技术领域technical field
本发明涉及生物技术领域中抗病相关蛋白在调控植物抗病性中的应用。The invention relates to the application of disease resistance-related proteins in regulating plant disease resistance in the field of biotechnology.
背景技术Background technique
水稻不仅是世界上最为重要的粮食作物，约有全球半数以上人口以此为食，它还是单子叶植物研究的模式植物。然而，在可耕地面积不断减少，人口数量不断膨胀的形势下，水稻产量已成为影响我国粮食安全的重要因素之一。虽然经历了绿色革命和杂交技术的广泛应用，但病虫害，恶劣的自然环境等因素依然严重地制约了水稻产量的增长。白叶枯病是水稻生产中的所遇到的非常严重的病害，水稻感染上白叶枯病后，一般减产20-30％，甚至绝收。鉴定和分离水稻抗白叶枯病基因，特别是生产中广泛应用的广谱抗病基因已成为水稻分子育种的重要环节。Rice is not only the most important food crop in the world, which is eaten by more than half of the world's population, it is also a model plant for the study of monocots. However, under the situation of decreasing arable land area and expanding population, rice yield has become one of the important factors affecting my country's food security. Although experienced the green revolution and the wide application of hybridization technology, factors such as pests and diseases and harsh natural environment still seriously restrict the growth of rice yield. Bacterial blight is a very serious disease encountered in rice production. After rice is infected with bacterial blight, the yield is generally reduced by 20-30%, or even no harvest. Identifying and isolating rice bacterial blight resistance genes, especially broad-spectrum resistance genes widely used in production, has become an important part of rice molecular breeding.
发明内容Contents of the invention
本发明所要解决的技术问题是如何提高植物的抗病性。The technical problem to be solved by the invention is how to improve the disease resistance of plants.
为解决上述技术问题，本发明首先提供了抗病相关蛋白在调控植物抗病性中的应用；所述抗病相关蛋白的名称为OsAPX8，是如下A1)、A2)或A3)：In order to solve the above-mentioned technical problems, the present invention firstly provides the application of the disease resistance-related protein in regulating plant disease resistance; the name of the disease resistance-related protein is OsAPX8, which is as follows A1), A2) or A3):
A1)氨基酸序列是序列2的蛋白质；A1) the amino acid sequence is the protein of sequence 2;
A2)将序列表中序列2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物抗病相关的蛋白质；A2) A protein in which the amino acid sequence shown in Sequence 2 in the sequence listing has been substituted and/or deleted and/or added by one or several amino acid residues and is related to plant disease resistance;
A3)在A1)或A2)的N端或/和C端连接标签得到的融合蛋白质。A3) A fusion protein obtained by linking a tag at the N-terminal or/and C-terminal of A1) or A2).
为了使A1)中的蛋白质便于纯化，可在由序列表中序列2所示的氨基酸序列组成的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。In order to make the protein in A1) easy to purify, the amino-terminal or carboxy-terminal of the protein consisting of the amino acid sequence shown in Sequence 2 in the Sequence Listing can be linked with the tags shown in Table 1.
表1、标签的序列Table 1. Sequence of tags
上述A2)中，所述一个或几个氨基酸残基的取代和/或缺失和/或添加为不超过10个氨基酸残基的取代和/或缺失和/或添加。In the above A2), the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.
上述A2)中OsAPX8可人工合成，也可先合成其编码基因，再进行生物表达得到。OsAPX8 in the above A2) can be synthesized artificially, or its coding gene can be firstly synthesized and then biologically expressed.
上述A2)中OsAPX8的编码基因可通过将序列1或序列4所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。The gene encoding OsAPX8 in the above A2) can be deleted by deleting one or several amino acid residue codons in the DNA sequence shown in Sequence 1 or Sequence 4, and/or performing missense mutations of one or several base pairs, And/or obtained by connecting the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.
为解决上述技术问题，本发明还提供了与OsAPX8相关的生物材料在调控植物抗病性中的应用；所述生物材料为下述B1)至B16)中的任一种：In order to solve the above technical problems, the present invention also provides the application of biological materials related to OsAPX8 in the regulation and control of plant disease resistance; the biological materials are any one of the following B1) to B16):
B1)编码OsAPX8的核酸分子；B1) a nucleic acid molecule encoding OsAPX8;
B2)含有B1)所述核酸分子的表达盒；B2) an expression cassette containing the nucleic acid molecule of B1);
B3)含有B1)所述核酸分子的重组载体；B3) a recombinant vector containing the nucleic acid molecule of B1);
B4)含有B2)所述表达盒的重组载体；B4) a recombinant vector containing the expression cassette described in B2);
B5)含有B1)所述核酸分子的重组微生物；B5) a recombinant microorganism containing the nucleic acid molecule of B1);
B6)含有B2)所述表达盒的重组微生物；B6) a recombinant microorganism containing the expression cassette described in B2);
B7)含有B3)所述重组载体的重组微生物；B7) a recombinant microorganism containing the recombinant vector described in B3);
B8)含有B4)所述重组载体的重组微生物；B8) a recombinant microorganism containing the recombinant vector described in B4);
B9)含有B1)所述核酸分子的转基因植物细胞系；B9) a transgenic plant cell line containing the nucleic acid molecule of B1);
B10)含有B2)所述表达盒的转基因植物细胞系；B10) a transgenic plant cell line containing the expression cassette of B2);
B11)含有B1)所述核酸分子的转基因植物组织；B11) a transgenic plant tissue containing the nucleic acid molecule of B1);
B12)含有B2)所述表达盒的转基因植物组织；B12) transgenic plant tissue containing the expression cassette described in B2);
B13)含有B1)所述核酸分子的转基因植物器官；B13) a transgenic plant organ containing the nucleic acid molecule of B1);
B14)含有B2)所述表达盒的转基因植物器官；B14) a transgenic plant organ containing the expression cassette described in B2);
B15)降低OsAPX8表达的核酸分子；B15) reduce the nucleic acid molecule of OsAPX8 expression;
B16)含有B15)所述核酸分子的表达盒、重组载体、重组微生物或转基因植物细胞系。B16) An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule of B15).
