WO2002002776A1 - Procede permettant d'augmenter le taux d'assimilation de l'azote par les plantes - Google Patents

Procede permettant d'augmenter le taux d'assimilation de l'azote par les plantes Download PDF

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WO2002002776A1
WO2002002776A1 PCT/CN2001/000294 CN0100294W WO0202776A1 WO 2002002776 A1 WO2002002776 A1 WO 2002002776A1 CN 0100294 W CN0100294 W CN 0100294W WO 0202776 A1 WO0202776 A1 WO 0202776A1
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gene
glutamate dehydrogenase
gdh
plants
neurospora
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PCT/CN2001/000294
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English (en)
French (fr)
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Bo Tian
Fang Wang
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Institute Of Microbiology, Chinese Academy Of Sciences
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Priority to AU2001246330A priority Critical patent/AU2001246330A1/en
Publication of WO2002002776A1 publication Critical patent/WO2002002776A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to a method for improving plant nitrogen assimilation efficiency.
  • Nitrogen is the first major nutrient required for plant growth. At present, only 30% -40% of the nitrogen fertilizer applied in agriculture is absorbed by plants, and most of it is wasted. Some are converted into nitrate and lost to the soil, causing environmental pollution. GDH-transformed crops can effectively increase plant nitrogen absorption and increase nitrogen fertilizer utilization rate, which can reduce the amount of nitrogen fertilizer applied, resulting in huge economic benefits. At the same time, GDH is widely present in animals and plants and microorganisms, so GDH-transformed plants do not cause harm to humans, animals and plants.
  • glutamate dehydrogenase transgenic plants are mainly carried out in the United States, Australia and other countries (US patents N0.5955651; 5985634; 5998700).
  • Their glutamate dehydrogenase genes are mainly derived from chlorella and E. coli, and model plants are tobacco, corn and other cash crops. It has been found through research that plants with transgenic glutamate dehydrogenase gene have improved nitrogen utilization rate, which is manifested in the larger and larger number of plant leaves.
  • the nitrogen content of different tissues of E. coli GDH-transformed plants was determined to be about 16% more than that of the control group. At the same time, the amino acid content of different tissues changed.
  • invention-The purpose of the present invention is to provide a method for improving nitrogen assimilation efficiency in plants.
  • the present invention provides a method for improving nitrogen assimilation efficiency in plants, including: (a) combining a fungal glutamate dehydrogenase (GDH) gene with a gene capable of directing the expression of a foreign gene in a plant
  • GDH fungal glutamate dehydrogenase
  • the promoter is connected to construct the chimeric gene: (b) The constructed chimeric gene is introduced into plant cells, and the transformed plant is selected and cultivated.
  • the promoter that can guide the expression of a foreign gene in a plant can be any promoter known in the art that can guide the expression of a foreign gene in a plant.
  • the glutamate dehydrogenase gene is preferably a co-NADP glutamate dehydrogenase gene or a co-NAD glutamate dehydrogenase gene.
  • the fungal glutamate dehydrogenase gene can be derived from a filamentous fungus of the genus Neurospora, including Neurospora intermediate, Neurospora edulis, and Neurospora crassa.
  • the glutamate dehydrogenase gene may also be derived from a yeast fungus, such as Saccharomyces cerevisiae.
  • the glutamate dehydrogenase gene may also be derived from a basidiomycete. For example, Gari bisporus).
  • the plant may be tobacco, corn, cotton or rice.
  • the glutamic acid dehydrogenase can be derived from a glutamic acid dehydrogenase of Neurospora intermedia (abbreviated as Ni), which has the sequence shown in SEQ ID NO: 1.
  • the gene encoding the above-mentioned glutamic acid dehydrogenase may have a sequence shown in SEQ ID NO: 2.
  • the glutamic acid dehydrogenase can be derived from Neurospora sitophila (Ns), which has the amino acid sequence shown in SEQ ID NO: 3.
  • Ns Neurospora sitophila
  • the gene encoding the above-mentioned glutamic acid dehydrogenase may have a sequence shown in SEQ ID NO: 4.
  • Ni-GDH, Ns-GDH, and Nc-GDH genes were cloned into E. coli BL21 (DE3) to induce expression, and purified Ni-GDH, Ns-GDH, and Nc-GDH were used for enzyme activity determination. It was found that the enzymatic activity of the three GDHs was higher than that of the other species. It has strong affinity and stability for ammonia.
  • These three GDH genes were subcloned into the plant expression vector pROKII, and tobacco, corn, cotton and other crops were transformed by Agrobacterium transformation, electroporation transformation, and pollen tube pathway method. After PCR, Southern, Northern and enzyme activity staining identification, P8 'sex transformants were screened.
