WO2022100031A1 - 水稻白叶枯病抗病基因Xa7及其应用 - Google Patents
水稻白叶枯病抗病基因Xa7及其应用 Download PDFInfo
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
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Definitions
- the invention belongs to the field of genetic engineering, in particular to a rice bacterial blight disease resistance gene Xa7 and its application.
- Bacterial blight of rice is caused by Xanthomonas oryzae pv.oryzae (Xoo), which is an important bacterial disease of rice. It occurs in rice producing areas all over the world, and can lead to a 20% reduction in yield all year round. -50%, abortion in severe cases. Breeding and planting resistant varieties is the most economical, effective and greenest measure to control bacterial blight.
- Xoo Xanthomonas oryzae pv.oryzae
- R genes correspond to the matched avirulence gene (avr) races of B. blight, showing a "gene-to-gene" resistance relationship between rice and the pathogen.
- avr gene of bacterial blight the products of the cloned and identified avr gene are all transcription factor effector (Transcription activator-like effector, TALE for short).
- TALE Transcription activator-like effector
- Pathogens transport the TALE protein into the rice nucleus through the three-type secretion system, and bind to the promoter of the rice R or S gene.
- the bound DNA sequence is called EBE (effector-binding element), which activates the expression of the R or S gene, resulting in Rice appears to be disease resistant or susceptible.
- the bacterial blight TALE protein has typical structural features: the N-terminal 280 amino acids (aa) contain secretion and translocation signals; the middle part is a repeating region (CRR) composed of 34aa repeating units, each TALE protein. The number of repeating units in the repeating region is different. The 12 and 13 aa of each repeating unit are highly variable, and the others are the same; the 12 and 13 highly variable amino acid residues are called RVDs, and each RVD binds 1 DNA base , determines the specificity with rice target genes; after CRR, there are 3 nuclear localization signals (NLS) and 1 acidic transcriptional activation domain (AD) at the C-terminus.
- NLS nuclear localization signals
- AD acidic transcriptional activation domain
- Identifying the corresponding relationship between rice disease resistance genes and bacterial blight TALE will help to deeply reveal the mechanism of rice susceptibility and the interaction mechanism between rice and pathogenic bacteria. Further breeding or breeding of related resistant varieties will not only be better It has important breeding value for rice gene function and rice disease resistance breeding.
- the technical problem to be solved by the present invention is: how to provide a rice bacterial blight disease resistance gene to overcome the shortage of existing rice resistance resources, so as to further select or cultivate related resistant varieties, better control and reduce white leaves Blight damage to rice.
- the technical solution provided by the present invention is to provide a rice bacterial blight resistance gene Xa7 and its application.
- the present invention provides rice bacterial blight resistance gene Xa7, the nucleotide sequence of which is shown in SEQ ID No.1.
- the present invention provides the protein encoded by the rice bacterial blight disease resistance gene Xa7, denoted as XA7 protein, which consists of 113 amino acids, and its amino acid sequence is shown in SEQ ID No. 2.
- Rice bacterial blight resistance gene Xa7 contains the EBE sequence combined with bacterial blight pathogenic effector AvrXa7 and PthXo3, its nucleotide sequence such as SEQ ID No.3 and its nucleotide sequence such as SEQ ID No. 3 ID No.4.
- the invention also provides the application of the rice bacterial blight resistance gene Xa7 in the breeding of rice bacterial blight resistance.
- the Xa7 gene is transferred into the susceptible rice variety to improve the rice resistance to bacterial blight. Blight ability.
- a susceptible rice variety may be a rice material that does not contain Xa7, such as Nipponbare.
- the present invention also provides a primer pair for cloning the rice bacterial blight resistance gene Xa7, the forward primer sequence is shown in SEQ ID No.5, and the reverse primer sequence is shown in SEQ ID No.6.
- the present invention also provides a method for cloning Xa7 with a primer pair of rice bacterial blight resistance gene Xa7, using the forward primer sequence shown in SEQ ID No.5 and the reverse primer sequence shown in SEQ ID No.6,
- the rice bacterial blight resistance gene Xa7 was obtained by PCR amplification and cloning from rice varieties IRBB7 and DV85, respectively.
