CN1225559C - Rice xanthomonad hrf3 gene, recombinant vector and method for plant transgenic breeding - Google Patents
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Abstract
本发明水稻黄单胞hrf3基因重组载体及转基因植物育种方法属于生物技术领域。本发明提供了hrf3基因并以hrf3基因作为目的基因构建在植物表达的Ti-质粒双元载体pBI121上,获得基因重组载体,将构建的重组载体pBI121∷hrf3转化于EHA105中;转化植物细胞,外植体在植物再生培养基中筛选被转化的再生植株(T0代),检测转化株系。hrf3基因转基因植物增强了植物广谱抗病虫能力并改善植物其他生产性状。图为hrf3基因的核苷酸序列。
The invention discloses a rice xanthomonad hrf3 gene recombination carrier and a transgenic plant breeding method, which belong to the field of biotechnology. The present invention provides the hrf3 gene and uses the hrf3 gene as the target gene to construct the Ti-plasmid binary vector pBI121 expressed in plants to obtain the gene recombination vector, and transform the constructed recombinant vector pBI121::hrf3 into EHA105; Plants were screened for transformed regenerated plants (T 0 generation) in the plant regeneration medium, and transformed lines were detected. The hrf3 gene transgenic plants enhanced the plant's broad-spectrum resistance to diseases and insect pests and improved other plant production traits. The picture shows the nucleotide sequence of the hrf3 gene.
Description
(一)技术领域(1) Technical field
本发明“水稻黄单胞hrf3基因、重组载体及用于植物转基因育种方法”属于生物技领域,专用于通过植物基因工程技术改善植物抗病性和其他有益生产性状。The invention "rice xanthomonad hrf3 gene, recombinant vector and method for plant transgenic breeding" belongs to the field of biotechnology, and is specially used to improve plant disease resistance and other beneficial production traits through plant genetic engineering technology.
(二)技术背景(2) Technical background
基因hrf存在于水稻黄单胞(Xanthomonas oryzae)两个致病变种(pv.oryzae和pv.oryzicola)和其他黄单胞细菌(Xanthomonas spp.)中,属于过敏反应和致病性(hypersensitiveresponse and pathogenicity,hrp)基因簇成员。大多数革兰氏阴性植物病原细菌都有hrp基因簇,其中编码诱导植物产生过敏反应的蛋白质(Harpin)的基因各不相同。目前已对革兰氏阴性4个属植物病原细菌中的hrp基因簇进行了全序列测定,并依据基因簇的结构,操纵子的组成和调节基因的共线性关系等特征将已知的4种类型hrp基因簇分为两组。第I组包括梨火疫欧文氏菌(E.amylovora)和丁香假单胞(Pseudomonas syringae pv.syringae);第II组包括青枯拉尔氏菌(Ralstonia solanacearum)和甘兰黑腐黄单胞(Xanthomonas campestrispv.campestris)。关于编码harpins的基因,在梨火疫病菌中是hrpN,在丁香假单胞中是hrpZ,在青枯拉尔氏菌中是pop1,在水稻白叶枯病菌(X.oryzae pv.oryzae)中是hrf3。不同植物病原细菌来源的编码Harpins基因的序列比较发现,获得hrf3的代表菌株为水稻黄单胞白叶枯致病变种的PXO86,这一菌株为公知公用菌株(陈功友等.2001,水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)化学诱变hrp基因突变体及相关性状的研究,植物病理学报.31(3):199~207)。The gene hrf exists in two pathogenic varieties (pv.oryzae and pv.oryzicola) of rice Xanthomonas oryzae and other Xanthomonas spp., and belongs to hypersensitive response and pathogenicity , hrp) gene cluster members. Most Gram-negative phytopathogenic bacteria have hrp gene clusters in which genes encoding proteins (Harpin) that induce allergic responses in plants vary. At present, the hrp gene clusters in four Gram-negative genera of plant pathogenic bacteria have been fully sequenced, and the four known hrp gene clusters have been classified according to the structure of the gene cluster, the composition of the operon, and the collinearity of the regulatory genes. The type hrp gene clusters were divided into two groups. Group I includes E. amylovora and Pseudomonas syringae pv. syringae; group II includes Ralstonia solanacearum and Xanthomonas solanacearum (Xanthomonas campestrispv. campestris). Regarding genes encoding harpins, hrpN in E. amylovora, hrpZ in Pseudomonas syringae, pop1 in Larsia solanacearum, X. oryzae pv. oryzae It is hrf3. Sequence comparison of genes encoding Harpins from different phytopathogenic bacteria found that the representative strain that obtained hrf3 was PXO86 of the pathogenic variety of Xanthomonas oryzae Bacteria blight, which is a known public strain (Chen Gongyou et al. Study on chemical mutagenesis of hrp gene mutants and related traits of pathogen (Xanthomonas oryzae pv. oryzae), Acta Phytopathology. 31(3): 199~207).
