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.     

农杆菌介导将白叶枯病抗性基因Xa21导入水稻获得转基因植株

利用农杆菌介导的高效遗传转化系统,将白叶枯病抗性基因Xa21转入黄淮稻区主栽品种豫粳6号的胚性愈伤组织,获得转基因植株,GUS染色和PCR分析证明Xa21基因已整合到水稻基因组中,其自交T1代植株经GUS染色和白叶枯病接种鉴定呈现3：1分离,研究为培育抗白叶枯病水稻品种奠定了基础。     

水稻抗白叶枯病基因Xa21的研究进展

Xa21是最早克隆的水稻抗病基因,作为类受体激酶类广谱抗病基因它受到广泛的关注。转基因Xa21材料,很可能成为世界上第一个被批准进行大田释放的水稻转基因材料。本文在简要回顾Xa21的发现、定位和克隆过程之后,总结了目前Xa21基因的抗病作用机理和育种应用研究现状,包括XA21蛋白质激酶的生化特性、AvrXa21的鉴别、Xa21介导的抗病途径、抗病机理等,并对今后的研究进行了展望。     

Breeding bacterial blight-resistant hybrid rice with the cloned bacterial blight resistance gene Xa21

The cloned bacterial blight (BB) resistance gene Xa21 was transferred into Minghui63, a widely used restorer line of indica hybrid rice in China, through an Agrobacterium-mediated system. Molecular and resistance analyses revealed that the Xa21 gene was integrated in the genomes of transgenic plants and their progeny inherited resistance stably. For the purpose of hybrid breeding, Xa21 transgenic homozygous restorer lines were selected through `within-lane' dosage comparison of hybridization signal in combination with PCR and resistance analyses. The selected transgenic restorer lines were then crossed with a commonly used sterile line, Zhenshan97A, to produce Xa21 transgenic hybrid rice, Shanyou63-Xa21. The hybrid rice plants with Xa21 displayed high broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo) races and maintained elite agronomic characters of Shanyou63. The propagation of this BB-resistant hybrid variety with Xa21 will benefit rice production.     

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。     

水稻转基因系CX8621中Xa21的整合和表达

农杆菌介导的转基因技术在植物中已被广泛应用,而目的基因能否发挥功能受到多种因素的影响。前期,通过农杆菌介导的转化,实验室创制了无选择标记、无载体骨架残留的水稻转Xa21基因系CX8621。截止目前,CX8621已稳定遗传16代,依然保持着对水稻白叶枯病的优良抗性。在此基础上,本研究对外源基因Xa21在CX8621中的整合和表达情况进行了分析。首先,通过在转化载体p BXa21的左右边界与Xa21基因序列设计嵌套引物,确定Xa21被完整地整合到CX8621中。随后,利用改良的Tail-PCR方法体外克隆了整合位点的边界序列,明确了Xa21被整合在CX8621的2号染色体上。然后,通过RT-PCR分析了Xa21在CX8621中不同时期和不同组织的表达情况,结果表明Xa21在CX8621中能稳定表达,其表达量的变化与之前报道的抗病性反应吻合。此外,还制备了天然XA21蛋白的抗体,对CX8621不同时期、不同组织中XA21蛋白的表达量进行了检测,结果发现在种子中检测不到XA21蛋白。由此,通过对外源基因Xa21的整合和表达分析,为CX8621的转基因生物安全评价提供了部分科学依据。     

水稻白叶枯病抗性基因Xa21和稻瘟病抗性基因Pi-d2激酶蛋白质在毕赤酵母中的表达及条件优化

【目的】白叶枯病和稻瘟病是最主要的水稻病害,Xa21是水稻白叶枯病抗性基因,Pi-d2是稻瘟病抗性基因,二者都编码类受体激酶蛋白质。本研究旨在毕赤酵母系统中表达XA21和PI-D2激酶蛋白质。【方法】用Xa21和Pi-d2的激酶区PCR产物,构建了pPICZαA-Xa21K、pPICZαA-Pi-d2K重组质粒,酶切及测序验证后,将重组质粒线性化,转化到毕赤酵母菌株中,系统地比较了不同酵母菌株(KM71、GS115、X33),不同甲醇浓度(1%、2%、3%),不同pH(pH5、pH6、pH7、pH8)值,不同诱导时间(24h、48h、72h)条件下激酶蛋白质的表达情况。【结果】XA21和PI-D2激酶蛋白质可以在毕赤酵母中表达,但表达的蛋白质不能分泌到培养基上清中,而只能在菌体中检测到,对表达条件的系统比较发现,毕赤酵母菌株KM71和X33、2%的甲醇诱导浓度、pH5和48h以上的诱导时间有利于激酶蛋白质的表达,最后我们在酵母裂解物上清中获得了纯化的考染可见的激酶蛋白质。【结论】在毕赤酵母中表达了XA21和PI-D2激酶蛋白质,为下一步生化特性研究奠定了基础。     