  • the primers used are as follows:
  • Primer 1 5 'GCTCAGAATGTCTAACCTTCCCTCTGAG 3'
  • the reverse transcription reaction conditions are:
  • GDH glutamic acid dehydrogenase
  • the glutamate dehydrogenase (GDH) gene amplified by RT-PCR was recovered by agarose gel electrophoresis. Take 3 microliters (ul) of the recovered product, add lul pGEM-T eas vector, 5ul 2xT4 ligase buffer, lul DNA ligase, and 4 'C enzyme overnight. The next day, the enzyme-linked product was transformed into E. coli 'DH50a for colony screening. The screening method was identified by colony PCR and digestion. The digestion sites were Xbal and Sacl. Digestion produced 3.0kb and 1.4kb bands were positive clones pT-GDH. Positive clones were picked and sequenced. 4. Sequencing of glutamic acid dehydrogenase (GDH) gene of beer yeast and Agaricus bisporus:
  • the glutamate dehydrogenase (GDH) gene amplified by RT-PCR was recovered by agarose gel electrophoresis. Take 3 microliters (ul) of the recovered product, add lul pGEM-T easy vector, 5ul 2xT4 ligase buffer, 1ul DNA ligase, and 4 ° C enzyme overnight. The next day, the enzyme-linked product was transformed into E. coli DH50a for colony screening. The screening method was identified by colony PCR and digestion. The digestion sites were Xbal and SacL digestion to produce 3.0kb and 1.4kb bands, which were positive clones pT-GDH. Positive clones were picked and sequenced.
  • the vector pT-GDH was digested with Xbal and Sad, and the GDH fragment was recovered by agarose electrophoresis.
  • the pDH-GDH was digested with pBluescript vector with the same digestion overnight at 4 ° C.
  • E. coli DH50a was transformed the next day, and the positive clone pBlueGDH was identified by colony PCR and digestion.
  • pBlueGDH was digested with ECoRV and SacI, and the GDH fragment was recovered by agarose electrophoresis.
  • the pDHGa was digested with the same EcoRV and Sad digested pET30a vector at 12 ° C overnight. The next day, E.
  • coli DH50a was transformed and identified by colony PCR and digestion.
  • the positive clone was pETGDH.
  • the next day, positive clones were picked and transformed into E. coli BL21 (DE3).
  • the cells were cultured to about OD 0.4, and induced by ImM IPTG for 4 hours, and the bacterial cells were harvested. After the bacteria were washed with deionized water, they were lysed by ultrasound. After centrifugation, the supernatant and pellet were subjected to 10% SDS-PAGE electrophoresis. The results proved that the expression products existed as inclusion bodies.
  • MCAC—40, MCAC—60, MCAC—80, MCAC—100, MCAC—200, MCAC—500 are prepared at the same time, that is, O.4mol / L, 0.6mol / L, O.8mol / L are added to MCAC-0, respectively. , 1mol / L, 2mol / K 5mol / L imidazole.
  • the renatured protein that was dialyzed overnight was centrifuged, and the supernatant was collected, and the protein concentration was determined by ultraviolet detection at 280 nm.
  • the concentration (mg / ml) A280 x 0.825 ⁇
  • the enzyme activity was measured in system A and system C, respectively.
  • the active unit is one unit for reducing one micromole of NADP + per minute or one unit for oxidizing one micromolar of NADPH per minute.
  • Ni-GDH activity was 109.92 UI mg in System A
  • Ni-GDH was 72.93U / mg in System C
  • Ns-GDH was 95.37U / mg in System A
  • It is 63013U / mg
  • Nc-GDH is 100.25U / mg in system A, and 65.00U / mg in system C.
  • the GDH gene fragment was cut from the pT-GDH vector by double digestion with Xbal and Saci, and was recovered by agarose electrophoresis. C enzyme overnight. The next day, E. coli DH5a was transformed.
  • the positive clone pROKII-GDH was identified by colony PCR and digestion, and the positive clones were selected for transformation into Agrobacterium LBA4404. 9. Saccharomyces cerevisiae and Agaricus bisporus glutamate dehydrogenase genes were subcloned into plant expression vector pROKII
  • GDH gene fragments were cut from the pT ⁇ GDH vector by Xbal and Sad double digestion, and recovered by agarose electrophoresis, and the pROKII vector digested with the same digestion was ligated overnight at 4 ⁇ . The next day, E. coli DH5a was transformed, and the positive clone pROKII-GDH was identified by colony PCR and digestion. The positive clones were selected and transformed into Agrobacterium LBA4404.
  • the surface of the tobacco seeds was sterilized and cultured on a hormone-free MS medium (MS salt 10g / L sucrose 10g / L agar). 25-28 ⁇ , 80uE (m 2 .S) light for 16 hours. With the growth of the seedlings (11.5 months later), the shoot tips were cut off and transferred to a new MS medium to generate plantlets.
  • a hormone-free MS medium MS salt 10g / L sucrose 10g / L agar. 25-28 ⁇ , 80uE (m 2 .S) light for 16 hours. With the growth of the seedlings (11.5 months later), the shoot tips were cut off and transferred to a new MS medium to generate plantlets.
