WO2021232551A1 - 一种高效人工根际联合固氮体系 - Google Patents

一种高效人工根际联合固氮体系 Download PDF

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WO2021232551A1
WO2021232551A1 PCT/CN2020/100411 CN2020100411W WO2021232551A1 WO 2021232551 A1 WO2021232551 A1 WO 2021232551A1 CN 2020100411 W CN2020100411 W CN 2020100411W WO 2021232551 A1 WO2021232551 A1 WO 2021232551A1
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nitrogen
ammonium
gene
fixing
plant
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柯秀彬
林敏�
燕永亮
战嵛华
陆伟
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中国农业科学院生物技术研究所
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Definitions

  • the invention belongs to the technical field of synthetic biology, and specifically relates to an efficient artificial rhizosphere joint nitrogen fixation system and its use.
  • Nitrogen fertilizer input is a necessary way for agricultural producers to obtain high yields.
  • the current nitrogen utilization efficiency is generally low, leading to excessive use.
  • Plant rhizosphere is the active interface of soil-plant material exchange, and it is also the main active area of interaction between host plants and microorganisms.
  • the combined nitrogen fixation system widely exists in the rhizosphere of non-leguminous crops such as rice and maize, but the natural combined nitrogen fixation system has many defects, such as being greatly affected by rhizosphere biological stress, low nitrogen fixation efficiency, and the resistance and tolerance of wild nitrogen-fixing mycorrhizal surfaces. Weak ammonium capacity, and weak nitrogen utilization capacity of traditional crop roots.
  • Synthetic biology is a convergence of cross-disciplines with the concept of systematic design and engineering construction, and it is the development frontier of a new generation of disruptive biotechnology.
  • Synthetic biology uses engineered design concepts to achieve a "bottom-up” design from components to modules to systems in living hosts as “chasses” or in vitro systems for transcription and translation.
  • the “regulation loop” or “intelligent system” realizes new biological functions that are more efficient, energy-saving or environmentally friendly.
  • the purpose of the present invention is to establish a new artificial rhizosphere joint nitrogen fixation system.
  • the artificial rhizosphere joint nitrogen fixation system of the present invention uses synthetic biology technology to artificially design and construct two brand-new functional modules: nitrogen-fixing ammonium gene module constructed by nitrogen-fixing microorganism chassis and nitrogen efficient utilization constructed by non-leguminous plant chassis Modules, and use seed coating and other inoculation techniques in the rhizosphere of crops to realize functional coupling of the above two modules.
  • a recombinant nitrogen-fixing engineered bacteria with a nitrogen-fixing and ammonium-secreting gene module is inoculated into a recombinant plant with a nitrogen-efficient functional module (nitrogen-efficient-utilizing plant chassis) to achieve the synergy of the two functions.
  • the aza-secret ammonium gene module includes the artificially designed nitrogen-fixation activation protein Neb (encoded by the DNA sequence shown in SEQ ID NO. 1) functional module and amtR (the gene of the DNA sequence shown in SEQ ID NO. 2) ammonium transport functional module.
  • the nitrogen efficient utilization module of the plant chassis is an artificially designed ammonium high affinity protein Ham functional module.
  • the present invention designs and constructs three kinds of plant chassis, specifically:
  • Ham is the coding gene of the ammonium high affinity protein Ham, which is artificially synthesized for the first time by the present invention, and its nucleotide sequence is shown in SEQ ID NO.3.
  • the construction method of the above-mentioned artificial rhizosphere joint nitrogen fixation system includes:
  • the ammonium secretion module of the nitrogen-fixing microorganism chassis and the nitrogen efficient utilization module of the plant chassis are functionally coupled through inoculation techniques such as seed coating in the rhizosphere of the crop.
  • the application method of the high-efficiency artificial rhizosphere combined nitrogen-fixing system of the present invention is to inoculate a recombinant nitrogen-fixing microorganism containing a nitrogen-fixing and ammonium-secreting gene module in the rhizosphere of a recombinant plant with a nitrogen-containing high-efficiency utilization module.