作为hrp基因簇成员hrf1对致病性有调节作用,但体外表达的蛋白质产物具有诱导抗病性、抗虫性和促进植物生长等有益效应。Hrf1基因编码的蛋白质HarpinXo具有Harpin蛋白家族的特征:蛋白质亲水性,对热稳定,等电点为酸性4.3左右。与报道的Harpins蛋白家族成员HarpinEa和HarpinPss不同之处在于HaprinXo含有一个半胱氨酸,氨基酸残基中有GGG-GG重复单元,3~5μg/ml可诱导植物产生细胞编程死亡(PCD)。本课题组的前一个发明“一种编码植物生长调节剂的基因表达产物及其用途”(专利申请号00135403.5;公开号CV 1300547A)已对从水稻黄单胞白叶枯致病变种(X.oryzae pv.oryzae)中获得的hrf1基因及其表达产物HarpinXoo在体外直接应用即以制剂形式进行浸种、拌种、蘸根和叶面喷雾以及用hrf1基因构建重组微生物的应用进行了专利保护。As a member of the hrp gene cluster, hrf1 has a regulatory effect on pathogenicity, but the protein product expressed in vitro has beneficial effects such as inducing disease resistance, insect resistance and promoting plant growth. The protein Harpin Xo encoded by the Hrf1 gene has the characteristics of the Harpin protein family: the protein is hydrophilic, stable to heat, and the isoelectric point is about 4.3. The difference from the reported Harpins protein family members Harpin Ea and Harpin Pss is that Haprin Xo contains a cysteine, and there are GGG-GG repeating units in the amino acid residues, and 3-5 μg/ml can induce plant programmed cell death (PCD ). The previous invention of our research group "a gene expression product encoding a plant growth regulator and its application" (patent application number 00135403.5; publication number CV 1300547A) has been used to treat the pathogenic variety of xanthomonad xanthomonas (X. oryzae pv.oryzae) and the direct application of the hrf1 gene and its expression product Harpin Xoo in vitro, that is, soaking seeds, dressing seeds, dipping roots and spraying leaves in the form of preparations, and using the hrf1 gene to construct recombinant microorganisms have been patented.
以hrf1基因的表达产物HarpinXoo作为制剂在植物体外直接喷雾后,可以激活植物防卫反应相关酶,如苯丙氨酸解氨酶(PAL)、过氧化物酶(PO)和多酚氧化酶(PPO)的活性,同时诱导病程相关蛋白基因如PR-1b和PR-1a基因的表达。其中PR-1b基因受茉莉酸分子的调控,PR-1a受水杨酸分子的调控(Eyal et al.,1993,Ohshima et al.,1990)。其中茉莉酸和水杨酸作为信号分子在诱导植物抗虫、抗病性中起作用。另外,在番茄上使用时,还能启动pto介导的信号反应,其中pti4/5/6和效应基因PR-1a1、PR-1b1、NP24、Pin2在不同时间有不同程度的增强表达。Harpin Xoo , the expression product of the hrf1 gene, can activate plant defense response-related enzymes, such as phenylalanine ammonia lyase (PAL), peroxidase (PO) and polyphenol oxidase ( PPO) activity, while inducing the expression of disease process-related protein genes such as PR-1b and PR-1a genes. Among them, PR-1b gene is regulated by jasmonic acid molecule, and PR-1a is regulated by salicylic acid molecule (Eyal et al., 1993, Ohshima et al., 1990). Among them, jasmonic acid and salicylic acid act as signal molecules in inducing plant resistance to insects and diseases. In addition, when used on tomato, it can also initiate pto-mediated signaling reactions, in which pti4/5/6 and effector genes PR-1a1, PR-1b1, NP24, and Pin2 have different degrees of enhanced expression at different times.