  • Agrobacterium tumefaciens containing the recombinant plasmid was inoculated in 5 ml of a medium containing kanamycin and rifampicin, cultured at 28 ° C, 200 rpm, and shaken overnight, and the cells were collected by centrifugation. Cut off the edges of the leaves of the sterile seedlings and the midrib with a scalpel. Cut the leaf into 5-8 mm wide strips along the midrib perpendicular direction. Immediately after the leaf is cut, put it in the Agrobacterium solution and soak it for 30-40 minutes.
  • the small shoots were cut with a scalpel and transferred to a rooting medium (MS inorganic salt 10 0.4 mg / 11 BA 10 100 mg I 1 kanamycin 10 30 g 1 1 sucrose 10 8 g / 1 agar) and cultured.
  • a rooting medium MS inorganic salt 10 0.4 mg / 11 BA 10 100 mg I 1 kanamycin 10 30 g 1 1 sucrose 10 8 g / 1 agar
  • Place the gene gun in a large clean bench to facilitate aseptic operation. Take 6ul of DNA-coated metal particle anhydrous ethanol suspension (about 0.6ug plasmid and 0.37ug metal particles) in the center of the microparticle carrier, and immediately dry it. Dry in a desiccator or blow dry on a bench. Place the target tissue to be transformed on the center of a 1-2 layer of filter paper or 9cm petri dish containing solid petri dish moistened with liquid medium, and evacuate when the vacuum reaches the desired value (660-760mmHg, lmmHg 133, 322Pa), the bombardment was carried out, and a shot was fired in about 12 seconds.
  • the bombarded explants are transferred to callus induction medium or bud differentiation medium, cultured in 28 'C, dark or low light, and no screening pressure such as antibiotics is added to the medium.
  • Transition culture is generally 1 to 2 weeks .
  • the explants after the transitional culture were selected and cultured on a medium containing appropriate kanaresin, and then transferred to a subculture medium in about 1 month.
  • the pROKII-GDH vector containing the GDH gene was dissolved in 1 X SSC solution.
  • the flowers with normal development were detasseled, and the bags were isolated.
  • the cotton filaments were trimmed the day before pollination, and a thin layer of pollen was germinated in a sterile Petri dish.
  • After collecting the freshly germinated fresh flower powder place it in the culture medium, and culture for about 3 minutes under 3CTC conditions.
  • When about 1/10 of the pollen has germinated, add 1/10 volume of The DNA solution is carefully mixed.
  • the final DNA concentration after mixing with the pollen is 5ug / ml.
  • the mixed solution of DNA and pollen is applied on the stigma. About 10mm 3 of the treated pollen is given a female ear. After pollination, it is repackaged and isolated to Seeds mature.
  • Agrobacterium tumefaciens containing recombinant GDH gene was inoculated in 5 ml of kanamycin and rifampicin-containing solution, cultured at 200 rpm at 28 ° C overnight, the cells were collected by centrifugation, and the sterile vaccine was cut and put into Agrobacterium After soaking in the solution for 30 minutes, the leaves were taken out, so the excess bacterial solution was aspirated on a sterile filter paper and transferred to a co-culture medium for cultivation. Incubate at 28 ° C for 48hr.
  • the total DNA of the extracted plant was digested overnight, and the digestion site was Xbal, Sad, and the next day was agarose gel electrophoresis.
  • the DNA fragments were transferred from the gel electrophoresis onto a nylon membrane, and the DNA fragments were fixed by UV irradiation for 3 minutes.
  • the probe labeled with digoxin (DIG) was hybridized with the DNA fixed on the nylon membrane, and the hybridization temperature was 68V for 20 hours. After the hybridization was completed, the nylon membrane was washed and reacted with anti-digoxigenin-resistant alkaline phosphatase (anti DIG-AP) for 30 minutes. After washing, NBT I BCIP was developed.
  • the plants After the plants have grown for 1 to 1.5 months, the plants are cleaned, dried and tested for dry weight. The results showed that the positive transformants had an increase of about 20% compared with the dry weight of the control group.
  • the nitrogen content of the transgenic plants was increased by 20- About 30%.
  • the nitrogen utilization rate of the plants grown in the MS medium was measured, and the nitrogen utilization rate of the transformed plants was increased by about 20-30% compared with the non-transformed plants.