- the present invention also provides a method for preparing rice containing the rice bacterial blight resistance gene Xa7.
- the rice bacterial blight resistance gene Xa7 is constructed on a transgenic vector, and transformed into a susceptible gene by an Agrobacterium-mediated method.
- diseased rice a regenerated rice plant containing the rice bacterial blight resistance gene Xa7 is obtained.
- the invention also protects rice containing the bacterial blight resistance gene Xa7.
- the nucleotide sequence of the Xa7 gene is cloned and obtained by the genetic engineering method, which encodes the rice XA7 protein, and has an important effect on the resistance of rice to bacterial blight.
- the present application obtained rice plants containing Xa7 gene by Agrobacterium-mediated transgenic method in bacterial blight-susceptible rice Japanese nitrile, and injected and cut leaves to inoculate white leaves Bacterial resistance was identified.
- the results of resistance identification showed that the rice plants containing the Xa7 gene were resistant to the infection of the bacterial blight bacteria containing the AvrXa7 and PthXo3 pathogenic protein-encoding genes, indicating that the Xa7 gene was positively correlated with the rice bacterial blight resistance.
- the transgenic rice plants of Xa7 gene did not show obvious changes in agronomic traits.
- the anti-bacterial blight gene of Xa7 has not been cloned, but the avirulence gene avrXa7 corresponding to bacterial blight has been cloned.
- the AvrXa7 protein contains 26 RVDs, and its deduced binding EBE was used to find its target genes in the rice germplasm bank genome.
- the present invention has the following advantages and beneficial effects:
- the Xa7 gene of the present invention is a disease resistance gene of rice bacterial blight, which is directly related to the bacterial blight race containing AvrXa7 and PthXo3 pathogenic protein encoding genes, and has no known other disease resistance genes of bacterial blight. homology. Genetic and molecular biological function analysis proved that Xa7 gene was dominant in resistance. Transplantation into susceptible varieties could change rice from susceptible to disease-resistant, and could significantly improve the resistance to bacterial blight. The rice plants transformed with the Xa7 gene did not have obvious changes in agronomic traits, that is, the Xa7 gene did not cause significant changes in rice agronomic traits.
- Figure 1 Schematic diagram of the structure of the rice bacterial blight resistance gene Xa7 and its encoded product.
- the sequence in the upper row represents the DNA sequence bound by the PthXo3 protein, and the sequence in the lower row represents the DNA sequence bound by the AvrXa7 protein.
- Figure 2 Determination of Xa7 resistance to bacterial blight after inoculation by leaf-cut inoculation into susceptible rice japonitrile.
- Nip-Xa7 indicates that Xa7 was transformed into Japanese nitrile rice, and AvrXa7 and PthXo3 indicate avirulence genes that activate Xa7 resistance.
- Figure 3 Determination of Xa7 resistance to bacterial blight after inoculation into susceptible rice japonitrile. Brown indicates disease resistance, and water-soaked indicates susceptible. Nip-Xa7 indicates that Xa7 was transformed into Japanese nitrile rice, and AvrXa7 and PthXo3 indicate avirulence genes that activate Xa7 resistance.
- PCR product was recovered and digested with endonucleases NcoRI and HindIII; pCAMBIA1300 plasmid DNA was digested with NcoRI and HindIII.
- the Xa7 gene contains only one exon.
- the numbers on the gene diagram represent the number of DNA bases, the sequence in the upper row represents the DNA sequence bound by the PthXo3 protein, and the sequence in the lower row represents the DNA sequence bound by the AvrXa7 protein.
- Agrobacterium infects rice callus
- the Xa7 gene in the regenerated rice plants was detected and sequenced by PCR, respectively. Referring to Figure 1, the results show that the Xa7 gene was transferred into Nipponbare rice material, named Nip-Xa7.