  • the present invention constructs the following artificial rhizosphere joint nitrogen fixation system:
  • the present invention has carried out the following work:
  • Pseudomonas stutzeri A1501 (Pseudomonas stutzeri A1501) was used as a model to combine nitrogen-fixing bacteria to construct a high-efficiency ammonium-secreting and nitrogen-fixing microbial chassis. Artificially designed high-efficiency ammonium transporter gene amtR, using a promoter under high ammonium conditions. Under nitrogen-limited conditions, ammonium high-affinity amtR protein transports extracellular ammonium into the cell.
  • the Neb function module and the amtR function module were transferred into the wild-type strain to obtain the engineered ammonium-secreting nitrogen-fixing bacteria.
  • Maize is used as a model plant, and a synthetic target gene is used to construct a maize chassis for efficient nitrogen utilization.
  • the method is: construct the target gene into the pCAMBIA 3300-bar expression vector (purchased from Biovector Science Lab, Inc., China Plasmid Vector Strains, Cell Line Gene Collection Center, Biovector Science Lab, Inc), and use maize HiII as the receptor, and use Agrobacterium-mediated maize immature embryos Obtained by transformation method.
  • Herbicide screening, PCR, RT-PCR, Southern blot and other techniques verify that the target gene is inserted into the maize genome, is stably expressed, and is inherited stably for multiple generations. In this way, nitrogen-efficient maize strains are obtained.
  • the target gene used in the nitrogen-efficient maize line is: the target gene Ham and the herbicide-resistant glufosinate-ammonium screening gene bar through the synthetic ammonium hyperphilic protein.
  • Rice is used as a model plant, and synthetic target genes are used to construct a rice chassis for efficient nitrogen utilization.
  • the method is: construct the target gene into the pCXK1301-hyg expression vector (purchased from Biovector Science Lab, Inc., China Plasmid Vector Strain and Cell Line Gene Collection Center, Biovector Science Lab, Inc), and use rice IR8 as the receptor to transform rice immature embryos through Agrobacterium-mediated transformation Method to obtain.
  • Antibiotic screening, PCR, RT-PCR, Southern blot and other techniques verify that the target gene is inserted into the rice genome, expressed stably, and inherited stably for multiple generations. In this way, nitrogen-efficient rice lines are obtained.
  • the target gene used in the nitrogen-efficient rice strain is: the target gene Ham and the hygromycin-resistant screening gene hyg are synthesized by artificially synthesized ammonium hyperphilic protein.
  • Wheat is used as a model plant, and synthetic target genes are used to construct a wheat chassis with efficient nitrogen utilization.
  • the method is: construct the target gene into the pCAMBIA1301-35S-NPT II expression vector (purchased from Biovector Science Lab, Inc., China Plasmid Vector Strain and Cell Line Gene Collection Center, Biovector Science Lab, Inc), using wheat Jing 411 as the recipient, and mediated by Agrobacterium Obtained by transformation of wheat immature embryos.
  • Antibiotic screening, PCR, RT-PCR, Southern blot and other techniques verify that the target gene is inserted into the wheat genome, expressed stably, and inherited stably for multiple generations. In this way, a nitrogen-efficient wheat line was obtained.
  • the target gene used in the nitrogen-efficient wheat line is: the target gene Ham and the kanamycin-resistant screening gene NPT II synthesized by artificially synthesized ammonium hyperphilic protein.
  • the method is to measure the plant growth and biological nitrogen fixation of the three artificial joint nitrogen fixation systems under greenhouse conditions.
  • the present invention adopts synthetic biology technology to construct a microbial nitrogen-fixing and ammonium secretion module and a plant’s nitrogen-efficient utilization module respectively, and complete the functional adaptation in the nitrogen-fixing bacteria chassis and the corn, rice and wheat chassis respectively, and secrete the nitrogen-fixing microorganisms.
  • the gene circuit is functionally coupled with the nitrogen efficient utilization module of the plant chassis to form a new artificial rhizosphere joint nitrogen fixation system. Compared with the control system, it has better nitrogen fixation efficiency and growth promotion effect, and has the potential to lose weight and increase production.