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Description

一种提高植物氮素同化效率的方法 技术领域
本发明涉及一种提高植物氮素同化效率的方法。
技术背景
氮素是植物生长所需的第一大营养元素。目前农业上所施用的氮肥仅有 30%-40% 被植物吸收利用， 大部分都浪费。 有一部分转化为硝酸盐流失到土壤中， 造成环境 污染。 转 GDH的作物能有效增加植物氮素的吸收， 提高氮肥利用率， 从而可以节 约氮肥的施用量， 起经济效益是巨大的。 同时， GDH广泛存在于动植物和微生物中， 因此转 GDH的植物对人和动植物不会产生危害。
现在主要有美国、 澳大利亚等国家进行谷氨酸脱氢酶转基因植物的研究 （美国 专利 N0.5955651 ; 5985634； 5998700)。 它们的谷氨酸脱氢酶基因主要来源于小球 藻和大肠杆菌， 模式植物为烟草、 玉米等经济作物。 经研究发现， 转谷氨酸脱氢酶 基因的植物其氮素利用率有所提高， 表现在植物叶片较大且数量较多。 转大肠杆菌 GDH的植物不同组织的含氮量经测定比对照组多 16%左右， 同时不同组织中氨基酸 含量有所变化。 较为显著的是， 谷氨酸含量明显较低， 而丙氨酸、 赖氨酸、 天冬氨 酸等含量增加较大。 转 GDH基因土豆的淀粉行量较对照组明显增多。 但目前为止所 使用的谷氨酸脱氢酶的活性多较低。
发明公幵 - 本发明的目的是提供一种提高植物氮素同化效率的方法。
为达到上述目的， 本发明提供了一种提高植物氮素同化效率的方法， 包括： (a) 将真菌谷氨酸脱氢酶（GDH) 基因与一种可在植物中引导外源基因表达的启动子相 连， 构建嵌合基因： （b) 将构建的嵌合基因导入植物细胞中， 筛选并培养出被转化 的植株。
在本发明的方法中， 所述的可在植物中引导外源基因表达的启动子可以是任何 一种现有技术中已知的可在植物中引导外源基因表达的启动子。 所述的谷氨酸脱氢 酶基因最好是辅基 NADP谷氨酸脱氢酶基因或辅基 NAD谷氨酸脱氢醵基因。 所述 的真菌谷氨酸脱氢酶基因可以来自丝状真菌的脉孢霉属真菌， 包括中间脉孢霉、 好 食脉孢霉、 粗糙脉孢霉。 所述的谷氨酸脱氢酶基因也可以来自酵母真菌， 例如啤酒 酵母 Saccharomyces cerevisiae )。 所述的谷氨酸脱氢酶基因还可以来自担子菌， 例 如双孢菇 garic bisporus )。 所述的植物可以是烟草、 玉米、 棉花或水稻。
在本发明的方法中， 所述的谷氨酸脱氢酶可以来自中间脉孢霉 Neurospora intermedia (简称 Ni)的谷氨酸脱氢酶， 它具有 SEQ ID NO: 1 所示的序列。 编码上 述的谷氨酸脱氢酶的基因可以具有 SEQ ID NO: 2所示的序列。
本发明的方法中， 所述的谷氨酸脱氢酶可以来自好食脉孢霉 Neurospora sitophila (简称 Ns)， 它具有 SEQ ID NO: 3所示的氨基酸序列。 编码上述的谷氨酸脱 氢酶基因可以具有 SEQ ID NO: 4所示的序列。
我们将 Ni-GDH, Ns-GDH, Nc-GDH三种脉孢霉属基因克隆进大肠杆菌 BL21 (DE3 ) 中进行诱导表达， 纯化的 Ni-GDH, Ns-GDH, Nc-GDH进行酶活性测定， 发 现三种 GDH的酶活性高于其他属的脉抱霉。 它对氨的亲和力和稳定性都较强。 将这 三种 GDH基因亚克隆到植物表达载体 pROKII中， 通过农杆菌转化， 电激法转化 和花粉管通道法转化烟草、 玉米、 棉花等作物。 经 PCR、 Southern, Northern和酶活 性染色鉴定， 筛选出 P8 '性转化子。 将其移至不同氮浓度的培养基中， 发现在低氮 5mM至更低的氨离子浓度下， 表达 GDH的烟草都能正常生长， 而未转化的对照组 的叶片黄化， 发育受阻， 呈现缺氮症状。 测定植物在低氮条件下的含氮总量及未利 用氮残留量， 发现转真菌 GDH植株的含氮量较未转化的高 20%以上， 氮残留量减 少 20— 30%。 测定植株在贫氮土壤中的总氮量， 结果较未转化植株高 40%左右。 同 时我们构建了转 GDH基因的玉米、 水稻、 棉花等经济作物， 发现其含氮量较末转化 作物高 20— 30%土壤中氮残留量减少 20%以上。 