上述应用中，B1)所述核酸分子可为如下b1)-b5)中任一种：In the above application, the nucleic acid molecule described in B1) can be any of the following b1)-b5):
b1)编码序列是序列表中序列4的cDNA分子或DNA分子；b1) The coding sequence is a cDNA molecule or a DNA molecule of sequence 4 in the sequence listing;
b2)核苷酸序列是序列表中序列1的cDNA分子或DNA分子；b2) The nucleotide sequence is a cDNA molecule or a DNA molecule of sequence 1 in the sequence listing;
b3)核苷酸序列是序列表中序列3的DNA分子；b3) The nucleotide sequence is the DNA molecule of sequence 3 in the sequence listing;
b4)与b1)或b2)或b3)限定的核苷酸序列具有75％或75％以上同一性，且编码OsAPX8的cDNA分子或基因组DNA分子；b4) It has 75% or more identity with the nucleotide sequence defined in b1) or b2) or b3), and encodes a cDNA molecule or genomic DNA molecule of OsAPX8;
b5)在严格条件下与b1)或b2)或b3)限定的核苷酸序列杂交，且编码OsAPX8的cDNA分子或基因组DNA分子；b5) hybridize to the nucleotide sequence defined by b1) or b2) or b3) under stringent conditions, and encode a cDNA molecule or a genomic DNA molecule of OsAPX8;
B15)所述核酸分子为与序列4所示的DNA分子中任一片段反向互补的DNA分子。B15) The nucleic acid molecule is a DNA molecule that is reverse complementary to any segment of the DNA molecule shown in Sequence 4.
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.
其中，序列1所示的DNA分子编码序列2所示的蛋白质。序列3来源于水稻，为OsAPX8基因的基因组序列，序列4为OsAPX8基因的cDNA序列。Wherein, the DNA molecule shown in sequence 1 encodes the protein shown in sequence 2. Sequence 3 is derived from rice and is the genome sequence of the OsAPX8 gene, and Sequence 4 is the cDNA sequence of the OsAPX8 gene.
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码OsAPX8的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明分离得到的OsAPX8的核苷酸序列75％或者更高同一性的核苷酸，只要编码OsAPX8且具有OsAPX8功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。Those skilled in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding OsAPX8 of the present invention. Those nucleotides that have been artificially modified and have 75% or higher identity with the nucleotide sequence of OsAPX8 isolated in the present invention, as long as they encode OsAPX8 and have the function of OsAPX8, are all derived from the nucleotide sequence of the present invention And is equivalent to the sequence of the present invention.
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码序列2所示的氨基酸序列组成的蛋白质的核苷酸序列具有75％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher, of the nucleotide sequence of the protein composed of the amino acid sequence shown in the coding sequence 2 of the present invention. Nucleotide sequences of higher identity. Identity can be assessed visually or with computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.
上述应用中，所述严格条件是在2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min；或，0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。In the above-mentioned application, the stringent conditions are in a solution of 2×SSC and 0.1% SDS, hybridize at 68° C. and wash the membrane twice, each time for 5 minutes, and then in a solution of 0.5×SSC and 0.1% SDS, in Hybridize and wash the membrane twice at 68°C, 15 min each time; or, hybridize and wash the membrane at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.
上述75％或75％以上同一性，可为80％、85％、90％或95％以上的同一性。The identity of 75% or more may be 80%, 85%, 90% or more.
上述应用中，B2)所述的含有编码OsAPX8的核酸分子的表达盒(OsAPX8基因表达盒)，是指能够在宿主细胞中表达OsAPX8的DNA，该DNA不但可包括启动OsAPX8基因转录的启动子，还可包括终止OsAPX8基因转录的终止子。进一步，所述表达盒还可包括增强子序列。可用于本发明的启动子包括但不限于：组成型启动子，组织、器官和发育特异的启动子，和诱导型启动子。启动子的例子包括但不限于：花椰菜花叶病毒的组成型启动子35S：来自西红柿的创伤诱导型启动子，亮氨酸氨基肽酶("LAP",Chao等人(1999)Plant Physiol 120：979-992)；来自烟草的化学诱导型启动子，发病机理相关1(PR1)(由水杨酸和BTH(苯并噻二唑-7-硫代羟酸S-甲酯)诱导)；西红柿蛋白酶抑制剂II启动子(PIN2)或LAP启动子(均可用茉莉酮酸甲酯诱导)；热休克启动子(美国专利5，187，267)；四环素诱导型启动子(美国专利5，057,422)；种子特异性启动子，如谷子种子特异性启动子pF128(CN101063139B(中国专利200710099169.7))，种子贮存蛋白质特异的启动子(例如，菜豆球蛋白、napin,oleosin和大豆beta conglycin的启动子(Beachy等人(1985)EMBO J.4：3047-3053))。它们可单独使用或与其它的植物启动子结合使用。此处引用的所有参考文献均全文引用。合适的转录终止子包括但不限于：农杆菌胭脂碱合成酶终止子(NOS终止子)、花椰菜花叶病毒CaMV 35S终止子、tml终止子、豌豆rbcS E9终止子和胭脂氨酸和章鱼氨酸合酶终止子(参见，例如：Odell等人(I985)Nature 313:810；Rosenberg等人(1987)Gene,56:125；Guerineau等人(1991)Mol.Gen.Genet,262:141；Proudfoot(1991)Cell,64:671；Sanfacon等人Genes Dev.,5:141；Mogen等人(1990)Plant Cell,2:1261；Munroe等人(1990)Gene,91:151；Ballad等人(1989)Nucleic Acids Res.17:7891；Joshi等人(1987)Nucleic AcidRes.,15:9627)。In the above-mentioned application, the expression cassette (OsAPX8 gene expression cassette) described in B2) containing the nucleic acid molecule encoding OsAPX8 refers to the DNA that can express OsAPX8 in the host cell, and the DNA can not only include the promoter that starts the transcription of the OsAPX8 gene, A terminator that terminates transcription of the OsAPX8 gene may also be included. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: Cauliflower Mosaic Virus Constitutive Promoter 35S: Wound-Inducible Promoter from Tomato, Leucine Aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato Protease inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoter (US Patent 5,187,267); tetracycline-inducible promoter (US Patent 5,057,422) ; Seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (for example, the promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine Synthase terminators (see, e.g.: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125;Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) ) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acids Res., 15:9627).