  • the problems of nitrogen limitation and low utilization rate in agricultural production systems provide a new method. This method also overcomes the shortcomings of traditional methods such as the long research cycle and the inability to carry out genetic modification and practice on plants and microorganisms at the same time.
  • SEQ ID NO. 1 The DNA sequence of the neb gene.
  • SEQ ID NO. 2 The DNA sequence of the amtR gene.
  • SEQ ID NO. 3 DNA sequence of ham gene.
  • FIG 1 Schematic diagram of artificial rhizosphere joint nitrogen fixation system.
  • the high-efficiency ammonium secretion and nitrogen fixation microbial chassis includes Neb+amtR ammonium secretion engineering bacteria.
  • Nitrogen-efficient crop trays include Ham+bar corn trays, Ham+hyg rice trays and Ham+NPY II wheat trays.
  • Figure 4 The ammonium secretion capacity of the high-efficiency ammonium secreting and nitrogen-fixing microbial chassis.
  • Nitrogen-fixing gene mutant strain nifH gene deletion mutant strain
  • Ammonium secretion engineering strain Neb+amtR ammonium secretion engineering strain
  • the constructed suicide recombinant plasmid was introduced into the wild-type A1501 strain by the method of three-parent combination, and the suicide plasmid was integrated into the chromosome by homologous recombination with the gene on the chromosome.
  • the single exchange strain was obtained by resistance screening and PCR verification. Then, according to the lethal characteristics of the amt gene under 10% sucrose selection pressure, the PCR-verified single exchange clones were coated on chloramphenicol containing 10% sucrose according to the dilution gradients of 10 -3 , 10 -4 and 10 -5.
  • the resistant LB plate was screened by double crossover, and the deletion mutant strain ⁇ amt of the target gene amt was verified by PCR.
  • Neb and amtR gene-transformed recombinant strains in P. stutzeri A1501 P. stutzeri A1501
  • the constructed E. coli Trans109 (pneb+amtR) donor plasmid was also introduced into the deletion mutant strain of amt by the method of tripartite binding.
  • Control strain wild-type Pseudomonas stutzeri A1501;
  • Nitrogen-fixing gene mutant strain nifH gene deletion mutant strain
  • Ammonium-secreting engineering strain Neb+amtR ammonium-secreting engineering strain
  • the method for determining the enzyme activity of nitrogen-fixing strains is as follows:
  • the wild-type, ammonium-secreting engineering bacteria and nifH gene deletion mutants of the tested strains were selected and inoculated into LB liquid medium containing corresponding antibiotics, and cultured overnight at 30°C and 220r/min with shaking.
  • the strains cultured overnight were collected by centrifugation at 4°C and 4000r/min for 10min, washed with 0.85% NaCl solution twice, transferred to A15 medium, adjusted initial OD600 to 0.1, 30°C, 200r/min shaking culture, every time Sampling was taken every 2h to determine the OD600 of the strain.
  • the acetylene reduction method was used to determine the nitrogenase activity of the strain.
  • the specific method is as follows:
  • the physiological activity of the ammonium secreting engineering strain is normal, and the nitrogenase activity and ammonium secreting ability are the best, which can be used for the subsequent construction of a high-efficiency combined nitrogen fixation system.
  • Example 3 Field application effect of high-efficiency combined nitrogen fixation system constructed by ammonium secreting engineering strain and corn
  • the experiment is carried out in a smart greenhouse, which can control temperature and humidity at all times.
  • the corn seeds were soaked and washed in sterile water for 30 minutes, and then transferred to a 5% NaClO solution soaked for 1 minute, then treated in 75% ethanol for 2 minutes, and finally washed with sterile water 5 times.
  • the seeds after surface disinfection were put into prepared various bacterial suspensions and sterilized wild-type suspensions (no inoculation control) and soaked for 30 minutes. Put the soaked corn seeds into a plastic pot (inner diameter 20cm, height 20cm) containing 2.5 kg of soil substrate (Klasmann-Deilmann), 4 seeds per pot, 8 replicates per group. The experiment is set to 8 replicates. After the corn emerges, thin the seedlings to 2 pieces/pot, and manage the corn as normal during the growth cycle. After 60 days of planting, the growth of corn above and below ground was measured.