检测表明， GDH在上述植物中得 到了高表达， 它加快了谷氨酸的氧化脱氨和 α—酮戊二酸（2— oxoglutarate)的还原氨 基化作用， 使植物中启动了一条新的氮利用途径， 从而提高了氨的利用率。 高等植 物中虽也有 GDH， 但其对氨的亲合力只有真菌 GDH的 1 / 10至 1 / 100, 故不能发 挥同化氨的作用。 真菌 GDH使谷氨酸的氧化脱氨伴随着大量 ATP的释放和 ct一酮 戊二酸 (2— oxoglutarate)的形成， 这给植物提供充足的能量和大量参与三羧酸循环的 碳水化合物。 氮素是植物生长所需的第一大营养元素， 目前农业上所施用的氮肥仅 有 30%— 40%为作物吸收利用， 大部分都流失， 造成环境污染。 而转 GDH 的作物 能够有效地提高氮素利用率， 从而可以节约氮肥的利用量， 减少环境污染， 其经济 效益是十分巨大。 实施例 实施例 1 . 脉孢霉属真菌、 啤酒酵母和双孢菇的培养及其谷氨酸脱氢酶的诱导
1. 将三种真菌由固体斜面转于麦芽汁培养基中， 250转 /分钟摇床培养 48 小时离 心收集菌丝体， 转入氨诱导培养基 (4%葡萄糖， 0.02MNH4AC, 0.04M N05) 中诱 导 3小时， 离心收集菌丝体。
2. 总 R A的抽提和逆转录一聚合酶链式反 (RT— PCR方法扩增谷氨酸脱氢酶基因： 诱导后的菌丝体经液氮研磨破碎后， 采用异硫氰酸胍法抽提总 RNA进行逆转录一聚 合酶链式反应。
所用引物如下：
引物 1： 5 ' GCTCAGAATGTCTAACCTTCCCTCTGAG 3 '
弓 1物 2: 5 ' GCGAGCTCTAGTCTTGGACCACCAGTCACC 3 '
逆转录反应条件是：
65 V 1分钟，
30°C 5分钟，
30°C— 65 °C 30分钟，
98 °C 5分钟，
5。C 5分钟。
聚合酶链式反应条件：
94 °C 3分钟， - 94 °C 1分钟
55Ό 1分钟 25个循环
72°C 2分钟
72 10分钟 3. 脉孢霉属谷氨酸脱氢酶 (GDH)基因序列测定：
将 RT— PCR方法扩增的谷氨酸脱氢酶 (GDH)基因经琼脂糖凝胶电泳回收。 取 3微 升 (ul)回收产物， 加入 lul pGEM-T eas 载体， 5ul 2xT4连接酶缓冲液， lul DNA连 接酶， 4' C酶连过夜。 次日， 将酶连产物转化大肠杆菌' DH50a进行菌落筛选。 筛选 方法釆用菌落 PCR和酶切鉴定， 酶切位点为 Xbal、 Sacl。 酶切产生 3.0kb和 1.4kb 两条带者为阳性克隆 pT— GDH。 挑取阳性克隆， 进行序列测定。 4. 啤酒酵母和双孢菇谷氨酸脱氢酶 (GDH)基因序列测定：
将 RT— PCR方法扩增的谷氨酸脱氢酶 (GDH)基因经琼脂糖凝胶电泳回收。 取 3微 升 (ul)回收产物， 加入 lul pGEM-T easy 载体， 5ul 2xT4连接酶缓冲液， lul DNA连 接酶， 4°C酶连过夜。 次日， 将酶连产物转化大肠杆菌 DH50a进行菌落筛选。 筛选 方法釆用菌落 PCR和酶切鉴定， 酶切位点为 Xbal、 SacL 酶切产生 3.0kb和 1.4kb 两条带者为阳性克隆 pT— GDH。 挑取阳性克隆， 进行序列测定。
5. 脉孢霉、 啤酒酵母和双孢菇谷氨酸脱氢酶基因在大肠杆菌中的表达
载体 pT-GDH经 Xbal、 Sad酶切， 琼脂糖电泳回收得到 GDH片段， 与经同样酶切 的 pBluescript载体在 4Ό酶连过夜。 次日转化大肠杆菌 DH50a， 经菌落 PCR和酶切 鉴定阳性克隆 pBlueGDH。pBlueGDH经 ECoRV、SacI酶切,琼脂糖电泳回收得到 GDH 片段， 与经同样 EcoRV、 Sad酶切的 pET30a载体在 12°C酶连过夜。 次日转化大肠 杆菌 DH50a， 经菌落 PCR和酶切鉴定， 阳性克隆为 pETGDH。次日， 挑取阳性克隆， 转化大肠杆菌 BL21(DE3)。 培养至 OD0.4左右， 经 ImM IPTG诱导 4hr,收获菌体。 菌体经去离子水洗涤后， 超声裂解。 离心， 上清和沉淀分别进行 10%SDS— PAGE 电泳。 结果证明表达产物以为包涵体形式存在。
6. 金属整合亲和层析纯化谷氨酸脱氢酶 .