- PXO99 A is a bacterial race that does not contain AvrXa7 and PthXo3 pathogenic proteins
- PXO99A (AvrXa7) and PXO99A (PthXo3) represent the white bacterial blight containing AvrXa7 and PthXo3 encoding genes, respectively.
- Leaf blight race
- Japanese nitrile rice is susceptible and also does not contain the Xa7 gene; on DV85 and IRBB7 rice, the leaves are inoculated with strains PXO99A (AvrXa7) and PXO99A (PthXo3) containing AvrXa7 or PthXo3, and the length of the lesions is basically not formed, while the inoculation does not contain AvrXa7 Or the PXO99A strain of PthXo3, the bacterial blight lesions were formed, and the length was more than 10 cm, indicating that the Xa7 gene was matched with AvrXa7 or PthXo3; on the Xa7 transgenic rice Nip-Xa7, inoculate the strain PXO99A (AvrXa7) containing AvrXa7 or PthXo3 and PXO99A (PthXo3) could not form a lesion length, while inoculation of PXO99A strain without AvrXa7
- the results of injection at the seedling stage showed that on rice containing Xa7 gene, whether it was rice IRBB7, DV85, or Xa7 transgenic rice Nip-Xa7, inoculated with PXO99A (AvrXa7) and PXO99A (PthXo3) strains containing AvrXa7 or PthXo3,
- the injected area produced a brown disease-resistant response (shown as a dark area in the figure). If the bacterial blight does not contain the AvrXa7 or PthXo3 pathogenic effector proteins, or if the rice does not contain the Xa7 gene, it shows a susceptible response to waterlogging symptoms (shown as a light-colored area in the figure).
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Abstract
涉及水稻抗白叶枯病基因Xa7及其应用，属于基因工程技术领域。Xa7基因编码113个氨基酸，启动子区域含有白叶枯病菌效应蛋白AvrXa7和PthXo3结合的EBE元件。Xa7基因转入感病水稻品种中可使水稻由感病转变为抗病。转入Xa7基因的水稻，没有明显的农艺性状变化。Xa7基因可用于抗白叶枯病水稻育种。
Description