作为与hrpN同,但其蛋白质产物明显不同的水稻黄单胞编码HarpinXo的基因hrp可能在植物基因工程中作为目的基因有应用价值。植物基因工程是以具有明确功能的外源基因作为目的基因,通过一定的技术路线转化植物后,随机整合到植物基因组中,使其在植物体内表达,来改善植物的抗病、抗虫和其他有益性状。如苏云金杆菌(Bacillus thuringiensis,Bt)杀虫蛋白(ICP)可以作为生物制剂喷洒植物进行害虫防治,基因转化棉花后,转基因棉花对棉铃虫也有抗性(A 01N63/02CN 95113498.1A);抗菌肽(Cecropin)基因转化烟草、马铃薯和水稻后,转基因植物表现对这些植物上的细菌病害有抗性(贾士荣,1993)。通过转基因技术还可以改善作物品质如耐贮性、耐盐性、氨基酸品质和植物脂质等。作为Harpin基因家族的一个成员,来自梨火疫病菌(Erwinia amylovora)的编码HarpinEa的基因hrpN已在美国研发生物农药Messenger中成功应用,作为目的基因用于转基因马铃薯和苹果,分别对马铃薯晚疫病(Phytophthora infestans)和梨火疫病(Erwinia amylovora)的抗病性有不同程度的改善。但是目前还没有报道hrf3基因及其在植物转基因育种工程技术中的得到应用。目前也没有有关以hrf基因为目的基因的hrf基因转基因育种方法的报道。As the same as hrpN, but its protein product is obviously different, the gene hrp of rice xanthomonad encoding Harpin Xo may have application value as the target gene in plant genetic engineering. Plant genetic engineering uses exogenous genes with definite functions as the target genes. After transforming plants through a certain technical route, they are randomly integrated into the plant genome and expressed in plants to improve plant resistance to diseases, insects and other diseases. Beneficial traits. For example, Bacillus thuringiensis (Bacillus thuringiensis, Bt) insecticidal protein (ICP) can be used as a biological agent to spray plants for pest control. After genetically transformed cotton, transgenic cotton also has resistance to cotton bollworm (A 01N63/02CN 95113498.1A); antimicrobial peptide ( Cecropin) gene transformed tobacco, potato and rice, the transgenic plants showed resistance to bacterial diseases on these plants (Jia Shirong, 1993). The quality of crops such as storability, salt tolerance, amino acid quality and plant lipids can also be improved through transgenic technology. As a member of the Harpin gene family, the gene hrpN encoding Harpin Ea from Erwinia amylovora has been successfully applied in the research and development of biopesticide Messenger in the United States. (Phytophthora infestans) and pear fire blight (Erwinia amylovora) were improved to varying degrees. But there is no report about the hrf3 gene and its application in plant transgenic breeding engineering technology. At present, there is no report about the hrf gene transgenic breeding method using the hrf gene as the target gene.
(三)发明内容(3) Contents of the invention
技术问题 本发明的目的是针对目前还没有报道hrf3基因及其在植物转基因育种工程技术中的得到应用的现状,提供水稻黄单胞hrf3基因、重组载体及转基因植物育种方法,将hrf3基因构建在植物表达的转化质粒,转化水稻、烟草获得转基因植株。其基因重组载体作为商业用途的品种、品系或作为基因资源在生产上直接使用或进行农业生物育种和转基因植物以提高作物抗病、抗虫性状和改善农作物其他生产性状。Technical problem The purpose of the present invention is to provide rice xanthomonad hrf3 gene, recombinant vector and transgenic plant breeding method for the current situation that hrf3 gene and its application in plant transgenic breeding engineering technology have not been reported at present, and hrf3 gene is constructed in The transformation plasmid expressed by the plant is used to transform rice and tobacco to obtain transgenic plants. Its genetic recombination vectors are used as varieties and strains for commercial use or as genetic resources for direct use in production or for agricultural biological breeding and transgenic plants to improve crop disease resistance, insect resistance traits and other production traits of crops.