可用现有的表达载体构建含有OsAPX8基因表达盒的重组载体。所述植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等。如pAHC25、pBin438、pCAMBIA1302、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa或pCAMBIA1391-Xb(CAMBIA公司)等。所述植物表达载体还可包含外源基因的3′端非翻译区域，即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸信号可引导聚腺苷酸加入到mRNA前体的3′端，如农杆菌冠瘿瘤诱导(Ti)质粒基因(如胭脂碱合成酶基因Nos)、植物基因(如大豆贮存蛋白基因)3′端转录的非翻译区均具有类似功能。使用本发明的基因构建植物表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所用植物表达载体进行加工，如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因(GUS基因、萤光素酶基因等)、抗生素的标记基因(如赋予对卡那霉素和相关抗生素抗性的nptII基因，赋予对除草剂膦丝菌素抗性的bar基因，赋予对抗生素潮霉素抗性的hph基因，和赋予对氨甲喋呤抗性的dhfr基因，赋予对草甘磷抗性的EPSPS基因)或是抗化学试剂标记基因等(如抗除莠剂基因)、提供代谢甘露糖能力的甘露糖-6-磷酸异构酶基因。从转基因植物的安全性考虑，可不加任何选择性标记基因，直接以逆境筛选转化植株。The existing expression vector can be used to construct the recombinant vector containing the OsAPX8 gene expression cassette. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopaline synthase gene Nos), plant gene (such as soybean The untranslated region transcribed at the 3′ end of the storage protein gene) has similar functions. When using the gene of the present invention to construct plant expression vectors, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc. The reading frames of the sequences are identical to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) genes, etc.), antibiotic marker genes (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, and the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene that confers resistance to methotrexate, the EPSPS gene that confers resistance to glyphosate) or the chemical resistance marker gene (such as the herbicide resistance gene), the mannose-6- that provides the ability to metabolize mannose Phosphate isomerase gene. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.
上述应用中，所述载体可为质粒、黏粒、噬菌体或病毒载体。所述质粒具体可为pCAMBIA1300或pANDA，也可为将pCAMBIA1300改造后得到的载体，如pCAMBIA1300-35S。In the above application, the vector can be a plasmid, cosmid, phage or viral vector. The plasmid can specifically be pCAMBIA1300 or pANDA, and can also be a vector obtained by transforming pCAMBIA1300, such as pCAMBIA1300-35S.
B3)所述重组载体可含有序列1、序列3或序列4所示的用于编码OsAPX8的DNA序列；进一步B3)所述重组载体具体可为pCAMBIA1300-35S-OsAPX8。所述pCAMBIA1300-35S-OsAPX8为将pCAMBIA1300-35S的EcoRI和XbaI识别位点间的DNA序列替换为序列4所示的DNA序列得到的表达序列2所示的OsAPX8的重组载体。B3) The recombinant vector may contain the DNA sequence for encoding OsAPX8 shown in Sequence 1, Sequence 3 or Sequence 4; further B3) The recombinant vector may specifically be pCAMBIA1300-35S-OsAPX8. The pCAMBIA1300-35S-OsAPX8 is a recombinant vector expressing OsAPX8 shown in Sequence 2 obtained by replacing the DNA sequence between EcoRI and XbaI recognition sites of pCAMBIA1300-35S with the DNA sequence shown in Sequence 4.
B16)所述重组载体可含有降低OsAPX8表达的核酸分子。在本发明的一个实施例中，所述重组载体为pANDA-OsAPX8i，pANDA-OsAPX8i的LB与RB间的结构如下：B16) The recombinant vector may contain a nucleic acid molecule that reduces the expression of OsAPX8. In one embodiment of the present invention, the recombinant vector is pANDA-OsAPX8i, and the structure between LB and RB of pANDA-OsAPX8i is as follows:
Ubiquitin启动子-反向APX492-gus linker-正向APX492-NOS终止子，pANDA-OsAPX8i的其他序列与pANDA相同。Ubiquitin promoter-reverse APX492 -gus linker-forward APX492 -NOS terminator, other sequences of pANDA-OsAPX8i are the same as pANDA.
上述应用中，所述微生物可为酵母、细菌、藻或真菌。所述细菌具体可为农杆菌，如农杆菌EHA105。In the above applications, the microorganisms can be yeast, bacteria, algae or fungi. Specifically, the bacteria can be Agrobacterium, such as Agrobacterium EHA105.