配制含 8M尿素的 MCAC— 0溶液中 (20mm/L Tris · CI， pH7.9, 0.5mol/L NaCl，
10%(v / v)甘油， ImmOl几 PMSF)。 同时配制 MCAC— 40， MCAC— 60， MCAC —80, MCAC— 100， MCAC— 200 , MCAC— 500， 即在 MCAC— 0 中分别添加 O.4mol/L、 0.6mol/L, O.8mol/L、 lmol/L、 2mol/K 5mol/L的咪唑。
将谷氨酸脱氢酶包涵体溶于含 8M尿素的 MCAC— 0溶液中， 上样于 NTA 层 析柱中， 以 20— 30ml / h的流速用 5ml 8M Urea— MCAC缓冲液洗柱， 弃流出液。 以分段洗脱的方式依次用 5ml下列缓冲液洗柱， 8MUrea— MCAC40, 8M Urea— MCAC60 , 8M Urea-MCAC80, 8M Urea-MCACIOO , 8Μ Urea-MCAC200 , 8Μ Urea-MCAC500, 分别收集流出液， 进行 10% SDS— PAGE电泳， 银染进行纯度鉴定。 收集 8M Urea "MCAC200以后的沈脱液， 进行透析复性。 透析缓冲液分两种， 分别 为 pH8.5和 pH7.4的 0.1mol/L Tris.HCl， lmM EDTA。 7. 谷氨酸脱氢酶活性测定
将透析过夜的复性蛋白离心， 收集上清， 经 280nm紫外检测测定蛋白浓度， 浓度 (mg / ml)=A280 x 0.825 ο 在体系 Α、 体系 C中分别测定酶活。
体系 A测定 GDH还原氨基化作用。
2.55ml 0.1MTris.HCl, ImMEDTA, pH7.4
0.1ml 0.1MNH4C1
0.15ml 0.2M a—酮戊二酸
0.2ml 0.15%(W / V)NADPH
2ul 复性蛋白， 25°C温育 10分钟， 在 25。 C恒温下测定体系 A在 340nm光吸收 值的变化。 体系 C， 测定 GDH氧化脱氨作用：
2.8ml 0.16M谷氨酸单钠盐溶于 0.1M Tris.HCl, ImM EDTA， pH 8.5的缓冲 液中， 0.2ml 0.2%NADP, 2ul复性蛋白， 37°C温育 10分钟后， 测定在 340mm光 吸收值的变化。 活性单位为每分钟还原每一微摩尔 NADP+为一单位或每分钟氧化每一微摩尔 NADPH为一单位。 - 结果测定为在体系 A中 Ni-GDH活性单位为 109.92U I mg,在体系 C中，为 Ni-GDH 为 72.93U / mg, Ns-GDH在体系 A中为 95.37U/mg,在体系 C 中为 63013U/mg, Nc-GDH在体系 A中为 100.25U/mg,在体系 C中为 65.00U/mg。 8. 脉孢霉谷氨酸脱氢酶基因亚克隆入植物表达载体 pROKII
从 pT— GDH载体上用 Xbal、 Saci双酶切将 GDH基因片段切下， 经琼脂糖电泳回 收后， 与经同样酶切的 pROKII载体在 4。 C酶连过夜。 次日转.化大肠杆菌 DH5a， 经 菌落 PCR和酶切鉴定阳性克隆 pROKII-GDH， 挑选阳性克隆转化入农杆菌 LBA4404 中。 9. 啤酒酵母、 双孢菇谷氨酸脱氢酶基因亚克隆入植物表达载体 pROKII
从 pT~GDH载体上用 Xbal、 Sad双酶切将 GDH基因片段切下， 经琼脂糖电泳回 收后， 与经同样酶切的 pROKII载体在 4Ό酶连过夜。 次日转化大肠杆菌 DH5a， 经 菌落 PCR和酶切鉴定阳性克隆 pROKII-GDH， 挑选阳性克隆转化入农杆菌 LBA4404 中。
10. 根癌农杆菌介导的叶盘转化法转化烟草
(1) 烟草无菌苗的培养
将烟草种子表面消毒后培养在无激素的 MS培养基 (MS盐十 15g/L蔗糖十 8g/L 琼脂)上培养。 25-28Ό , 80uE(m2. S)光照 16小时， 随着小苗的生长 (1一 1.5个月以 后)， 将茎尖切下， 转到新的 MS培养基生成小植株。
(2) 烟草叶盘共培养
含重组质粒的根癌农杆菌接种于 5ml含卡那霉素和利福平的培养基中， 28°C 200 转 /分摇床培养过夜， 离心收集菌体。 将无菌苗叶片边缘和中脉用手术刀切去， 沿 中脉垂直方向将叶片切成 5— 8mm宽的叶条， 叶片切割后立即放入农杆菌液中浸泡 30-40分钟。 用镊子取出共培养的叶片， 放到无菌的滤纸上吸去过多的菌液， 叶条 移入含共培养培养基 (MS无机盐十 0.6mg 1 12， 4一 D十 30g / 1蔗糖十 8g 11琼脂) 的平皿中。 用膜将平皿封口， 以减少水分蒸发和污染。 28°C暗培养 48hr。