  • the average plant height of the artificially designed high-efficiency nitrogen fixation system was significantly higher than that of the control system (Table 1).
  • the average plant height of corn after inoculation with ammonium secreting engineering bacteria increased by 1.9% compared with the fertilization treatment, and it increased by 4.7% compared with the control without inoculation.
  • the artificial combined nitrogen fixation system constructed by the high-efficiency ammonium secreting and nitrogen-fixing microbial chassis and the nitrogen-efficient plant chassis can significantly increase plant growth and has the best growth-promoting effect on corn.
  • the 15N dilution method was used to evaluate the nitrogen-fixing ability of nitrogen-fixing bacteria in the artificially designed rhizosphere joint nitrogen-fixing system (Table 2).
  • Table 2 The data analysis of the artificial combined nitrogen fixation system constructed by the Neb+amtR ammonium engineered bacteria chassis and the Ham+bar corn chassis showed that compared with the control system, the ammonium engineered bacteria significantly increased the total nitrogen content of the corn plant and fixed it from the air.
  • the percentage of nitrogen in the total plant nitrogen bio-nitrogen fixation efficiency
  • the amount of biological nitrogen fixation was used to evaluate the nitrogen-fixing ability of nitrogen-fixing bacteria in the artificially designed rhizosphere joint nitrogen-fixing system.
  • the total nitrogen content of the plant after inoculation with the ammonium secreting engineering strain increased by 40.6% relative to the control system, and the contribution rate of biological nitrogen fixation was 21.5%.
  • the biological nitrogen fixation amount was 0.8g/plant.
  • the estimated amount of biological nitrogen fixation per hectare is 48kg.
  • the estimated fertilizer saving rate is about 25.6% (Table 2).
  • the contribution rate of biological nitrogen fixation is 10.1%. After conversion, the biological nitrogen fixation amount is about 0.23g/plant. The estimated biological nitrogen fixation per hectare is 13.8kg, and the fertilizer saving rate is about 7.3% (Table 2) .
  • the artificial combined nitrogen fixation system constructed by the high-efficiency ammonium-secreting and nitrogen-fixing microbial chassis and the high-efficiency nitrogen utilization plant chassis can enable plants to obtain higher biological nitrogen fixation efficiency and nitrogen fixation.
  • the effect of fertilizer saving is predicted to be the best. It is estimated that about 25.6% of fertilizer can be saved per hectare. , And the control system is 7.3%.
  • Example 4 Field application effect of high-efficiency combined nitrogen fixation system constructed by ammonium secreting engineering strain and rice
  • the experiment is carried out in a smart greenhouse, which can control temperature and humidity at all times.
  • the rice seeds were soaked and washed in sterile water for 30 minutes, then transferred to 5% NaClO solution for 1 minute, then treated in 75% ethanol for 2 minutes, and finally washed with sterile water 5 times.
  • the seeds after surface disinfection were put into prepared various bacterial suspensions and sterilized wild-type suspensions (no inoculation control) and soaked for 30 minutes. Put the soaked rice seeds into a plastic pot (inner diameter 20cm, height 20cm) containing 2 kg of soil substrate (Klasmann-Deilmann), 4 seeds per pot, 8 replicates per group. The experiment is set to 8 replicates. After the rice emerges, thin the seedlings to 2 seeds/pot, and manage the rice as normal during the growth cycle. After 60 days of planting, the aboveground and underground growth of rice were measured.
  • the plant height, aboveground and underground parts and total biomass were significantly higher than those of the control (Table 1).
  • the total biomass of plants inoculated with ammonium secretion engineering strains was increased by 6.6% compared with the fertilization treatment, and increased compared with the control without inoculation. 10.9%.
  • the above-ground biomass increased by 10.4% compared with the fertilization treatment, and 20.6% compared with the non-inoculated control; the root weight increased by 2.4% compared with the fertilized treatment, and 2.6% compared with the non-inoculated control.
  • the average plant height of the artificially designed high-efficiency nitrogen fixation system was significantly higher than that of the control system (Table 1).