上述应用中，所述转基因植物细胞系、转基因植物组织和转基因植物器官均不包括繁殖材料。In the above applications, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs do not include propagation materials.
上述应用中，B15)所述核酸分子具体可为与序列表中序列4的第1108-1599位核苷酸所示的DNA片段反向互补的DNA分子。In the above application, the nucleic acid molecule described in B15) can specifically be a DNA molecule that is reverse complementary to the DNA fragment shown in nucleotides 1108-1599 of Sequence 4 in the Sequence Listing.
为解决上述技术问题，本发明还提供了OsAPX8或所述生物材料的下述任一应用：In order to solve the above technical problems, the present invention also provides any of the following applications of OsAPX8 or the biological material:
H1、在培育抗病性增加或降低植物中的应用；H1. Application in breeding plants with increased or decreased disease resistance;
H2、在制备提高或降低植物抗病性产品中的应用。H2. Application in the preparation of products for improving or reducing plant disease resistance.
为解决上述技术问题，本发明还提供了下述G1或G2：In order to solve the above technical problems, the present invention also provides the following G1 or G2:
G1、OsAPX8或所述生物材料；G1, OsAPX8 or said biological material;
G2、调控植物抗病性产品，所述产品含有OsAPX8或所述生物材料。G2. A product for regulating plant disease resistance, said product containing OsAPX8 or said biological material.
上述产品中，所述调控植物抗病性产品可以以OsAPX8或所述生物材料作为活性成分，还可以将OsAPX8或所述生物材料与其它抗病性物质进行组合得到的组合物作为活性成分。Among the above products, the product for regulating plant disease resistance may use OsAPX8 or the biological material as an active ingredient, or a composition obtained by combining OsAPX8 or the biological material with other disease-resistant substances as an active ingredient.
为解决上述技术问题，本发明还提供了下述M1)或M2)：In order to solve the above technical problems, the present invention also provides the following M1) or M2):
M1)培育抗病性转基因植物的方法，包括向受体植物中导入下述1)或2)得到转基因植物；M1) A method for cultivating a disease-resistant transgenic plant, comprising introducing the following 1) or 2) into a recipient plant to obtain a transgenic plant;
1)OsAPX8的编码基因；1) the coding gene of OsAPX8;
2)含有OsAPX8的编码基因的表达盒；2) an expression cassette containing the gene encoding OsAPX8;
所述转基因植物与所述受体植物相比抗病性增加；The transgenic plant has increased disease resistance compared to the recipient plant;
M2)培育抗病性降低的转基因植物的方法，包括降低目的植物中OsAPX8的编码基因的表达，得到抗病性低于所述目的植物的转基因植物。M2) A method for cultivating transgenic plants with reduced disease resistance, comprising reducing the expression of the gene encoding OsAPX8 in the target plant to obtain a transgenic plant with lower disease resistance than the target plant.
上述方法中，降低目的植物中OsAPX8的编码基因的表达可通过将B15)所述核酸分子导入所述目的植物实现。In the above method, reducing the expression of the gene encoding OsAPX8 in the target plant can be achieved by introducing the nucleic acid molecule described in B15) into the target plant.
上述方法中，OsAPX8的编码基因可为B1)所述核酸分子；所述表达盒为B2)所述表达盒。In the above method, the gene encoding OsAPX8 can be the nucleic acid molecule described in B1); the expression cassette is the expression cassette described in B2).
在本发明的实施例中，所述OsAPX8的编码基因(即序列1所示的DNA分子)通过含有OsAPX8基因表达盒的OsAPX8基因重组表达载体导入目的植物中。所述OsAPX8基因表达盒中，启动OsAPX8基因转录的启动子为35S。In an embodiment of the present invention, the gene encoding OsAPX8 (ie, the DNA molecule shown in Sequence 1) is introduced into the target plant through an OsAPX8 gene recombinant expression vector containing an OsAPX8 gene expression cassette. In the OsAPX8 gene expression cassette, the promoter for initiating the transcription of the OsAPX8 gene is 35S.