技术方案Technical solutions
本发明所提供的水稻黄单胞hrf3基因序列如下:The rice xanthomonad hrf3 gene sequence provided by the present invention is as follows:
如序列表SEQ ID NO.1所示;As shown in the sequence table SEQ ID NO.1;
上述水稻黄单胞hrf3基因重组载体是通过以下方法构建而成:The above-mentioned rice xanthomonad hrf3 gene recombination vector is constructed by the following method:
水稻黄单胞hrf3基因序列:如序列表SEQ ID NO.1所示Rice xanthomonad hrf3 gene sequence: as shown in the sequence table SEQ ID NO.1
以hrf3基因作为目的基因构建在植物表达的Ti-质粒双元载体pBI121上:用于构建双元转化—表达载体的质粒的是由T-DNA改造而来,其中除T-DNA 25bp重复序列(LB和RB),胭脂碱合成酶基因的启动子(P-nos)和终止子(T-nos)外,还有在原核生物(细菌)中作为选择标记使用的nptI和在真核生物(植物)中作为选择标记使用的nptII,用于选择的抗生素均为Km和G-418;另外在pBI121中还有在真核生物表达的花椰菜花叶病毒的启动子35S(p35S)和β-葡糖醛酸酶(Gus)基因uidA;作为目的基因使用的hrf3基因插入在pBI121质粒的p35S和uid之间的多酶切克隆位点上,所用内切酶为XbaI和BamHI,即为重组质粒。The hrf3 gene was used as the target gene to construct the Ti-plasmid binary vector pBI121 for plant expression: the plasmid used to construct the binary transformation-expression vector was transformed from T-DNA, except for the T-DNA 25bp repeat sequence ( LB and RB), in addition to the promoter (P-nos) and terminator (T-nos) of the nopaline synthase gene, nptI used as a selection marker in prokaryotes (bacteria) and nptI used in eukaryotes (plants) ) used as a selectable marker, the antibiotics used for selection are Km and G-418; in addition, in pBI121, there are also the promoter 35S (p35S) and β-glucose of the cauliflower mosaic virus expressed in eukaryotes The aldolase (Gus) gene uidA; the hrf3 gene used as the target gene is inserted into the multi-enzyme cloning site between p35S and uid of the pBI121 plasmid, and the endonucleases used are XbaI and BamHI, which is the recombinant plasmid.
本发明所提供的水稻黄单胞hrf3基因重组载体的转基因植物育种方法为:The transgenic plant breeding method of the rice xanthomonad hrf3 gene recombination carrier provided by the present invention is:
1)构建的双元转化重组载体质粒pBI121::hrf3,可以直接转化于含vir区辅助质粒不带Km1) The constructed binary transformation recombinant vector plasmid pBI121::hrf3 can be directly transformed into the helper plasmid containing the vir region without Km
抗性的pTiBo542衍生质粒的感受态根癌土壤杆菌(Agrobacterium tumefaciens)EHA105中;也可以用含pBI121::hrf3的大肠杆菌DH5α,在帮助质粒pRK2013存在情况下,通过三亲交配转移到EHA105中。EHA105是EHA101(C58/pTiBo542)的Km敏感衍生株。The resistant pTiBo542-derived plasmid was competent for Agrobacterium tumefaciens EHA105; E. coli DH5α containing pBI121::hrf3 could also be transferred to EHA105 by triparental mating in the presence of the helper plasmid pRK2013. EHA105 is a Km-sensitive derivative of EHA101 (C58/pTiBo542).
2)同时含有辅助质粒pTiBo542(Kms)和转化质粒(pBI121::hrf3)的根癌土壤杆菌EHA105用于植物细胞的转化,重组根癌土壤杆菌转化植物细胞的方法用叶碟法、胚转化法或花粉管导入方法。2) Agrobacterium tumefaciens EHA105 containing the helper plasmid pTiBo542 (Km s ) and transformation plasmid (pBI121::hrf3) at the same time is used for the transformation of plant cells, and the method of transforming plant cells with recombinant Agrobacterium tumefaciens uses the leaf disc method and embryo transformation method or pollen tube introduction method.
3)外植体在含Km的植物再生培养基中筛选被转化的再生植株(T0代):T1代种子催芽时用3) The explants are selected in the plant regeneration medium containing Km for the transformed regeneration plant (T 0 generation): when the T 1 generation seeds are accelerated to germination, use
Km(150μg/ml)浸泡12小时进行筛选转化植株,对T1代植株按株系检测转化频率以及植株中hrf3基因的表达。Soak in Km (150 μg/ml) for 12 hours to screen the transformed plants, and detect the transformation frequency and the expression of hrf3 gene in the T1 generation plants according to the line.
4)转化株系的检测方法:用PCR和Southern印迹杂交法检测转化植株叶片中hrf3基因和P-35S启动子,hrf3基因的表达用定量RT-PCR方法检测植株叶片中hrf3基因的RNA的积累。4) Detection method of transformed strains: detection of hrf3 gene and P-35S promoter in leaves of transformed plants by PCR and Southern blot hybridization method, the expression of hrf3 genes was detected by quantitative RT-PCR method for the accumulation of RNA of hrf3 genes in leaves of plants .