  • the average plant height of rice after inoculation with ammonium secreting engineering bacteria increased by 4.3% compared with the fertilization treatment, and increased by 6.8% compared with the control without inoculation.
  • the artificial combined nitrogen fixation system constructed by the high-efficiency ammonium secreting and nitrogen-fixing microbial chassis and the nitrogen-efficient plant chassis can significantly increase plant growth and has the best growth-promoting effect on rice.
  • the 15N dilution method was used to evaluate the nitrogen-fixing ability of nitrogen-fixing bacteria in the artificially designed rhizosphere joint nitrogen-fixing system (Table 2).
  • Table 2 The data analysis of the artificial combined nitrogen fixation system constructed by the Neb+amtR secretion engineering bacteria chassis and the Ham+hyg rice chassis shows that compared with the control system, the ammonium secretion engineering bacteria significantly increase the total nitrogen content of rice plants and fix it from the air.
  • the percentage of nitrogen in the total plant nitrogen bio-nitrogen fixation efficiency
  • the amount of biological nitrogen fixation is used to evaluate the nitrogen-fixing ability of nitrogen-fixing bacteria in the artificially designed rhizosphere joint nitrogen-fixing system.
  • the total nitrogen content of the plant after inoculation with the ammonium secreting engineering strain increased by 48.2% compared to the control system, and the contribution rate of biological nitrogen fixation was 18.5%.
  • the biological nitrogen fixation amount was 0.14g/plant, assuming the yield of corn With 210,000 plants, the estimated amount of biological nitrogen fixation per hectare is 29.4kg. According to the normal fertilization rate of rice is 120kg N/ha, the estimated fertilizer saving rate is about 24.5% (Table 2).
  • the contribution rate of biological nitrogen fixation is 9.0%. After conversion, the biological nitrogen fixation is about 0.06g/plant. The estimated biological nitrogen fixation per hectare is 12.6kg, and the fertilizer saving rate is about 10.5% (Table 2) .
  • the artificial combined nitrogen fixation system constructed by the high-efficiency ammonium secreting and nitrogen-fixing microbial chassis and the nitrogen-efficient plant chassis can enable plants to obtain higher biological nitrogen fixation efficiency and nitrogen fixation.
  • the effect of fertilizer saving is predicted to be the best. It is estimated that about 24.5% of fertilizer can be saved per hectare , And the control system is 10.5%.
  • Example 5 Field application effect of high-efficiency combined nitrogen fixation system constructed by ammonium secreting engineering strains and wheat
  • the experiment is carried out in a smart greenhouse, which can control temperature and humidity at all times.
  • the wheat seeds were soaked and washed in sterile water for 30 minutes, and then transferred to a 5% NaClO solution soaked for 1 minute, then treated in 75% ethanol for 2 minutes, and finally washed with sterile water 5 times.
  • the seeds after surface disinfection were put into prepared various bacterial suspensions and sterilized wild-type suspensions (no inoculation control) and soaked for 30 minutes. Put the soaked wheat seeds into a plastic pot (inner diameter 20cm, height 20cm) containing 2.5 kg of soil substrate (Klasmann-Deilmann), 4 seeds per pot, 8 replicates per group. The experiment is set to 8 replicates. According to normal management during the wheat growth cycle. After 60 days of planting, the aboveground and underground growth of wheat were measured.
  • the average plant height of the artificially designed high-efficiency nitrogen fixation system was significantly higher than that of the control system (Table 1).
  • the average plant height of wheat after inoculation with ammonium secreting engineering bacteria increased by 1.9% compared with the fertilization treatment, and it increased by 4.7% compared with the control without inoculation.
  • the artificial combined nitrogen fixation system constructed by the high-efficiency ammonium-secreting and nitrogen-fixing microbial chassis and the nitrogen-efficient plant chassis can significantly increase plant growth and has the best growth-promoting effect on wheat.
  • the 15N dilution method was used to evaluate the nitrogen-fixing ability of nitrogen-fixing bacteria in the artificially designed rhizosphere joint nitrogen-fixing system (Table 2).