上述方法中，其中所述OsAPX8基因可先进行如下修饰，再导入受体种子植物中，以达到更好的表达效果：In the above method, the OsAPX8 gene can be modified as follows first, and then introduced into the recipient seed plant to achieve better expression effect:
1)根据实际需要进行修饰和优化，以使基因高效表达；例如，可根据受体植物所偏爱的密码子，在保持本发明所述OsAPX8基因的氨基酸序列的同时改变其密码子以符合植物偏爱性；优化过程中，最好能使优化后的编码序列中保持一定的GC含量，以最好地实现植物中导入基因的高水平表达，其中GC含量可为35％、多于45％、多于50％或多于约60％；1) modify and optimize according to actual needs, so that the gene can be expressed efficiently; for example, according to the codon preferred by the recipient plant, its codon can be changed to meet the plant preference while maintaining the amino acid sequence of the OsAPX8 gene of the present invention In the optimization process, it is best to keep a certain GC content in the optimized coding sequence, so as to best realize the high-level expression of the introduced gene in the plant, wherein the GC content can be 35%, more than 45%, more than more than 50% or more than about 60%;
2)修饰邻近起始甲硫氨酸的基因序列，以使翻译有效起始；例如，利用在植物中已知的有效的序列进行修饰；2) modifying the gene sequence adjacent to the starting methionine to allow efficient initiation of translation; for example, using sequences known to be effective in plants for modification;
3)与各种植物表达的启动子连接，以利于其在植物中的表达；所述启动子可包括组成型、诱导型、时序调节、发育调节、化学调节、组织优选和组织特异性启动子；启动子的选择将随着表达时间和空间需要而变化，而且也取决于靶物种；例如组织或器官的特异性表达启动子，根据需要受体在发育的什么时期而定；尽管证明了来源于双子叶植物的许多启动子在单子叶植物中是可起作用的，反之亦然，但是理想地，选择双子叶植物启动子用于双子叶植物中的表达，单子叶植物的启动子用于单子叶植物中的表达；3) Linking with various plant-expressed promoters to facilitate its expression in plants; said promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters ; the choice of promoter will vary with the temporal and spatial requirements of expression, and also depends on the target species; e.g. a tissue or organ-specific expression promoter, depending on what stage of development the recipient is desired; although proven source Many promoters for dicots are functional in monocots and vice versa, but ideally, dicot promoters are chosen for expression in dicots and monocot promoters are used for Expression in monocots;
4)与适合的转录终止子连接，也可以提高本发明基因的表达效率；例如来源于CaMV的tml，来源于rbcS的E9；任何已知在植物中起作用的可得到的终止子都可以与本发明基因进行连接；4) Linking with suitable transcription terminators can also improve the expression efficiency of the gene of the present invention; for example, tml derived from CaMV, E9 derived from rbcS; any available terminators known to work in plants can be combined with The gene of the present invention is connected;
5)引入增强子序列，如内含子序列(例如来源于Adhl和bronzel)和病毒前导序列(例如来源于TMV,MCMV和AMV)。5) Introduce enhancer sequences, such as intron sequences (eg derived from Adhl and bronze) and viral leader sequences (eg derived from TMV, MCMV and AMV).
所述OsAPX8基因表达载体可通过使用Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、显微注射、电导、农杆菌介导、基因枪等常规生物学方法转化植物细胞或组织，并将转化的植物组织培育成植株。The OsAPX8 gene expression vector can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium-mediated, gene gun, and transform plant tissues grown into plants.
所述方法还包括从导入序列1、序列3或序列4所示的OsAPX8的编码基因的植株中筛选表达所述编码基因的植株，得到转基因植物。The method also includes screening the plants expressing the coding gene from the plants introduced with the coding gene of OsAPX8 shown in sequence 1, sequence 3 or sequence 4 to obtain transgenic plants.
在本发明的一个实施例中，与序列表中序列4的第1108-1599位核苷酸所示的DNA片段反向互补的DNA分子通过pANDA载体导入所述目的植物中。In one embodiment of the present invention, the DNA molecule reverse complementary to the DNA fragment shown in nucleotides 1108-1599 of Sequence 4 in the Sequence Listing is introduced into the target plant through the pANDA vector.
本发明中，所述转基因植物理解为不仅包含将所述OsAPX8基因转化目的植物得到的第一代转基因植物和降低OsAPX8表达的转基因植物，也包括其子代。对于转基因植物，可以在该物种中繁殖该基因，也可用常规育种技术将该基因转移进入相同物种的其它品种，特别包括商业品种中。所述转基因植物包括种子、愈伤组织、完整植株和细胞。In the present invention, the transgenic plant is understood to include not only the first-generation transgenic plant obtained by transforming the target plant with the OsAPX8 gene and the transgenic plant with reduced OsAPX8 expression, but also its progeny. For transgenic plants, the gene can be propagated in that species, or transferred into other varieties of the same species, particularly including commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.
本发明中，所述植物可为单子叶植物或双子叶植物；所述目的植物和所述受体植物均可为单子叶植物或双子叶植物。所述单子叶植物可为禾本科植物。所述禾本科植物可为水稻。In the present invention, the plant may be a monocotyledon or a dicotyledon; both the target plant and the recipient plant may be a monocotyledon or a dicotyledon. The monocotyledonous plant may be a grass plant. The grass plant can be rice.
本发明中，所述抗病性可为抗白叶枯病。所述白叶枯病由白叶枯病菌引起，如白叶枯病菌菲律宾小种PXO99引起的白叶枯病。In the present invention, the disease resistance may be resistance to bacterial blight. The bacterial blight is caused by bacterial blight, such as bacterial blight caused by bacterial blight philippine race PXO99.
实验证明，本发明的OsAPX8及其编码基因可以调控植物的抗病性，提高OsAPX8的表达可以提高植物的抗病性，降低OsAPX8的表达可以降低植物的抗病性：利用白叶枯病侵染OsAPX8表达升高的植物(转基因植株甲)与OsAPX8表达降低的植物(转基因植株乙)后发现，接种病菌5天后转基因植株甲的病斑长度为0.5±0.3cm，显著短于野生型植株的病斑长度(1.5±0.3cm)，转基因植株乙的病斑长度为2.3±0.2cm，显著长于野生型植株的病斑长度(1.5±0.3cm)。说明，可以利用OsAPX8及其编码基因调控植物的抗病性。Experiments have proved that OsAPX8 of the present invention and its coding gene can regulate the disease resistance of plants, increasing the expression of OsAPX8 can improve the disease resistance of plants, and reducing the expression of OsAPX8 can reduce the disease resistance of plants: using bacterial blight infection Plants with elevated expression of OsAPX8 (transgenic plant A) and plants with reduced expression of OsAPX8 (transgenic plant B) found that the lesion length of transgenic plant A was 0.5±0.3 cm 5 days after inoculation, which was significantly shorter than that of wild-type plants. Lesion length (1.5±0.3cm), the lesion length of the transgenic plant B was 2.3±0.2cm, which was significantly longer than that of the wild type plant (1.5±0.3cm). It shows that OsAPX8 and its coding gene can be used to regulate the disease resistance of plants.