本发明属于基因工程领域，尤其是涉及一种水稻白叶枯病抗病基因Xa7及其应用。
水稻白叶枯病由稻黄单胞菌稻致病变种(Xanthomonas oryzae pv.oryzae，Xoo)引起，是一种重要的水稻细菌病害，世界各地水稻产区均有发生，常年可导致减产20％-50％，严重时绝产。培育和种植抗病品种是防治白叶枯病最经济、最有效和最绿色的措施。
水稻不同品种对白叶枯病菌侵染的抗性不同，至今从水稻不同品种和材料中鉴定了40多个抗病(R)基因，但仅有少数抗病基因被分离和克隆，有些是显性抗病基因，如Xa1、Xa4/Xa26、Xa10、Xa21、Xa23、Xa27等，有些是隐性抗病基因，如xa5、xa13和xa41(t)等。水稻隐性抗病基因的等位基因是感病(S)基因。多数R基因，例如Xa7等，虽然已知广谱抗病，至今未被克隆。这些R基因对应白叶枯病菌匹配的无毒基因(avr)的小种，表现为水稻和病原菌间的“基因-对-基因”抗性关系。就白叶枯病菌的avr基因而言，目前克隆和鉴定的avr基因的产物均为转录因子类效应物(Transcription activator-like effector，简称TALE)。病原菌通过三型分泌系统将TALE蛋白转运注入水稻细胞核中，结合在水稻R或S基因的启动子上，结合的DNA序列称为EBE(effector-binding element)，激活R或S基因表达，从而导致水稻表现为抗病或感病。研究发现，白叶枯病菌TALE蛋白具有典型的结构特征：N-端280氨基酸(aa)含有分泌和转位信号；中间部分是由34aa的重复单元构成的重复区(CRR)，每个TALE蛋白重复区的重复单元数不一样，每个重复单元的12和13位aa高度变异，其他均相同；12和13位高度变异的氨基酸残基称之为RVD，每个RVD结合1个DNA碱基，决定与水稻靶标基因的专一性；CRR后在C-端有3个核定位信号(NLS)和1个酸性转录激活域(AD)。
鉴定水稻抗病基因与白叶枯病菌TALE间的对应关系，有助于深入揭示水稻感病机理以及水稻与病原菌间的互作机制，进一步的选育或者培育相关的抗性品种，不仅更好地控制白叶枯病的危害，同时对水稻基因功能和水稻抗病育种都具有重要 的育种价值。
发明内容
本发明所要解决的技术问题为：如何提供水稻白叶枯病抗病基因，克服现有水稻抗性资源的不足，以便进一步选育或者培育相关的抗性品种，更好地控制和降低白叶枯病菌对水稻的危害。
基于上述技术问题，本发明提供的技术方案是：提供水稻白叶枯病抗病基因Xa7及其应用。
本发明的目的可以通过以下技术方案来实现：
本发明提供水稻白叶枯病抗病基因Xa7，其核苷酸序列如SEQ ID No.1所示。
本发明提供水稻白叶枯病抗病基因Xa7编码的蛋白，记为XA7蛋白，由113个氨基酸组成，其氨基酸序列如SEQ ID No.2所示。
水稻白叶枯病抗病基因Xa7，其启动子中含有白叶枯病菌致病效应子AvrXa7和PthXo3结合的EBE序列，其核苷酸序列如SEQ ID No.3和其核苷酸序列如SEQ ID No.4所示。
本发明还提供水稻白叶枯病抗病基因Xa7在培育水稻抗白叶枯病育种上的应用。
在本发明提供的一个关于水稻白叶枯病抗病基因Xa7在培育水稻抗白叶枯病育种上的应用技术方案中，通过在感病水稻品种中转入Xa7基因，来提高水稻抗白叶枯病的能力。
感病水稻品种可以是不含有Xa7的水稻材料，例如日本腈(Nipponbare)。
本发明还提供克隆所述水稻白叶枯病抗病基因Xa7的引物对，其正向引物序列如SEQ ID No.5所示，反向引物序列如SEQ ID No.6所示。
本发明还提供水稻白叶枯病抗病基因Xa7的引物对克隆Xa7的方法，使用如SEQ ID No.5所示的正向引物序列和如SEQ ID No.6所示的反向引物序列，分别从水稻品种IRBB7和DV85水稻中进行PCR扩增和克隆获得所述水稻白叶枯病抗病基因Xa7。
本发明还提供制备含有所述水稻白叶枯病抗病基因Xa7的水稻的方法，将所述水稻白叶枯病抗病基因Xa7构建在转基因载体上，通过农杆菌介导的方法转入感病水稻中，获得含有所述水稻白叶枯病抗病基因Xa7的再生水稻植株。