根癌土壤杆菌EHA105为公知公用菌株,基因转化和转化细胞的植株再生方法为转基因植物的常规方法(Sambrook J,Russell D.W.,2001,Molelcular cloning,A LaboratoryManual(3rd ed),Spring Harbor Laboratory Press;闻伟刚、王金生,2001,水稻白叶枯病菌harpin基因的克隆与表达,植物病理学报,31(4):295~300)。Agrobacterium tumefaciens EHA105 is a known public strain, and the plant regeneration method of gene transformation and transformed cells is a conventional method for transgenic plants (Sambrook J, Russell DW, 2001, Molelcular cloning, A Laboratory Manual (3 rd ed), Spring Harbor Laboratory Press; Wen Weigang, Wang Jinsheng, 2001, Cloning and expression of the harpin gene of Xanthomonas oryzae, Acta Phytopathology, 31(4): 295-300).
有益效果 本发明提供了一种水稻黄单胞hrf3基因重组载体及转基因植物育种方法,突破了hrf3基因还没有在植物转基因育种工程技术中的得到应用的现状,首次将hrf基因构建在植物表达的转化质粒,以双元转化—表达载体pBI121::hrf3转化植物及hrf3基因在转基因植物中的表达根癌土壤杆菌(Agrobacterium tumefaciens)介导的方法,转化水稻、烟草获得了转基因植株。在温室和大田鉴定转基因植株的抗病性对植物上的真菌病害、细菌病害和病毒病害的抗病性均有明显增强。试验表明,hrf3基因转基因水稻的T1代对稻瘟病免疫和高抗的单株各占20%,对水稻白叶枯病表现中抗的单株约占15%,与对照相比,防病效果分别达到80~100%和70~80%。水稻的转Harpin基因植株还表现耐弱光、耐低温、株型矮化、多分蘖等优良生产性状。hrf3基因转基因烟草的T1代植株90%以上表现出抗烟草花叶病毒(TMV),烟草青枯病细菌(Ralstonia solanacearum)和烟草赤星病菌(Alternariaalternata)3种病害,达到高抗和中抗水平,效果分别为84%、70%和54%。在转基因植物株系可以检测到GST1、PR-1a、PR-1b等防卫相关基因的表达,而在亲本品系中不表达。已获得hrf3转基因棉花、小麦、玉米和番茄,并检测到hrf3基因和相关防卫基因的表达。Beneficial effects The present invention provides a rice xanthomonad hrf3 gene recombination vector and a transgenic plant breeding method, which breaks through the current situation that the hrf3 gene has not been applied in plant transgenic breeding engineering technology, and constructs the hrf gene in a plant-expressed plant for the first time Transform the plasmid, transform rice and tobacco with the method of binary transformation—expression vector pBI121::hrf3 to transform plants and express hrf3 gene in transgenic plants Agrobacterium tumefaciens-mediated method, and obtain transgenic plants. The disease resistance of the transgenic plants identified in the greenhouse and the field has significantly enhanced disease resistance to fungal diseases, bacterial diseases and viral diseases on the plants. The test showed that the T1 generation of hrf3 gene transgenic rice had 20% immunity to rice blast and 20% high resistance to rice blast, and about 15% of rice bacterial blight resistance. Compared with the control, the disease prevention effect 80-100% and 70-80% respectively. Rice transgenic plants with Harpin gene also exhibit excellent production traits such as low light tolerance, low temperature tolerance, dwarf plant type, and multiple tillers. More than 90% of the T1 generation plants of hrf3 gene transgenic tobacco showed resistance to tobacco mosaic virus (TMV), tobacco bacterial wilt bacteria (Ralstonia solanacearum) and tobacco red spot bacteria (Alternaria alternata), reaching high and medium resistance levels, The effects are 84%, 70% and 54%, respectively. The expression of defense-related genes such as GST1, PR-1a and PR-1b can be detected in the transgenic plant lines, but not expressed in the parental lines. hrf3 transgenic cotton, wheat, maize and tomato have been obtained, and the expression of hrf3 gene and related defense genes has been detected.