附图说明Description of drawings
图1为转基因植株的半定量RT-PCR结果。其中，泳道1-4分别为转基因植株甲的四个株系，泳道5-8分别为转基因植株丁的四个株系，Marker表示DNA分子量标准。Figure 1 is the semi-quantitative RT-PCR results of the transgenic plants. Among them, lanes 1-4 are four strains of transgenic plant A, lanes 5-8 are four strains of transgenic plant D respectively, and Marker represents a DNA molecular weight standard.
图2为各转基因水稻的抗病性检测结果。其中，tAPX8OX表示转基因水稻甲，tAPX8Ri表示转基因水稻乙，TP309-CK表示水稻品种“TP309”。Figure 2 is the detection results of disease resistance of each transgenic rice. Among them, tAPX8OX means transgenic rice A, tAPX8Ri means transgenic rice B, and TP309-CK means rice variety "TP309".
具体实施方式detailed description
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention.
下述实施例中的实验方法，如无特殊说明，均为常规方法。The experimental methods in the following examples are conventional methods unless otherwise specified.
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
下述实施例中的白叶枯病菌菲律宾小种PXO99，为文献(水稻白叶枯病数量抗性座位定位及其小种专化性.作物学报，2006，32(11)：1611-1617))中的菲律宾小种P6，经中国农科院作物科学研究所周永力同意后，公众可从申请人处获得，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。The bacterial blight Philippine race PXO99 in the following examples is a document (Location of rice bacterial blight quantitative resistance loci and its race specialization. Acta Crops, 2006, 32(11): 1611-1617) The Philippine race P6 in ) can be obtained from the applicant by the public with the consent of Zhou Yongli, Institute of Crop Science, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
实施例1、OsAPX8可以调控水稻的抗病性Example 1, OsAPX8 can regulate the disease resistance of rice
本发明提供了来源于水稻品种“TP309”的抗病相关蛋白，其名称为OsAPX8。OsAPX8的氨基酸序列如序列表中序列2所示，OsAPX8编码基因的基因组序列如序列3所示，OsAPX8的编码序列如序列1所，OsAPX8基因cDNA序列如序列表中序列4所示，序列4的第31-1467位与序列1相同。The present invention provides a disease resistance-related protein derived from rice variety "TP309", and its name is OsAPX8. The amino acid sequence of OsAPX8 is shown in sequence 2 in the sequence listing, the genome sequence of the gene encoding OsAPX8 is shown in sequence 3, the coding sequence of OsAPX8 is shown in sequence 1, the cDNA sequence of OsAPX8 gene is shown in sequence 4 in the sequence listing, and the sequence 4 is Positions 31-1467 are identical to sequence 1.
一、转基因水稻的构建1. Construction of transgenic rice
1、重组载体的构建1. Construction of recombinant vector
将pCAMBIA1300-35S的EcoRI和XbaI识别序列间的DNA片段替换为序列4所示的DNA分子，保持载体的其他序列不变，得到重组载体，将该重组载体命名为pCAMBIA1300-35S-OsAPX8。pCAMBIA1300-35S-OsAPX8能表达序列2所示的抗病相关蛋白OsAPX8。The DNA fragment between the EcoRI and XbaI recognition sequences of pCAMBIA1300-35S was replaced with the DNA molecule shown in Sequence 4, and other sequences of the vector were kept unchanged to obtain a recombinant vector, which was named pCAMBIA1300-35S-OsAPX8. pCAMBIA1300-35S-OsAPX8 can express the disease resistance related protein OsAPX8 shown in sequence 2.
其中，pCAMBIA1300-35S按照如下方法制备：利用BstXI和KpnI内切酶将双元载体pEZR-LN(由英国Glasgow大学John Christie教授赠送)(The Plant Cell,2003，Vol.15,1781–1794)上的含35S启动子的1692bp片段替换掉pCAMBIA1300的BstXI和KpnI之间的268bp片段，构建成含EcoRI-KpnI-SmaI-BamHI-XbaI多克隆位点的pCAMBIA1300-35S载体。其中，pCAMBIA1300为cambia产品。Among them, pCAMBIA1300-35S was prepared according to the following method: the binary vector pEZR-LN (gifted by Professor John Christie, University of Glasgow, UK) (The Plant Cell, 2003, Vol. The 1692bp fragment containing the 35S promoter replaced the 268bp fragment between BstXI and KpnI of pCAMBIA1300 to construct a pCAMBIA1300-35S vector containing EcoRI-KpnI-SmaI-BamHI-XbaI multiple cloning sites. Among them, pCAMBIA1300 is a cambia product.
提取水稻品种“TP309”的总RNA并反转录为cDNA，以cDNA为模板，用F2和R2组成的引物对进行PCR扩增(F2：5′-CCAGCCGTCGAAGAGAAGGATCC-3′；R2：5′-CACATGCCCATCCTTTTACCTC-3′)，将得到的492bp的PCR扩增产物APX492，克隆到pENTRTM/SD/载体(赛默飞世尔科技公司(Thermo Fisher Scientific)产品)，将序列正确的重组载体命名为pENTR-OsAPX8i；用LR Clonase II Plus Enzyme Mix，将pENTR-OsAPX8i质粒与pANDA载体(Proc Natl Acad Sci U S A 103,10503-10508(2006))重组，将序列正确的重组载体命名为pANDA-OsAPX8i。pANDA-OsAPX8i的LB与RB间的结构如下：Ubiquitin启动子-反向APX492-gus linker-正向APX492-NOS终止子，pANDA-OsAPX8i的其他序列与pANDA相同。The total RNA of the rice variety "TP309" was extracted and reverse-transcribed into cDNA. Using the cDNA as a template, the primer pair composed of F2 and R2 was used for PCR amplification (F2: 5′-CCAGCCGTCGAAGAGAAGGATCC-3′; R2: 5′-CACATGCCCATCCTTTTACCTC -3′), the resulting 492bp PCR amplification product APX492 was cloned into pENTRTM /SD/ Vector (Thermo Fisher Scientific products), the recombinant vector with the correct sequence was named pENTR-OsAPX8i; LR Clonase II Plus Enzyme Mix was used to combine the pENTR-OsAPX8i plasmid with the pANDA vector (Proc Natl Acad Sci USA 103, 10503-10508 (2006)) recombination, and the recombinant vector with correct sequence was named pANDA-OsAPX8i. The structure between LB and RB of pANDA-OsAPX8i is as follows: Ubiquitin promoter-reverse APX492 -gus linker-forward APX492 -NOS terminator, other sequences of pANDA-OsAPX8i are the same as pANDA.