本发明还保护含有白叶枯病抗病基因Xa7的水稻。
本发明利用基因工程方法克隆获得了Xa7基因的核苷酸序列，编码水稻XA7蛋白，对水稻抗白叶枯病具有重要作用。
为了进一步确认Xa7基因的抗病功能，本申请在感白叶枯病水稻日本腈中，通过农杆菌介导的转基因方法，获得了含有Xa7基因的水稻植株，对其注射和剪叶接种白叶枯病菌进行抗性鉴定。抗性鉴定结果显示，含有Xa7基因的水稻植株，抗分别含有AvrXa7和PthXo3致病蛋白编码基因的白叶枯病菌的侵染，说明Xa7基因与水稻白叶枯病抗性呈正相关。同时发现，Xa7基因的转基因水稻植株并没有出现明显的农艺性状变化。
在本发明以前，Xa7抗白叶枯病基因没有被克隆，但其对应白叶枯病菌的无毒基因avrXa7已被克隆。AvrXa7蛋白含有26个RVD，利用其推导结合的EBE在水稻种质资源库基因组中，来发现其作用的靶标基因。
与现有技术相比，本发明具有以下优点及有益效果：
本发明的Xa7基因是水稻白叶枯病的抗病基因，与含有AvrXa7和PthXo3致病蛋白编码基因的白叶枯病菌小种直接关联，与已知的白叶枯病的其他抗病基因没有同源性。遗传和分子生物学功能分析证明，Xa7基因呈显性抗性作用，转入感病品种中可使水稻由感病转变为抗病，可显著提高对白叶枯病的抗性。转入Xa7基因的水稻植株没有明显的农艺性状变化，即Xa7基因不会造成明显的水稻农艺性状改变。
图1：水稻白叶枯病抗病基因Xa7和其编码产物的结构示意图。上面一行的序列示意PthXo3蛋白结合的DNA序列，下面一行的序列示意AvrXa7蛋白结合的DNA序列。
图2：剪叶接种方法测定Xa7转入感病水稻日本腈中后对白叶枯病的抗性。Nip-Xa7示意Xa7转入日本腈后的水稻，AvrXa7和PthXo3示意激活Xa7抗性的无毒基因。
图3：注射接种测定Xa7转入感病水稻日本腈中后对白叶枯病的抗性。褐色示意抗病，水渍状示意感病。Nip-Xa7示意Xa7转入日本腈后的水稻，AvrXa7和PthXo3示意激活Xa7抗性的无毒基因。
下面结合附图和具体实施例对本发明进行详细说明。
以下实施例中没有做详细说明的均是采用本领域常规实验手段就能实现。
实施例1
水稻白叶枯病抗病基因Xa7的克隆
1)、IRBB7和DV85水稻的基因组DNA提取
2)、引物设计：
正向：5′-TAC
GAATTCAACCAATATATAACCCCCCCCCCCCCA-3′(SEQ ID No.5)
反向：5′-TCC
AAGCTTTCATTAATTGCCACCGATGAGGTAATC-3′(SEQ ID No.6)
3)、Xa7基因全长扩增
4)PCR产物回收，经核酸内切酶NcoRI和HindIII消化；pCAMBIA1300质粒DNA经NcoRI和HindIII消化。
5)产物连接及转化。
6)提取含有分别来自IRBB7和DV85基因组DNA的质粒。
7)利用正向引物SEQ ID No.5和反向引物SEQ ID No.6进行测序。
8)对测序结果进行分析，确定Xa7基因及其编码产物的结构特征。
如图1所示，图1中Xa7基因的结构示意图。Xa7基因仅含有1个外显子。基因示意图上的数字代表DNA碱基数，上面一行的序列示意PthXo3蛋白结合的DNA 序列，下面一行的示意AvrXa7蛋白结合的DNA序列。
实施例2 Xa7基因转入感病水稻日本腈(Nipponbare)中的制备
Xa7基因转入日本腈的制备步骤与方法：
1)、提取含有Xa7基因的载体pCAMBIA1300质粒DNA；
2)、转化农杆菌EHA105；
3)、农杆菌感染水稻愈伤组织；
4)、在含潮霉素抗生素培养基上获得水稻再生植株。
利用正向引物(SEQ ID No.5)和反向引物(SEQ ID No.6)，分别PCR检测和测序水稻再生植株中Xa7基因。参考图1，结果显示，Xa7基因转入日本腈(Nipponbare)水稻材料中，命名为Nip-Xa7。
实施例3
含有Xa7基因的水稻抗病性鉴定：
1)准备苗期和扬花期的水稻，品种分别为Nipponbare、IRBB7、DV85和、Nip-Xa7。
2)准备生长良好的白叶枯病菌，PXO99