(四)附图说明(4) Description of drawings
图1 hrf3基因的核苷酸序列Figure 1 Nucleotide sequence of hrf3 gene
图2 hrf3基因编码蛋白质的氨基酸序列特征Figure 2 The amino acid sequence characteristics of the protein encoded by the hrf3 gene
图3 35S::hrf3基因重组载体构建图Figure 3 Construction diagram of 35S::hrf3 gene recombination vector
H:Hind III;S:Sph I;P:Pst I;X:Xba I;B:BamH I;Sm:Sma I;Sa:Sac I;E:EcoR IH: Hind III; S: Sph I; P: Pst I; X: Xba I; B: BamH I; Sm: Sma I; Sa: Sac I; E: EcoR I
(五)具体实施方式(5) Specific implementation methods
1.在植物中表达hrf3基因的双元转化—表达载体pBIH(pBI121::hrf3)的构建1. The binary transformation of expressing hrf3 gene in plants—the construction of expression vector pBIH (pBI121::hrf3)
用于构建双元转化—表达载体的质粒的是由T-DNA改造而来,其中除T-DNA 25bp重复序列(LB和RB),胭脂碱合成酶基因的启动子(P-nos)和终止子(T-nos)外,还有在原核生物(细菌)中作为选择标记使用的nptI和在真核生物(植物)中作为选择标记使用的nptII,用于选择的抗生素均为Km和G-418。另外在pBI121中还有在真核生物表达的花椰菜花叶病毒的启动子35S(p35S)和β-葡糖醛酸酶(Gus)基因uidA。作为目的基因使用的hrf3基因插入在pBI121质粒的p35S和uid之间的多酶切克隆位点上(图3),本发明所用内切酶为XbaI和BamHI。双元载体pBI121为公知公用载体(Jefferson,R.A.,Kavanash,T.A.& Bevan,1987,M.W.EMBO J.,6,3901-3907)。The plasmid used to construct the binary transformation-expression vector is transformed from T-DNA, except for the T-DNA 25bp repeat sequence (LB and RB), the promoter (P-nos) and termination of the nopaline synthase gene In addition to T-nos, there are nptI used as a selection marker in prokaryotes (bacteria) and nptII used as a selection marker in eukaryotes (plants). The antibiotics used for selection are both Km and G- 418. Also in pBI121 are the promoter 35S (p35S) of the cauliflower mosaic virus expressed in eukaryotes and the uidA of the β-glucuronidase (Gus) gene. The hrf3 gene used as the target gene is inserted into the multi-enzyme cloning site between p35S and uid of the pBI121 plasmid ( FIG. 3 ), and the endonucleases used in the present invention are XbaI and BamHI. The binary vector pBI121 is a well-known public vector (Jefferson, R.A., Kavanash, T.A. & Bevan, 1987, M.W. EMBO J., 6, 3901-3907).
双元转化—表达载体pBI121::hrf3转化植物及hrf3基因在转基因植物中的表达:Binary transformation—expression vector pBI121::hrf3 transformed plants and expression of hrf3 gene in transgenic plants:
构建的双元转化载体质粒pBI121::hrf3,可以直接转化含vir区辅助质粒不带Km抗性的pTiBo542衍生质粒的感受态根癌土壤杆菌(Agrobacterium tumefaciens)EHA105中。EHA105是EHA101(C58/pTiBo542)的Km敏感衍生株。也可以用含pBI121::hrf3的大肠杆菌DH5α,在帮助质粒pRK2013存在情况下,通过三亲交配转移到EHA105中。同时含有辅助质粒pTiBo542(Kms)和转化质粒(pBI121::hrf1)的根癌土壤杆菌EHA105用于植物细胞的转化。重组根癌土壤杆菌转化植物细胞的方法用叶碟法、胚转化法或花粉管导入方法。外植体在含Km的植物再生培养基中筛选被转化的再生植株(T0代)。T1代种子催芽时用Km(150μg/ml)浸泡12小时进行筛选转化植株。The constructed binary transformation vector plasmid pBI121::hrf3 can be directly transformed into the competent Agrobacterium tumefaciens EHA105 containing the pTiBo542 derived plasmid of the vir region helper plasmid without Km resistance. EHA105 is a Km-sensitive derivative of EHA101 (C58/pTiBo542). E. coli DH5α containing pBI121::hrf3 can also be transferred into EHA105 by triparental mating in the presence of helper plasmid pRK2013. Agrobacterium tumefaciens EHA105 containing both helper plasmid pTiBo542 (Km s ) and transformation plasmid (pBI121::hrf1) was used for transformation of plant cells. The method for transforming plant cells by recombining Agrobacterium tumefaciens uses leaf disk method, embryo transformation method or pollen tube introduction method. The explants were selected for transformed regenerated plants (T 0 generation) in plant regeneration medium containing Km. The T 1 generation seeds were soaked in Km (150 μg/ml) for 12 hours to select transformed plants when they germinated.