将pCAMBIA1300-35S-OsAPX8导入农杆菌菌株EHA105(Plant MolecularBiology,2003,52:957-966)中，将得到的重组菌命名为E-pCAMBIA1300-35S-OsAPX8，将pCAMBIA1300-35S导入农杆菌菌株EHA105中，将得到的重组菌命名为E-pCAMBIA1300-35S；将pANDA-OsAPX8i导入农杆菌菌株EHA105中，将得到的重组菌命名为E-pANDA-OsAPX8i，将pANDA导入农杆菌菌株EHA105中，将得到的重组菌命名为E-pANDA。Introduce pCAMBIA1300-35S-OsAPX8 into Agrobacterium strain EHA105 (Plant Molecular Biology, 2003, 52:957-966), and name the recombinant bacteria obtained as E-pCAMBIA1300-35S-OsAPX8, and introduce pCAMBIA1300-35S into Agrobacterium strain EHA105 , the obtained recombinant bacteria were named E-pCAMBIA1300-35S; pANDA-OsAPX8i was introduced into the Agrobacterium strain EHA105, and the obtained recombinant bacteria were named E-pANDA-OsAPX8i, and pANDA was introduced into the Agrobacterium strain EHA105, and the obtained The recombinant bacteria were named E-pANDA.
2、转基因水稻的构建2. Construction of transgenic rice
通过农杆菌介导的转基因方法(Kumar et al.,2005)利用步骤1的E-pCAMBIA1300-35S-OsAPX8转化水稻品种“TP309”的成熟胚愈伤组织,采用50mg/L潮霉素筛选抗性愈伤组织，然后经过预分化、分化、生根后得到T0代转OsAPX8基因植株，将其命名为T0代转基因水稻甲。利用E-pCAMBIA1300-35S转化水稻品种“TP309”，得到T0代转空载体水稻，将其命名为T0代转基因水稻丙，作为对照。Using the E-pCAMBIA1300-35S-OsAPX8 in step 1 to transform the mature embryo callus of rice variety "TP309" by Agrobacterium-mediated transgenic method (Kumar et al., 2005), use 50mg/L hygromycin to select resistance The callus was then pre-differentiated, differentiated, and rooted to obtain the T0 generation transgenic OsAPX8 gene plant, which was named as the T0 generation transgenic rice A. The rice variety "TP309" was transformed with E-pCAMBIA1300-35S to obtain the T0 generation transgenic rice, which was named as the T0 generation transgenic rice C, as a control.
通过农杆菌介导的转基因方法(Kumar et al.,2005)利用步骤1的E-pANDA-OsAPX8i转化水稻品种“TP309”的成熟胚愈伤组织,采用50mg/L潮霉素筛选抗性愈伤组织，然后经过预分化、分化、生根后得到T0代OsAPX8基因RNAi植株，将其命名为T0代转基因水稻乙。利用pANDA转化水稻品种“TP309”，得到T0代转空载体水稻，将其命名为T0代转基因水稻丁，作为对照。The mature embryo callus of rice variety "TP309" was transformed with E-pANDA-OsAPX8i in step 1 by Agrobacterium-mediated transgenic method (Kumar et al., 2005), and the resistant callus was selected with 50mg/L hygromycin Tissues, and then pre-differentiated, differentiated, and rooted to obtain T0 generation OsAPX8 gene RNAi plants, which were named T0 generation transgenic rice B. The rice variety "TP309" was transformed with pANDA to obtain the T0 generation transgenic rice, which was named the T0 generation transgenic rice D, as a control.
利用潮霉素抗性基因上的引物对(Hyg_F：5′-GACGGTGTCGTCCATCACAGTTT-3′与Hyg_R：5′-ACTCACCGCGACGTCTGTCGAGAA-3′)对T0代转基因水稻甲、T0代转基因水稻乙、T0代转基因水稻丙与T0代转基因水稻丁进行基因组水平上的鉴定，将水稻品种“TP309”作为阴性对照，结果显示，T0代转基因水稻甲、T0代转基因水稻乙、T0代转基因水稻丙与T0代转基因水稻丁中均含有潮霉素抗性基因，水稻品种“TP309”无含目的DNA的载体片段。The primer pair on the hygromycin resistance gene (Hyg_F: 5′-GACGGTGTCGTCCATCACAGTTT-3′ and Hyg_R: 5′-ACTCACCGCGACGTCTGTCGAGAA-3′) was used to test the transgenic rice A of the T0 generation, the transgenic rice B of the T0 generation, and the T0 generation Transgenic rice C and T0 transgenic rice D were identified at the genome level, and the rice variety "TP309" was used as a negative control. The results showed that T0 transgenic rice A, T0 transgenic rice B, and T0 Both the transgenic rice D and the T0 generation contained the hygromycin resistance gene, and the rice variety "TP309" had no vector fragment containing the target DNA.