Transgenic elite Indica rice varieties,resistant to Xanthomonas oryzae pv. oryzae

The agronomically important Indica (group 1) rice varieties IR64, IR72, hybrid restorer line Minghui 63, and BG90-2 were co-transformed by microbombardment of embryogenic suspensions with plasmids that contain the Xa21 gene which confers resistance to Xanthomonas oryzae pv. oryzae and the hph gene for resistance to hygromycin B. Six of the 55 transgenic R0 plant lines containing the Xa21 gene displayed high levels of resistance to the pathogen, and no partial resistance was observed. The trait was stably inherited in subsequent generations, and transgenic plants are currently in field tests. The ability to transfer agronomically important genes into elite Indica rice varieties demonstrates the applicability of genetic engineering for the agronomic improvement of rice.     

Bacterial leaf blight resistance genes (Xa21, xa13 and xa5) pyramiding through molecular marker assisted selection into rice cultivars

Marker assisted selection was employed to pyramid three bacterial blight resistance genes Xa21, xa13 and xa5 into high yielding susceptible rice cultivars ADT43 and ADT47. With the assistance of PCR markers, homozygous and heterozygous genotypes were identified in F₂ generation of two crosses (ADT43 × IRBB60 and ADT47 × IRBB60) and goodness of fit was tested. Eighty nine plants from F₃ generation of ADT43 × IRBB60 were also screened for resistance genes. The genotypes carrying resistance genes in different combinations were identified. The pyramided lines showed a wider spectrum and higher level of resistance against two Xoo isolates under field conditions.     

Introduction of bacterial blight resistance into Triguna,a high yielding,mid-early duration rice variety

Bacterial blight (BB) is a serious disease of rice in India. We have used molecular marker-assisted selection in a backcross breeding program to introgress three genes (Xa21, xa13, and xa5) for BB resistance into Triguna, a mid-early duration, high yielding rice variety that is susceptible to BB. At each generation in the backcross program, molecular markers were used to select plants possessing these resistance genes and to select plants that have maximum contribution from the Triguna genome. A selected BC3F1 plant was selfed to generate homozygous BC₃F₂ plants with different combinations of BB resistance genes. Plants containing the two-gene combination, Xa21 and xa13, were found to exhibit excellent resistance against BB. Single plant selections for superior agronomic characteristics were performed on the progeny of these plants, from BC₃F₃ generation onwards. The selected plants were subjected to yield trials at the BC₃F₈ generation and were found to have a significant yield advantage over Triguna. The newly developed lines are being entered into national multi-location field trials. This work represents a successful example of the application of molecular marker-assisted selection for BB resistance breeding in rice.     

Identification and expression analysis of components involved in rice Xa21‐mediated disease resistance signalling

Rice Xa21 gene encodes a receptor‐like kinase that confers broad‐spectrum resistance against Xanthomonas oryzae pv. oryzae (Xoo). Recently, a number of genes involved in the Xa21‐mediated disease resistance pathway have been identified. Based on our previous data and the literature, we chose 16 candidate proteins and made corresponding antibodies. Using Western blotting, we systematically investigated the expression profile of the proteins in Xa21‐mediated disease resistance response. We found nine proteins with altered expression. We further compared their expression in resistance, susceptible and mock responses, and found that GST expression was up‐regulated during the resistance process, indicating GST is a positive regulator in resistance responses. ATPsB expression was down‐regulated during both the resistance and susceptible response processes, although it was higher in the resistance response than that in the susceptible response. The total amount of MYB, GAPDH, CatB, Trx and NB‐ARC proteins was lower in the resistance than in the susceptible response, but their abundance per unit bacteria in the resistance response was still higher than in the susceptible response, suggesting that these proteins might be positive regulators in the resistance response. In addition, expression of another ERF was induced by inoculation with bacterial blight pathogen, and expression of Zf‐LSD1 was activated by wounding stress alone. Interestingly, most proteins showed similar altered expression patterns in the resistance and susceptible responses, but differed to some extents, implying that both responses might share common molecular mechanisms. This study revealed evidence of resistance‐related protein expression, providing a foundation for better understanding of their functions.     