  • Table 2 The data analysis of the artificial joint nitrogen fixation system constructed by the Neb+amtR ammonium-secreting engineering bacteria chassis and the Ham+NPT II wheat chassis showed that compared with the control system, the ammonium-secreting engineering bacteria significantly increased the total nitrogen content of wheat plants and removed the nitrogen from the air.
  • the percentage of fixed nitrogen in total plant nitrogen bio-nitrogen fixation efficiency
  • the amount of biological nitrogen fixation was used to evaluate the nitrogen-fixing ability of nitrogen-fixing bacteria in the artificially designed rhizosphere joint nitrogen-fixing system.
  • the total nitrogen content of the plant after inoculation with the ammonium secretion engineering strain increased by 42.6% compared to the control system, and the biological nitrogen fixation contribution rate was 14.3%. After conversion, the biological nitrogen fixation amount was 0.02g/plant. The amount of nitrogen fixation is 80kg, and the fertilizer saving rate is about 66.6% (Table 2).
  • the contribution rate of biological nitrogen fixation is 8.5%. After conversion, the biological nitrogen fixation is about 0.01g/plant. Assuming that the wheat yields 4 million plants per hectare, the estimated biological nitrogen fixation per hectare is 40kg. The normal fertilization rate is 120kg N/ha. The estimated fertilizer saving rate is about 33.3% (Table 2).
  • the artificial combined nitrogen fixation system constructed by the high-efficiency ammonium-secreting and nitrogen-fixing microbial chassis and the nitrogen-efficient plant chassis can enable plants to obtain higher biological nitrogen fixation efficiency and nitrogen fixation.
  • the effect of fertilizer saving is predicted to be the best. It is estimated that about 66.6% of chemical fertilizers can be saved per hectare , While the control system is 33.3%.
  • the measurement indicators of the experimental results include the amount of plant growth and the amount of biological nitrogen fixation. in:
  • Table 1 The growth rate of plant traits and biomass of artificial rhizosphere combined nitrogen fixation system relative to the control system
  • the amount of nitrogen fertilizer applied in the fertilization control system is 50 mg nitrogen per kilogram of soil.
  • the 15N stable isotope dilution method is a general method for determining the amount of biological nitrogen fixation.
  • the growth evaluation of corn, rice, and wheat is basically the same as the 15N method for determining the amount of biological nitrogen fixation.
  • the specific operation and calculation method are:
  • the experiment set up 4 replicates.
  • the contribution rate of biological nitrogen fixation to plant nitrogen nutrition is calculated as the percentage (%Ndfa) of nitrogen fixed from the air to the total plant nitrogen.
  • the calculation method is as follows:
  • %Ndfa (1-atom%15NFexcess/%15NNFexcess) ⁇ 100%
  • the calculation result is expressed as the arithmetic mean of 3 independent measurement results obtained under repeatability conditions.
  • Nt-total plant nitrogen content (%), unit %;
  • Biomass the sum of the aboveground and underground biomass of the plant, in grams per plant;
  • the calculation result is expressed as the arithmetic mean of 3 independent measurement results obtained under repeatability conditions.