11. 转化植株的筛选 - 将共培养叶片转移到愈伤组织诱导培养基 (MS无机盐十 0.6mg 1 12,4-D十 300mg 1 1卡那霉素十 500mg / 1援节青霉素十 30g 1 1蔗糖十 8g 1 1琼脂)上， 使转化叶片 充分接触培养基有利于营养和激素的吸收， 在愈伤组织诱导培养基上培养两周后， 将叶片转移到芽培养基 (MS无机盐， lmg / 1 IAA十 lmg / 16— BA十 300mg I 1卡 那霉素十 500mg 11羧苄青霉素十 30g 1 1蔗糖十 8g I 1琼脂)上培养。 用解剖刀切下 小芽转移到生根培养基 (MS无机盐十 0.4mg / 11BA十 lOOmg I 1卡那霉素十 30g 1 1 蔗糖十 8g / l琼脂)上培养。
12. 基因枪法转化玉米
将基因枪放置于一较大超净台中， 以利于无菌操作。 取 6ul包被 DNA的金属颗 粒无水乙醇悬浮液 (约 0.6ug质粒和 0.37ug金属颗粒)点于微粒子载体中心， 立即在干 燥器中干燥， 或在工作台上吹干。 将欲转化的靶组织中铺在一个由液体培养基润湿 过的 1-2层滤纸或含固体培养皿的 9cm培养皿中心，抽真空，当真空度达到所需值 (660 — 760mmHg, lmmHg=133, 322Pa)时， 进行轰击， 约 12秒钟打一枪。 将轰击后的 外植体转入愈伤组织诱导培养基或芽分化培养基， 28' C, 黑暗或弱光下培养， 该培 养基中不加入抗生素等筛选压力， 过渡培养一般 1一 2周。 过渡培养后的外植体在含 适当卡那雷素的培养基上进行选择培养， 1个月左右转入继代扩繁培养基中培养。
13. 花粉管通道法介导真菌 GDH基因转化棉花
含 GDH基因的 pROKII— GDH载体溶于 1 X SSC溶液中， 选择发育正常的花去雄， 套袋隔离， 授粉前一天将棉花花丝剪齐， 在无菌培养皿中加入一薄层花粉萌发培养 基， 采集末萌发的新鲜花粉置于培养基中， 在 3CTC条件下培养 3分钟左右， 每 10ml 培养基中加入 30mm3花粉， 当约 1 / 10的花粉已经萌发时， 加入 1 / 10体积的 DNA 溶液小心混勾， 与花粉混合后 DNA的终浓度为 5ug / ml, 将 DNA与花粉的混合液 涂于柱头上， 约 10mm3处理后的花粉授一个雌穗， 授粉后重新套袋隔离至种子成熟。
14. 农杆菌介导的真菌 GDH基因转化水稻
含重组 GDH基因的根癌农杆菌接种于 5ml含卡那霉素和利福平液中， 28°C 200转 /分培养过夜， 离心收集菌体， 将无菌苗切割后立即放入农杆菌液中浸泡 30分钟， 取出叶片， 故到无菌的滤纸上吸去过多的菌液， 转入共培养基中培养。 28°C暗培养 48hr。
15. 转真菌 GDH基因植物阳性转化子的筛选和鉴定
(1) 从植物中抽提 DNA方法
取 lg植物叶片，加液氮研磨成粉末。加入 700以 2xCTAB提取液 (2 %(W I V)CTAB (十六垸基三乙基溴化铵)， lOOmmol / lTris ' CI , pH8.0, 20mmol / L EDTA, 1.4mol / INaCI), 轻轻摇匀， 65°C水浴 30分钟， 其间不时摇动。 加入 700ul酚 /氯仿 /异 戊醇 (25: 24： 1)， 并轻摇至溶液呈乳化状态， 7000转 /分离心 5分钟。 取上清液继 续用酚 /氯仿 /异戊醇 (25: 24： 1)抽提 2-3次， 加 2倍体积无水乙醇， .混匀于一 20 °C沉淀过夜。 7000转 /分离心 10分钟， 收集核酸沉淀， 溶于去离子水中。 -20°C贮 存备用。 (2) 核酸杂交法鉴定阳性转化子
将抽提的植物总 DNA酶切过夜， 酶切位点为 Xbal, Sad, 次日进行琼脂糖凝 胶电泳。 将 DNA片段从凝胶电泳中转移至尼龙膜上， 紫外照射 3分钟使 DNA片段 固定。 将用地高辛 (DIG)标记好的探针与尼龙膜上固定的 DNA进行杂交， 杂交温度 68V , 时间 20小时。 杂交完成后， 清洗尼龙膜， 和抗地高辛的碱性磷酸酶 (anti DIG 一 AP)反应 30分钟， 清洗后进行 NBT I BCIP显色。
结果， 在 1400bp左右位置出现一条杂交带， 和阳性对照相同， 表明 GDH基因 已经整合入植物基因组中。
16. 转化植株的移栽和后代分析
小心移去植株根部的琼脂， 将植株转移至含 MS无机盐溶液的较大容器里， 将 盖子打开， 使气体扩散几小时， 同时加入一些无菌水以补充蒸发的液体， 之后把植 株移栽到湿润的土壤里。 所结种子以核酸杂交和 GDH酶活染色测定后代分离， 直至 获得纯合的转基因品系。
17. 凝胶分析和谷氢酸脱氢酶活性染色
在液氨中研磨， 提取植株叶片蛋白， 进行 5%非变性凝胶电泳， i20V72 小时。 将凝胶浸泡在下述染色液中（50mMTrispH9.3 , 8mg / ml 谷氨酸， 0.04mg I ml NADP, 0.04mg / ml MTT, 0.04mg / ml硫酸的吩咳， 0.08mg /-mICaC12)。 凝胶在染 色液中浸泡后， 在含 GDH的部位有一条带， 表明转化的 GDH在植株体内表达出有 活性谷氨酸脱氢酶。