Markers for selection of the rice Xa21 disease resistance gene

Six molecular markers were mapped to a 7.4-cM region of rice chromosome 11 containing the Xa21 gene, which confers resistance to the pathogen Xanthomonas oryzae pv oryzae. Three markers, RG103, 248 and 818, co-segregated with Xa21 in a population of 1141 plants. Multiple copies of all marker loci were present within the region that was introgressed from Oryza longistaminata into O. sativa. The marker loci were cloned and primers were designed that defined sequence-tagged sites. Physical mapping of the three tightly linked central markers revealed that RG103, the marker that hybridizes to the Xa21 gene, resides on a separate DNA fragment from the other two markers.Disclaimer: Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

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。     

云南30个水稻品种对水稻白叶枯病的抗性评价

【目的】水稻白叶枯病是一种严重危害水稻的细菌性病害,培育抗性品种是治理该病害的重要途径。因此,明确云南水稻材料对检疫性病害水稻白叶枯病的抗性,可以为该病害的防治与监测、水稻栽培的合理布局和良好抗性资源的获取提供依据。【方法】采用剪叶接种法测定云南稻区30个品种对7个不同致病型白叶枯病菌的抗性。【结果】在供试的30个云南水稻品种中,2个品种(玉粳16和JS42糯稻)对7个不同致病型菌株均表现为抗性;15个品种对7个致病型菌株均表现感病;对HEN11、SCYC-6、YN7、YN11、FUJ、YN241和PX099等7个致病型菌株表现抗性的水稻品种分别占26.67%、16.67%、23.33%、13.33%、6.67%、10.00%和20.00%。此外,区试材料的抗性比例高于主栽品种,地方稻未发现抗性品种。【结论】现在生产上的大部分水稻品种对优势致病型病原菌入侵的抵抗能力降低甚至丧失。针对云南地区的优势致病小种FUJ筛选得到2个抗性品种:玉粳16和JS42糯稻。     

Rice NRR, a negative regulator of disease resistance, interacts with Arabidopsis NPR1 and rice NH1

Arabidopsis NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR), which confers lasting broad-spectrum resistance. Over-expression of Arabidopsis NPR1 or the NPR1 homolog 1 (NH1) in rice results in enhanced resistance to the pathogen Xanthomonasoryzae pv. oryzae (Xoo), suggesting the presence of a related defense pathway in rice. We investigated this pathway in rice by identifying proteins that interact with NH1. Here we report the isolation and characterization of a rice cDNA encoding a novel protein, named NRR (for negative regulator of resistance). NRR interacts with NPR1 in the NPR1-interacting domain (NI25) consisting of 25 amino acids. NRR also interacts with NH1; however, NI25 was not sufficient for a strong interaction, indicating a difference between the rice and the Arabidopsis proteins. Silencing of NRR in rice had little effect on resistance to Xoo. When constitutively over-expressed in rice, NRR affected basal resistance, age-related resistance and Xa21-mediated resistance, causing enhanced susceptibility to Xoo. This phenotype was correlated with elevated NRR mRNA and protein levels and increased Xoo growth. Over-expression of NRR suppressed the induction of defense-related genes. NRR:GFP (green fluorescent protein) protein was localized to the nucleus, indicating that NRR may act directly to suppress the activation of defense genes. The fact that NRR compromises Xa21-mediated resistance indicates cross-talk or overlap between NH1- and Xa21-mediated pathways.     

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。     

Identification and fine mapping of a new bacterial blight resistance gene,Xa43(t), in Zhangpu wild rice (Oryza rufipogon)

• Bacterial blight (BB) is currently considered one of the most serious rice diseases and is caused by Xanthomonas oryzae pv. oryzae (Xoo). Numerous studies have shown that breeding resistant rice varieties is one of the most effective methods to prevent BB, and it is important to identify and isolate more BB resistance (R) genes from different rice resources.
• Using a map-based approach, we identified a new QTL/gene, Xa43(t), from ZhangPu wild rice, which was highly resistant to the BB isolate PX099. We performed bulked segregant analysis combined with candidate gene prediction to identify the candidate gene.
• The Xa43(t) gene was narrowed down to a 29-kb region containing four putative genes. More importantly, the candidate gene Xa43(t) did not affect the main agronomic traits of rice. We also identified a widely applicable molecular marker, namely Inde1-18, which co-segregates with the Xa43(t) gene.
• The Xa43(t) gene is a new broad-spectrum BB resistance gene without identified alleles and has good application prospects for rice disease resistance breeding.

     

Marker assisted selection of bacterial blight resistance genes in rice

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae is one of the most important diseases affecting rice production in Asia. We were interested in surveying rice genotypes that are popularly used in the Indian breeding program for conferring resistance to bacterial blight, using 11 STMS and 6 STS markers. The basis of selection of these DNA markers was their close linkage to xa5, xa13, and Xa21 genes and their positions on the rice genetic map relative to bacterial blight resistance genes. Eight lines were found to contain the xa5 gene while two lines contained Xa21 gene and none of the lines contained the xa13 gene with the exception of its near-isogenic line. Using the polymorphic markers obtained in the initial survey, marker-assisted selection was performed in the F3 population of a cross between IR-64 and IET-14444 to detect lines containing multiple resistance genes. Of the 59 progeny lines analyzed, eight lines contained both the resistance genes, xa5 and Xa4.