提取T0代转基因植株甲、T0代转基因植株乙、T0代转基因植株丙、T0代转基因植株丁和水稻品种“TP309”的叶片总RNA进行半定量RT-PCR分析，引物为F:5’-gctgcgaaatactcctacg-3’和R:5’-AGAGGAGGTCATCAGACCATCG-3’，采用Actin作为内参。结果显示，转基因植株丙、转基因植株丁和水稻品种“TP309”中的OsAPX8基因的表达水平无显著差异，OsAPX8基因在转基因植株甲中的表达显著高于水稻品种“TP309”(图1)，转基因植株乙中几乎检测不到OsAPX8基因的表达(图1)，转基因植株乙中OsAPX8基因的表达受到抑制。The total RNA of the leaves of transgenic plant A of T0 generation, transgenic plant B of T0 generation, transgenic plant C of T0 generation, transgenic plant D of T0 generation and rice variety "TP309" were extracted for semi-quantitative RT-PCR analysis, and the primers were F: 5'-gctgcgaaatactcctacg-3' and R:5'-AGAGGAGGTCATCAGACCATCG-3', using Actin as internal reference. The results showed that there was no significant difference in the expression level of OsAPX8 gene in transgenic plant C, transgenic plant D and rice variety "TP309", and the expression level of OsAPX8 gene in transgenic plant A was significantly higher than that in rice variety "TP309" (Fig. 1). The expression of OsAPX8 gene was hardly detected in plant B (Fig. 1), and the expression of OsAPX8 gene in transgenic plant B was suppressed.
二、转基因水稻抗病性检测2. Detection of disease resistance of transgenic rice
用白叶枯病菌菲律宾小种PXO99侵染正常培养至分蘖期的T0代转基因植株甲、T0代转基因植株乙、T0代转基因植株丙、T0代转基因植株丁和水稻品种“TP309”，检测各水稻的抗病性，实验重复三次，具体方法如下：The T0 transgenic plant A, T0 transgenic plant B, T0 transgenic plant C, T0 transgenic plant D and the rice variety "TP309" were infected with bacterial blight PXO99. , to detect the disease resistance of each rice, the experiment was repeated three times, the specific method is as follows:
将保存-70℃的白叶枯菌菲律宾小种PXO99菌种于PSA培养基(马铃薯300g/L，Ca(NO3)2·4H2O 0.5g/L，Na2HPO4·12H2O 2.0g/L，蔗糖15g/L，琼脂粉15g/L)上复壮，保存于4℃冰箱内备用。在接种前2天在PSA平板培养基上划线培养，在28℃培养72h，待细菌长的浓密而均匀，用纯净水洗脱菌体，调节浓度至109个细胞/ml进行接种。具体接种方法参考Kauffman等(1973年)的接菌方法人工剪叶接种，每种水稻接种3-5个叶片，每个叶片减去顶端3-5cm，接种两周后当病斑长度明显且稳定时进行调查，每一植株测量3-5个叶片。Put the Bacteria philippines race PXO99 strain stored at -70°C in PSA medium (potato 300g/L, Ca(NO3 )2 4H2 O 0.5g/L, Na2 HPO4 12H2 O 2.0 g/L, sucrose 15g/L, and agar powder 15g/L) for rejuvenation, and stored in a 4°C refrigerator for subsequent use. Streak culture on PSA plate medium 2 days before inoculation, culture at 28°C for 72 hours, until the bacteria grow dense and uniform, wash the bacteria with pure water, adjust the concentration to 109 cells/ml for inoculation. For the specific inoculation method, refer to the inoculation method of Kauffman et al. (1973) and manually cut leaves for inoculation. Each type of rice is inoculated with 3-5 leaves, and the top 3-5 cm of each leaf is subtracted. Two weeks after inoculation, when the lesion length is obvious and stable During the investigation, each plant measured 3-5 leaves.
结果(图2)显示，T0代转基因植株丙、T0代转基因植株丁和水稻品种“TP309”的病斑长度无显著差异，水稻品种“TP309”的病斑长度为1.5±0.3cm；T0代转基因植株甲的病斑长度为0.5±0.3cm，显著短于水稻品种“TP309”的病斑长度；T0代转基因植株乙的病斑长度为2.3±0.2cm，显著长于水稻品种“TP309”的病斑长度。说明，提高OsAPX8基因的表达水平可以提高水稻对白叶枯病菌引起的白叶枯病的抗性，降低OsAPX8基因的表达水平可以降低水稻对白叶枯病菌引起的白叶枯病的抗性，表明，OsAPX8基因可以调控水稻对白叶枯病的抗性。The results (Fig. 2) showed that there was no significant difference in the lesion length of the T0 transgenic plant C, the T0 transgenic plant D, and the rice variety "TP309", and the lesion length of the rice variety "TP309" was 1.5 ± 0.3 cm; T The lesion length of0 generation transgenic plant A was 0.5±0.3cm, which was significantly shorter than that of rice variety “TP309”; the lesion length of T0 transgenic plant B was 2.3±0.2cm, significantly longer than that of rice variety “TP309” "The lesion length. It shows that increasing the expression level of OsAPX8 gene can improve the resistance of rice to bacterial blight caused by bacterial blight, and reducing the expression level of OsAPX8 gene can reduce the resistance of rice to bacterial blight caused by bacterial blight, indicating that, OsAPX8 gene can regulate rice resistance to bacterial blight.
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