对T1代植株按株系检测转化频率以及植株中hrf3基因的表达。转化株系的检测方法是用PCR和Southern印迹杂交法检测转化植株叶片中hrf3基因和P-35S启动子,hrf3基因的表达用定量RT-PCR方法检测植株叶片中hrf3基因的RNA的积累。The transformation frequency and the expression of hrf3 gene in the T1 generation plants were detected by line. The detection method of the transformed strain is to detect the hrf3 gene and the P-35S promoter in the leaf of the transformed plant by PCR and Southern blot hybridization method, and the RNA accumulation of the hrf3 gene in the leaf of the plant is detected by the quantitative RT-PCR method for the expression of the hrf3 gene.
根癌土壤杆菌EHA105为公知公用菌株,基因转化和转化细胞的植株再生方法为转基因植物的常规方法(Clark MS,1997,Plant Molecular Biology,A Laboratory Manual,Springer,Berlin Germany)。Agrobacterium tumefaciens EHA105 is a known public bacterial strain, and the plant regeneration method of gene transformation and transformed cells is a conventional method for transgenic plants (Clark MS, 1997, Plant Molecular Biology, A Laboratory Manual, Springer, Berlin Germany).
2.hrf3基因转基因水稻的表型特征2. Phenotypic characteristics of hrf3 gene transgenic rice
将1所得到的hrf3基因转基因水稻的T1代种子分别播种在温室和病害流行区隔离田块中,用人工接种和诱发行感染法测定hrf3基因转基因水稻株系对稻瘟病(Magnaporthegrisea)和水稻白叶枯病(Xanthomonas oryzae pv.oryzae)的抗病型。人工接种法用常规喷雾(稻瘟病菌)和剪叶接种法(白叶枯病菌)。诱发行感染法是用稻瘟病菌人工喷雾接种感病品种籼优63,发病后移栽到病害鉴定圃的四周及畦间。畦面宽1.8米,株行距均为15厘米。诱发行水稻中病斑上的孢子可以随风雨均匀随机传播。感病株系和出发品种100%被感染发病。通过34个转基因水稻株系约3000个单株的鉴定结果,对稻瘟病免疫和高抗的单株各占20%,对水稻白叶枯病表现中抗的单株约占15%,与对照相比,防病效果分别达到80~100%和70~80%。用于转基因的亲本水稻品种有优质粳稻R109,籼稻3103和杂交稻恢复系明恢63。除抗病表型有明显改善外,转基因株系还表现明显的耐弱光、耐低温和株型矮化等特点。转基因水稻品系命名为转Hrp基因抗优稻(Harice)。The T1 generation seeds of the hrf3 gene transgenic rice obtained in 1 were respectively sown in the greenhouse and the isolated field in the disease endemic area, and the resistance of the hrf3 gene transgenic rice lines to rice blast (Magnaporthegrisea) and rice white was determined by artificial inoculation and induced infection. A resistant form of leaf blight (Xanthomonas oryzae pv. oryzae). The artificial inoculation method uses conventional spraying (Pyricularia grisea) and leaf cutting inoculation (bacteria bacterial blight). The induced row infection method is to artificially spray and inoculate the susceptible variety Xianyou 63 with Magnaporthe grisea, and transplant it to the periphery and between the borders of the disease identification nursery after the onset of the disease. The width of the border is 1.8 meters, and the spacing between plants and rows is 15 centimeters. The spores on the diseased spots in the induced row rice can spread evenly and randomly with the wind and rain. 100% of the susceptible strains and starting varieties were infected. According to the identification results of about 3,000 individual plants of 34 transgenic rice lines, 20% of the individual plants are immune to rice blast and highly resistant to rice blast, and about 15% of the individual plants are moderately resistant to rice bacterial blight. In contrast, the disease prevention effects reached 80-100% and 70-80% respectively. The parental rice varieties used for transgenesis include high-quality japonica rice R109, indica rice 3103 and hybrid rice restorer line Minghui 63. In addition to the obvious improvement of the disease resistance phenotype, the transgenic lines also showed obvious characteristics such as resistance to low light, low temperature and dwarfing plant type. The transgenic rice line was named the transgenic Hrp gene-resistant rice (Harice).
进行抗病性鉴定的水稻白叶枯病菌和稻瘟病菌均为公知公用。转基因亲本水稻品种R109、3103和明恢63是生产上使用的品种,公知公用。The bacterial blight of rice and the rice blast fungus for disease resistance identification are known and public. The transgenic parent rice varieties R109, 3103 and Minghui 63 are varieties used in production and are known and used for public use.