A为不含有AvrXa7和PthXo3致病蛋白的白叶枯病菌小种，PXO99A(AvrXa7)和PXO99A(PthXo3)，分别代表含有AvrXa7和PthXo3编码基因的白叶枯病菌小种。
3)制备接种用的白叶枯病菌孢子液。
4)苗期注射接种，3天后观察注射区域的褐色反应。
5)扬花期剪叶接种，14天后测量病斑长度(cm)。
水稻生长条件：温度28℃，湿度90-95％，光照14h，黑暗10h。
试验结果如图2所示，感病水稻日本腈，无论接种PXO99
A菌株，还是PXO99A(AvrXa7)和PXO99A(PthXo3)菌株，扬花期剪叶接种，病斑长度在10cm以 上(图2)，说明日本腈水稻感病，也不含有Xa7基因；在DV85和IRBB7水稻上，剪叶接种含有AvrXa7或PthXo3的菌株PXO99A(AvrXa7)和PXO99A(PthXo3)，基本不形成病斑长度，而接种不含有AvrXa7或PthXo3的PXO99A菌株，则形成白叶枯病病斑，长度在10cm以上，说明Xa7基因与AvrXa7或PthXo3匹配；在Xa7转基因水稻Nip-Xa7上，接种含有AvrXa7或PthXo3的菌株PXO99A(AvrXa7)和PXO99A(PthXo3)，不能形成病斑长度，而接种不含有AvrXa7或PthXo3的PXO99A菌株，则能形成10cm以上的病斑长度。
苗期注射结果显示(图3)，在含有Xa7基因的水稻上，无论是水稻IRBB7、DV85，还是转Xa7水稻Nip-Xa7，接种含有AvrXa7或PthXo3的PXO99A(AvrXa7)和PXO99A(PthXo3)菌株，注射区域产生褐色的抗病反应(图中示为深色的区域)。如果白叶枯病菌不含有AvrXa7或PthXo3致病效应蛋白，或者水稻中不含有Xa7基因，则显示水渍症状的感病反应(图中示为浅色的区域)。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (10)
- 水稻白叶枯病抗病基因Xa7，其特征在于，其核苷酸序列如SEQ ID No.1所示。
- 根据权利要求1所述水稻白叶枯病抗病基因Xa7，其特征在于，其启动子中含有被白叶枯病菌致病效应蛋白AvrXa7所结合的DNA序列，该序列如SEQ ID No.3所示，含有被白叶枯病菌致病效应蛋白PthXo3所结合的DNA序列，该序列如SEQ ID No.4所示。
- 根据权利要求1所述水稻白叶枯病抗病基因Xa7，其特征在于，其通过引物对被克隆，其正向引物序列如SEQ ID No.5所示，反向引物序列如SEQ ID No.6所示。
- 如权利要求1所述水稻白叶枯病抗病基因Xa7编码的蛋白，其特征在于，其氨基酸序列如SEQ ID No.2所示。
- 克隆如权利要求1所述水稻白叶枯病抗病基因Xa7的引物对，其特征在于，其正向引物序列如SEQ ID No.5所示，反向引物序列如SEQ ID No.6所示。
- 克隆如权利要求1所述水稻白叶枯病抗病基因Xa7的方法，其特征在于，使用如SEQ ID No.5所示的正向引物序列和如SEQ ID No.6所示的反向引物序列，分别从水稻品种IRBB7和DV85水稻中进行PCR扩增和克隆获得如权利要求1所述水稻白叶枯病抗病基因Xa7。
- 如权利要求1所述水稻白叶枯病抗病基因Xa7在培育水稻抗白叶枯病育种上的应用。
- 根据权利要求7所述水稻白叶枯病抗病基因Xa7在培育水稻抗白叶枯病育种上的应用，其特征在于，通过在感病水稻品种中转入所述水稻白叶枯病抗病基因Xa7，用以提高水稻抗白叶枯病的能力。
- 根据权利要求8所述水稻白叶枯病抗病基因Xa7在培育水稻抗白叶枯病育种上的应用，其特征在于，所述感病水稻品种为不含有Xa7的水稻材料。
- 制备含有如权利要求1所述水稻白叶枯病抗病基因Xa7的水稻的方法，其特征在于，将如权利要求1所述水稻白叶枯病抗病基因Xa7构建在转基因载体上，通过农杆菌介导的方法转入感病水稻中，获得含有如权利要求1所述水稻白叶枯病抗病基因Xa7的再生水稻植株。
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