18. 转基因植株的生长和氮素利用率测试 - 表达真菌 GDH阳性植物收获种子后， 进行无菌苗的繁殖 (同 8(1))。 随着小苗的 生长，将茎尖切下，转到不同氮含量的 MS培养基上。氨的浓度分别为 20mM, 10mM, 5m , 2.5mM, 以测试其生长状况。 结果， 在 20mM， 10mM的 MS培养基上， 阳 性转化子和对照组后代未显出明显区别。 在 5mM和 2.5mM培养基上， 阳性转化子 生长状况显著优于对照组， 而对照则出现叶片萎黄等缺氮症状。 等植物生长 1一 1.5 个月后， 将植株清洁干净， 烘干， 测试干重。 结果显示， 阳性转化子比对照组干重 增加 20%左右。 同时利用凯氏定氮法， 测得转基因植株的含氮率比对照组增加 20— 30%左右。 同时测定生长在 MS培养基中植物的氮素利用率， 转化植株较未转化植 物的氮素利用率提高 20-30%左右。
序列表
<110> Institute of Microbiology, Chinese Academy of Sciences <120>一种提高植物氮素同化效率的方法
<160> 4
<210>SEQID O: 1
<211>454氨基酸
<212>蛋白质
<213> 中间脉孢霉 Neurospora intermedia
<400>
Figure imgf000011_0001
ELPSLVAGSNIAGFV VAQAMHDQGDWWSKN
<210>SEQIDNO:2
<211> 1365碱基对
<212> DNA
<213> 中间脉孢霉 Neurospora intermedia
<400>
AGGGTGACTGGTGGTCCAAGAACTAA
<210>SEQIDNO:3
<211>454氨基酸
<212>蛋白质
<213>好食脉抱霉 Neurospora sitophila
<400>
MSNLPSEPEFEQAY ELAYTLENSSLFQ HPEYRTALAVASIPERVIQFRWWEDDNGNVQ GELPSLVAGSNIAGFVKVPQAMHDQGDWWSKN
<210>SEQID O:4
<211> 1365碱基对
<212> DNA
<213>好食脉抱霉 Neurospora sitophila <400>
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Figure imgf000014_0001
^6J00/10N3/X3d 9 0 OAV

Claims

权 利 要 求
1 . 一种提高植物氮素同化效率的方法， 包括： （a)将真菌谷氨酸脱氢酶（GDH) 基因与一种可在植物中引导外源基因表达的启动子相连， 构建嵌合基因： （b )将构 建的嵌合基因导入植物细胞中， 筛选并培养出被转化的植株。
2.按照权利要求 1所述的方法，其中，所述的真菌谷氨酸脱氢酶基因是辅基 NADP 谷氨酸脱氢酶基因或辅基 NAD谷氨酸脱氢酶基因。
3. 按照权利要求 1所述的方法， 其中， 所述的真菌谷氨酸脱氢酶基因来自丝状 真菌的脉孢霉属真菌。
4. 按照权利要求 3所述的方法， 其中， 所述的丝状真菌的脉孢霉属真菌包括中 间脉孢霉、 好食脉孢霉、 粗糙脉孢霉。
5. 按照权利要求 1所述的方法， 其中， 所述的谷氨酸脱氢酶基因来自酵母真菌。
6.按照权利要求 5所述的方法，其中
Figure imgf000015_0001
cerevisiae )。
7. 按照权利要求 1所述的方法， 其中， 所述的谷氨酸脱氢酶基因来自担子菌。
8 . 按照权利要求 7 所述的方法， 其中， 所述的担子菌是双孢菇 〔Aga,'iCUS bisporus )。
9. 按照权利要求 1所述的方法， 其中， 所述的植物是烟草、 玉米、 棉花或水稻。
10. 按照权利要求 1 所述的方法， 其中， 所述的谷氨酸脱氢酶来自中间脉孢霉 Neurospora intermedia的谷氨酸脱氢酶， 它具有 SEQ ID NO: 1所示的序列。
11.按照权利要求 10所述的方法，其中，所述的谷氨酸脱氢酶的基因具有 SEQ ID NO.- 2所示的序列。
12. 按照权利要求 1 所述的方法， 其中， 所述的谷氨酸脱氢酶来自好食脉孢霉 Neurospora sitophila, 它具有 SEQ ID NO: 3所示的序列。
13. 按照权利要求 12所述的方法， 其中， 所述的谷氨酸脱氢酶基因具有 SEQ ID NO: 4所示的序列。
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