稻白叶枯病抗性基因Xa21的分子生物学研究进展

由黄单胞杆菌水稻致病变种Xanthomonas oryzae pv.oryzae(Xoo)引起的白叶枯病是水稻重要细菌性病害之一。迄今,已有7个水稻白叶枯病抗性基因被克隆。Xa21是第一个被克隆的白叶枯病抗性基因,因具有广谱抗性而受到广泛的关注。对Xa21的发现、定位及克隆、表达特征、编码产物XA21的生化特性、作用与调控以及XA21介导的免疫反应模式等方面的研究结果进行综述,并对今后的研究方向进行展望。     

水稻Xa1基因启动子的克隆及功能分析

Xa1是一个能对日本白叶枯病优势小种(小种1号)产生专化性抗性的R基因,虽已有该基因克隆、表达和功能方面的研究,但对其表达调控分子机制还不很清楚。本研究利用Xa1启动子与GUS报告基因的转基因T1株系,研究了Xa1启动子的时空表达及对不同外源激素的应答特征。结果表明,Xa1启动子驱动的GUS基因在水稻根中的表达量明显高于茎和叶,且在根部的中柱区GUS的表达量明显高于周围组织;在外源MeJA作用下GUS的表达显著增强,在SA和ABA处理下也有一定程度的增强,这些结果暗示Xa1的抗病作用与其在根系中柱的组织特异性表达存在一定的相关性,MeJA对Xa1启动子的活性起重要的调控作用。     

利用竞争性PCR筛选无标记Xa21转基因水稻

PCR是一种简单、迅速、灵敏的检测方法,但假阳性与假阴性却影响了它在常规应用中的准确性。本研究利用竞争性PCR解决无标记Xa21转基因水稻PCR检测中的假阳性与假阴性问题。标记基因潮霉素基因(Hygromycin phosphotransferase,hpt)的竞争模板是外加的日本晴hpt转基因植株基因组DNA,抗白叶枯病基因Xa21的竞争模板是待测水稻内源的位于第11染色体上的Xa21同源基因序列。利用这一方法对双右边界T-DNA载体转化产生的转基因T1代植株进行分析,可以有效地减少或排除假阳性或假阴性样品,选出真正的转基因阳性植株。与常规PCR相比竞争性PCR提高了无标记Xa21转基因植株筛选的准确性。对获得的无标记Xa21转基因植株进行白叶枯抗病鉴定与潮霉素抗性鉴定证实了该方法的可靠性。     

水稻Xa21基因启动子序列分析及其在不同条件下的表达特征

以水稻品种‘日本晴’(Oryza sativa‘Nipponbare’)为实验材料,根据GenBank上公布的同品种水稻的基因组DNA序列设计1对引物,对水稻Xa21基因启动子进行克隆并测序,通过PCR扩增获得的Xa21基因启动子序列长1 982 bp,其中除包含启动子基本元件外,还包含一些与逆境信号相关的元件(GCC-box、A-box、TC-rich repeats、MBS、LTR和W-box等)。利用GUS组织化学染色和定量分析方法,研究了转基因水稻T1代株系不同器官和发育阶段Xa21基因启动子的表达特异性及其在不同逆境和激素处理条件下的表达特征,结果显示:在转基因水稻的叶、茎和根部均能检测到GUS活性,但根部GUS活性最高,特别是在根尖的中柱区活性最强;随苗龄增长(3叶期、5叶期和8至9叶期)叶片中GUS活性逐渐增加,8至9叶期GUS活性最高;机械损伤和100μmol.L-1茉莉酸甲酯(MeJA)处理可使叶片中GUS活性显著或极显著提高,而干旱、500μmol.L-1水杨酸(SA)和100μmol.L-1脱落酸(ABA)处理则对叶片中GUS活性无明显影响。研究结果表明:外界逆境胁迫对水稻Xa21基因启动子的表达有诱导作用;该启动子的表达受水稻发育阶段的调控并具有一定的器官组织特异性,在根中的表达量最高;其介导的抗病反应依赖于茉莉酸(JA)信号通路。     

The molecular basis of disease resistance in rice

The rice gene Xa21 conferring resistance to Xanthomonas oryzae pv. oryzae (Xoo), was isolated using a map-based cloning strategy. Compared with previously cloned genes, the structure of Xa21 represents a novel class of plant disease R genes encoding a putative receptor kinase (RK). This article proposes a model for the mode of action of Xa21 and summarizes our current knowledge of the modular basis of resistance in rice to bacterial leaf blight and blast.     

