稻瘟菌激活蛋白基因pemG1在水稻和烟草中的表达及功能研究
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摘要
蛋白激发子是诱导植物抗病性的重要信号分子,其与植物的受体蛋白识别后能诱发植物的防御反应。研究表明,来源于稻瘟菌(Magnaporthe grisea)的激活蛋白PemG1能促进种子萌发和幼苗生长,提高水稻对稻瘟病的抗性。为探讨PemG1蛋白在植物中诱导抗病性和促进生长的功能,本研究将稻瘟菌激活蛋白基因pemG1转化水稻和烟草,检测该基因在受体植物内的整合,转录和表达,分析转基因植株的抗病性和农艺性状。
本研究获得的主要结果如下:
(1)构建了稻瘟菌激活蛋白基因pemG1的植物表达载体,载体含有控制pemG1基因表达的玉米泛素启动子和章鱼碱合成酶基因终止子以及卡那霉素抗性基因nptⅡ(neomycin phosphotransfersⅡ)。对载体进行了酶切和测序检测,通过冻融法将载体转化了根癌农杆菌(Agrobactrium tumefaciens)菌株AGL-1。
(2)比较了B6、MS和NB三种培养基的培养效果,结果表明,NB培养基最适合于日本晴(Nipponbare)成熟胚的组织培养。通过根癌农杆菌介导转化,将pemG1基因转化日本晴和烟草(Nicotiana tabacum),获得了转基因水稻和烟草植株,并将其繁育到T2代。
(3)通过PCR检测确认了阳性转化株,用Southern杂交进一步证实了pemG1基因已整合到受体植物基因组,用RT-PCR和Northern杂交验证了pemG1基因在植物细胞中的转录,用Western杂交证实了pemG1基因在植物细胞中的表达。遗传分析表明,pemG1基因在转基因后代中的分离符合3∶1的理论比例。
(4)比较了T0代转基因水稻植株和非转基因对照的农艺性状。转基因植株的平均株高为60.0 cm,非转基因对照的平均株高为43.6 cm。转基因植株平均收获到63.8颗饱满的种子,非转基因对照平均收获到42.6颗饱满的种子。
(5)鉴定了T0代和T2代转基因水稻植株对稻瘟病的抗性。离体叶片接种试验,稻瘟菌孢子悬浮液喷雾接种试验和台盼兰染色的结果表明,和非转基因对照相比,转基因水稻植株对稻瘟病的抗性提高了36～56%。
(6)鉴定了T2代转基因三生烟(Nicotiana tobacum cv. Samsun NN)对烟草花叶病毒(Tobacco Mosaic Virus)的抗性,转基因植株对枯斑的抑制率为35～47%。Northern杂交结果表明,pemG1基因的转录水平与枯斑抑制率存在相关性。
Protein elicitors are important signal molecules that trigger plants disease resistance. Defence responses will be induced once the elicitors were recognized by acceptors in plants. Research shows that PemG1 activator protein from Magnaporthe grisea is able to promote seeds germination and seedlings growth, and also induce rice resistance against rice blast fungus. To study PemG1’s functions in plants, pemG1 gene was transferred into rice and tobacco. The transgene’s integration, transcription and expression were identified. Disease resistance and agronomic traits of transgenic plants were also studied. Key results are as follows:
(1) Plant expression vectors were constructed. The maize ubiquitin promoter/octopine synthase terminator system and kanamycin-resistant gene nptⅡ(neomycin phosphotransfersⅡ) were used for constitutive expression of the pemG1 gene. DNA constructs were verified by restriction enzyme digestion and sequencing and then introduced into Agrobacterium tumefaciens strain AGL-1 by freeze-thaw method.
(2) Performances of N6 medium, MS medium and NB medium in tissue culture were evaluated. Results revealed that NB medium was the best for culture of Nipponbare scutellum tissue. Then, pemG1 gene was transferred into Nipponbare and tobacco (Nicotiana tabacum) by Agrobacterium-mediated transformation. Primary rice and tobacco transformants were obtained. So far T2 progenies have been harvested.
(3) Transgenic plants were confirmed to be electropositive by polymerase chain reaction (PCR). The pemG1 gene integration was further confirmed by Southern blot. The transgene transcription was verified by reverse transcription polymerase chain reaction (RT-PCR) and Northern blot. PemG1 protein expression was detected by Western blot. Genetic analysis showed that the transgenes were segregated normally in the progenies.
(4) Differences of agronomic traits between primary transgenic rice plants and non-transformed Nipponbare control were compared. The average height of transgenic plants was 60.0 cm and that of control plants was 43.6 cm. Transgenic plants harvested 63.8 full seeds on average; control plants harvested an average of 42.6.
(5) Transgenic rice plants of T0 and T2 generation were tested for resistance against rice blast disease by detached leaves assay, spores spray inoculation and lacto phenol blue staining. Results indicated that the transgenic plants increased resistance to rice blast fungus by 36～56%.