3.hrf3基因转基因烟草的表型特征3. Phenotypic characteristics of hrf3 gene transgenic tobacco
将1所得到的hrf3基因转基因烟草的T1代种子,播种在温室盆钵中,2叶期单株移栽至直径20厘米的盆钵中,4~5片真叶时人工接种烟草花叶病毒(TMV),烟草青枯病细菌(Ralstonia solanacearum)和烟草赤星病菌(Alternaria alternata)。TMV接种用叶面摩擦接种,青枯病用细菌悬浮液茎注射法,赤星病菌用孢子悬浮液液滴法叶面接种。转基因烟草株系T1代种子经Km(150μg/ml)筛选后的植株90%以上表现出抗以上3种病害,达到高抗和中抗水平,效果分别为84%、70%和54%。但其他性状与亲本烟草品种Xanthi(nc)相同。转Harpin基因烟草品系命名为转Hrp基因抗优烟(Hartob)。Sow the T1 generation seeds of the hrf3 gene transgenic tobacco obtained in 1 in a pot in the greenhouse, transplant a single plant at the 2-leaf stage into a pot with a diameter of 20 cm, and artificially inoculate tobacco mosaic virus when there are 4 to 5 true leaves (TMV), Ralstonia solanacearum and Alternaria alternata. TMV inoculation was inoculated by foliar rubbing, bacterial wilt was inoculated by stem injection of bacterial suspension, and Alternaria spp. was inoculated by spore suspension droplet method. More than 90% of the plants of T1 generation seeds of transgenic tobacco lines screened by Km (150 μg/ml) showed resistance to the above three diseases, reaching high and medium resistance levels, and the effects were 84%, 70% and 54% respectively. But other traits are the same as the parent tobacco variety Xanthi (nc). The tobacco line transgenic for Harpin was named Hartob transgenic Hrp gene.
进行烟草抗病性鉴定的烟草花叶病毒、青枯病细菌和赤星病菌在田间经常发生,为公知公用。Tobacco mosaic virus, bacterial wilt bacteria and Alternaria rubella for identification of tobacco disease resistance often occur in the field and are known and used.
4.表达hrf3基因的转基因植物品系启动植物防卫反应的特征4. Characteristics of plant defense response initiated by transgenic plant lines expressing hrf3 gene
从水稻T1代植株中有约70%的单株,烟草T1植株中有95%的单株可以用PCR和Southern印迹杂交方法检测到hrf3基因的全长插入序列和外源p35S启动子。hrf3基因转基因水稻和烟草的转基因品系T1代植株均不表现细胞编程死亡(PCD)现象。用定量RT-PCR方法测定hrf3基因和抗病防卫相关基因表达,PCR、Southern印迹杂交和RT-PCR方法是分子生物学常规研究方法。在转基因植物株系可以检测到GST1、PR-1a、PR-1b等防卫相关基因的表达,而在亲本品系中不表达。From about 70% of the single plants of the T1 plants of rice and 95% of the single plants of the T1 plants of tobacco, the full-length insertion sequence of the hrf3 gene and the exogenous p35S promoter can be detected by PCR and Southern blot hybridization methods. The T1 generation plants of transgenic rice and tobacco transgenic lines with hrf3 gene did not show the phenomenon of programmed cell death (PCD). Quantitative RT-PCR was used to measure the expression of hrf3 gene and genes related to disease resistance and defense. PCR, Southern blot hybridization and RT-PCR were routine research methods in molecular biology. The expression of defense-related genes such as GST1, PR-1a and PR-1b can be detected in the transgenic plant lines, but not expressed in the parental lines.
运用相同方法,已获得hrf3转基因棉花、小麦、玉米和番茄,并检测到hrf3基因和相关防卫基因的表达。Using the same method, hrf3 transgenic cotton, wheat, corn and tomato have been obtained, and the expression of hrf3 gene and related defense genes has been detected.
双元载体pBI121和根癌土壤菌EHA105,水稻品种R109,明恢63,烟草品种Xanthi(nc);公知公用。Binary vector pBI121 and Agrobacterium tumefaciens EHA105, rice variety R109, Minghui 63, tobacco variety Xanthi (nc); public knowledge.
作为目的基因使用的hrf3基因的来源菌株PXO86公知公用(Weiguang Zhu,et al. 2000,White identification of Two Novel hrp-Associated Genes in the hrp Gene Cluster of Xanthomonasoryzae pv.oryzae,Journal of Bacteriology,182(7):1844-1853)。The source strain PXO86 of the hrf3 gene used as the target gene is known and public (Weiguang Zhu, et al. 2000, White identification of Two Novel hrp-Associated Genes in the hrp Gene Cluster of Xanthomonasoryzae pv.oryzae, Journal of Bacteriology, 182 (7) : 1844-1853).
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