Introduction of a rice blight resistance gene,Xa21, into five Chinese rice varieties through anAgrobacterium-mediated system

A cloned gene, Xa21 was transferred into five widely-used Chinese rice varieties through an Agrobacterium-mediated system, and over 110 independent transgenic lines were obtained. PCR and Southern analysis of transgenic plants revealed the integration of the whole Xa21 gene into the host genomes. The integrated Xa21 gene was stably inherited, and segregated in a 3 : 1 ratio in the selfed T1 generation when one copy of the gene was integrated in the transfor-mants. Inoculation tests displayed that transgenic T0 plants and Xa21 PCR-positive T1 plants were highly resistant to bacterial blight disease. The selected Xa21 homozygous resistant transgenic lines with desirable qualities may be propagated as new varieties or utilized in hybrid rice breeding.     

Introduction of a rice blight resistance gene, Xa21, into five Chinese rice varieties through an Agrobacterium-mediated system

A cloned gene, Xa21 was transferred into five widely-used Chinese rice varieties through an Agrobacterium-mediated system, and over 110 independent transgenic lines were obtained. PCR and Southern analysis of transgenic plants revealed the integration of the whole Xa21 gene into the host genomes. The integrated Xa21 gene was stably inherited, and segregated in a 3∶1 ratio in the selfed T1 generation when one copy of the gene was integrated in the transformants. Inoculation tests displayed that transgenic T0 plants and Xa21 PCR-positive T1 plants were highly resistant to bacterial blight disease. The selected Xa21 homozygous resistant transgenic lines with desirable qualities may be propagated as new varieties or utilized in hybrid rice breeding.     

水稻白叶枯病抗性基因Xa23鉴别菌株突变体库的构建及无毒基因突变体的筛选

利用in vivo转座技术构建了白叶枯病抗性基因Xa23鉴别菌株的突变体库,特异性引物PCR扩增和转座子插入位点旁侧序列分析结果表明转座子插入到白叶枯病菌的基因组中。经人工接种鉴定,筛选到4个毒力发生变化的突变体。为进一步克隆Xa23无毒基因提供了条件。     

Analysis of Pathogenic Diversity of the Rice Bacterial Blight Pathogen (Xanthomonas oryzae pv. oryzae) in the Andaman Islands and Identification of Effective Resistance Genes

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of rice in the tropics for which genetic resistance in the host plants is the only effective solution. This study aimed at identification of resistance gene combinations effective against Xoo isolates and fingerprinting of the Xoo isolates of Andaman Islands (India). Here, we report the reaction of 21 rice BB differentials possessing Xa1 to Xa21 genes individually and in different combinations to various isolates of pathogen collected from Andaman Islands. Pathological screening results of 14 isolates revealed that among individual genes tested across 2 years, Xa4, Xa7 and Xa21 conferred resistance reaction across all isolates, whereas among combinations, IRBB 50 (Xa4 + xa5), IRBB 52 (Xa4 + Xa21) and IRBB 60 (Xa4 + xa5 + xa13 + Xa21) conveyed effective resistance against tested isolates. The nature of genetic diversity among four isolates selected on the basis of geographical isolation in the islands was studied through DNA finger printing. The RAPD primers S111, S119, S1117, S1109, S1103, S109 and S105 were found to be better indicators of molecular diversity among isolates than JEL primers. The diversity analysis grouped 14 isolates into three major clusters based on disease reaction wherein isolate no. 8 was found the most divergent as well as highly virulent. The remaining isolates were classified into two distinct groups. The importance of the study in the context of transfer of resistance gene(s) in the local cultivars specifically for tropical island conditions is presented and discussed.     

Transgenic rice variety ‘IR72’ with Xa21 is resistant to bacterial blight

 An elite indica rice variety, ‘IR72’, was transformed with a cloned gene, Xa21, through particle bombardment. Molecular analysis of transgenic plants revealed the presence of a 3.8-kb EcoRV-digested DNA fragment corresponding to most of the Xa21 coding region and its complete intron sequence, indicating the integration of Xa21 into the genome of ‘IR72’. In the T₁ generation, the transgene was inherited and segregated in a 3:1 ratio. After inoculation with the prevalent races 4 and 6 of Xanthomonas oryzae pv. oryzae (Xoo), T₁ plants positive for the transgene were found to be resistant to bacterial blight (BB). We also observed that the level of resistance to race 4 of Xoo was higher due to the pyramiding of Xa21 and Xa4 present in ‘IR72’. Since the inactivation of the transgene Xa21 occurred in the two transgenic T₁ plants, a larger progeny should be obtained for selecting homozygous line with a consistently higher level of resistance to the BB pathogen. Received: 13 October 1997 / Accepted: 21 October 1997