(6) Transgenic tobacco plants (Nicotiana tobacum cv. Samsun NN) of T2 generation were challenged with TMV (Tobacco Mosaic Virus). In comparison with TMV-infected wild-type SNN control, transgenic plants reduced hypersensitive response lesions by 35～47%. Northern blot results revealed that accumulation level of pemG1 steady-state transcripts corresponds to the inhibition rate of local lesions.
本研究获得的主要结果如下:
(1)构建了稻瘟菌激活蛋白基因pemG1的植物表达载体,载体含有控制pemG1基因表达的玉米泛素启动子和章鱼碱合成酶基因终止子以及卡那霉素抗性基因nptⅡ(neomycin phosphotransfersⅡ)。对载体进行了酶切和测序检测,通过冻融法将载体转化了根癌农杆菌(Agrobactrium tumefaciens)菌株AGL-1。
(2)比较了B6、MS和NB三种培养基的培养效果,结果表明,NB培养基最适合于日本晴(Nipponbare)成熟胚的组织培养。通过根癌农杆菌介导转化,将pemG1基因转化日本晴和烟草(Nicotiana tabacum),获得了转基因水稻和烟草植株,并将其繁育到T2代。
(3)通过PCR检测确认了阳性转化株,用Southern杂交进一步证实了pemG1基因已整合到受体植物基因组,用RT-PCR和Northern杂交验证了pemG1基因在植物细胞中的转录,用Western杂交证实了pemG1基因在植物细胞中的表达。遗传分析表明,pemG1基因在转基因后代中的分离符合3∶1的理论比例。
(4)比较了T0代转基因水稻植株和非转基因对照的农艺性状。转基因植株的平均株高为60.0 cm,非转基因对照的平均株高为43.6 cm。转基因植株平均收获到63.8颗饱满的种子,非转基因对照平均收获到42.6颗饱满的种子。
(5)鉴定了T0代和T2代转基因水稻植株对稻瘟病的抗性。离体叶片接种试验,稻瘟菌孢子悬浮液喷雾接种试验和台盼兰染色的结果表明,和非转基因对照相比,转基因水稻植株对稻瘟病的抗性提高了36～56%。
(6)鉴定了T2代转基因三生烟(Nicotiana tobacum cv. Samsun NN)对烟草花叶病毒(Tobacco Mosaic Virus)的抗性,转基因植株对枯斑的抑制率为35～47%。Northern杂交结果表明,pemG1基因的转录水平与枯斑抑制率存在相关性。
Protein elicitors are important signal molecules that trigger plants disease resistance. Defence responses will be induced once the elicitors were recognized by acceptors in plants. Research shows that PemG1 activator protein from Magnaporthe grisea is able to promote seeds germination and seedlings growth, and also induce rice resistance against rice blast fungus. To study PemG1’s functions in plants, pemG1 gene was transferred into rice and tobacco. The transgene’s integration, transcription and expression were identified. Disease resistance and agronomic traits of transgenic plants were also studied. Key results are as follows:
(1) Plant expression vectors were constructed. The maize ubiquitin promoter/octopine synthase terminator system and kanamycin-resistant gene nptⅡ(neomycin phosphotransfersⅡ) were used for constitutive expression of the pemG1 gene. DNA constructs were verified by restriction enzyme digestion and sequencing and then introduced into Agrobacterium tumefaciens strain AGL-1 by freeze-thaw method.
(2) Performances of N6 medium, MS medium and NB medium in tissue culture were evaluated. Results revealed that NB medium was the best for culture of Nipponbare scutellum tissue. Then, pemG1 gene was transferred into Nipponbare and tobacco (Nicotiana tabacum) by Agrobacterium-mediated transformation. Primary rice and tobacco transformants were obtained. So far T2 progenies have been harvested.
(3) Transgenic plants were confirmed to be electropositive by polymerase chain reaction (PCR). The pemG1 gene integration was further confirmed by Southern blot. The transgene transcription was verified by reverse transcription polymerase chain reaction (RT-PCR) and Northern blot. PemG1 protein expression was detected by Western blot. Genetic analysis showed that the transgenes were segregated normally in the progenies.
(4) Differences of agronomic traits between primary transgenic rice plants and non-transformed Nipponbare control were compared. The average height of transgenic plants was 60.0 cm and that of control plants was 43.6 cm. Transgenic plants harvested 63.8 full seeds on average; control plants harvested an average of 42.6.
(5) Transgenic rice plants of T0 and T2 generation were tested for resistance against rice blast disease by detached leaves assay, spores spray inoculation and lacto phenol blue staining. Results indicated that the transgenic plants increased resistance to rice blast fungus by 36～56%.
(6) Transgenic tobacco plants (Nicotiana tobacum cv. Samsun NN) of T2 generation were challenged with TMV (Tobacco Mosaic Virus). In comparison with TMV-infected wild-type SNN control, transgenic plants reduced hypersensitive response lesions by 35～47%. Northern blot results revealed that accumulation level of pemG1 steady-state transcripts corresponds to the inhibition rate of local lesions.
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