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Abstract

一种人工根际联合固氮体系，包括转化了固氮激活蛋白Neb和铵转运蛋白amtR编码基因的重组固氮工程菌，以及转化了铵高亲蛋白Ham编码基因的重组植物，并在作物根际通过种子包衣接种技术，实现上述两者功能偶联。

Description

一种高效人工根际联合固氮体系 技术领域
本发明属于合成生物学技术领域，具体涉及一种高效人工根际联合固氮体系及其用途。
背景技术
氮肥投入是农业生产者获得高产的必要途径。但是目前氮利用效率普遍不高，导致过度使用。植物根际是土壤-植物进行物质交换的活跃界面，也是宿主植物和微生物相互作用的主要活动区。联合固氮体系广泛存在于水稻、玉米等非豆科作物根际，但是天然的联合固氮体系有许多缺陷，如受根际生物逆境影响大、固氮效率低下以及野生固氮菌根表抗逆能力和耐铵能力弱、传统农作物根系氮利用能力弱。
合成生物学是以系统化设计和工程化构建为理念的汇聚交叉学科，是新一代颠覆性生物技术的发展前沿。合成生物学利用工程化的设计理念，在作为“底盘”的活体宿主或用于转录和翻译的体外系统中，实现从元件到模块再到系统的“自下而上”设计。利用生物系统的DNA、RNA、蛋白质等生物大分子作为候选“元器件”进行人工设计和智能改造，利用转录调控、代谢调控等生物功能将这些人工元器件关联起来，创建人工“功能模块”、“调控回路”或“智能系统”，实现更高效、更节能或更环保的全新生物功能。
传统的增强植物氮素利用效率的研究主要围绕两方面技术开展：一是通过对植物底盘的性能改良，人工设计氮高效利用功能模块，使植物对氮素的亲和力增强；二是人工设计固氮菌固氮及耐铵泌铵等模块，提高根际固氮微生物的固氮效率，使固氮微生物为植物提供更多生物固定的氮素。
但是，传统的方法存在研究周期长、微生物和植物的改良技术和应用都相对独立等诸多缺陷。因此，利用合成生物学技术建立一种全新的人工根际联合固氮体系，对于提高氮肥利用率并降低农业生产中对氮肥的依赖具有十分重要的意义。
发明内容
本发明的目的是建立一种全新的人工根际联合固氮体系。
本发明的人工根际联合固氮体系，是运用合成生物学技术，分别人工设计并构建两种全新的功能模块：固氮微生物底盘构建的固氮泌铵基因模块和非豆科植物底盘构建的 氮高效利用模块，并在作物根际通过种子包衣等接种技术，将上述两种模块实现功能偶联。
即，在具有氮高效利用功能模块的重组植物(氮高效利用植物底盘)中接种具有固氮泌铵基因模块的重组固氮工程菌(固氮微生物底盘)，以实现二者功能的协同。
所述固氮泌铵基因模块，是包括人工设计的固氮激活蛋白Neb(由SEQ ID NO.1所示的DNA序列编码)功能模块和amtR(SEQ ID NO.2所示DNA序列的基因)铵转运功能模块。
所述植物底盘的氮高效利用模块，是人工设计的铵高亲蛋白Ham功能模块。本发明设计并构建了三种植物底盘，具体为：
含有基因Ham和基因bar的重组玉米；
含有基因Ham和基因hyg的重组水稻；
含有基因Ham和基因NPT II的重组小麦。
其中，Ham是铵高亲蛋白Ham的编码基因，由本发明首次人工合成，其核苷酸序列为SEQ ID NO.3所示。
上述人工根际联合固氮体系的构建方法，包括：
1)获得固氮泌铵模块，将所述固氮泌铵模块导入固氮微生物中，获得含所述固氮泌铵模块的重组微生物；
2)获得氮高效利用模块，将所述氮高效利用模块导入植物中，获得含所述氮高效利用模块的重组植物。
3)在作物根际通过种子包衣等接种技术，将固氮微生物底盘泌铵模块与植物底盘的氮高效利用模块实现功能偶联。
本发明的高效人工根际联合固氮体系的应用方法，是在含氮高效利用模块的重组植物根际中接种含有固氮泌铵基因模块的重组固氮微生物。
因此，本发明构建了以下人工根际联合固氮体系：
neb+amtR泌铵工程菌与Ham+bar玉米品系
neb+amtR泌铵工程菌与Ham+hyg水稻品系
neb+amtR泌铵工程菌与Ham+NPT II小麦品系
经测定植物生长量和生物固氮量，评价促生效果和固氮效率发现，本发明提供的上述人工根际联合固氮体系的固氮效果均非常显著。
具体来讲，本发明进行了如下工作：
1、构建微生物高效固氮泌铵基因模块
用施氏假单胞菌A1501(Pseudomonas stutzeri A1501)作为模式联合固氮菌，构建高效泌铵固氮微生物底盘。人工设计高效铵转运蛋白基因amtR，采用高铵条件下的启动子。在限氮条件下，铵高亲和力amtR蛋白将细胞外的铵转运到细胞内。
通过以下人工设计的功能模块，构建提高固氮酶的耐铵和泌铵能力的工程菌：
通过对野生型A1501进行人工设计固氮激活Neb功能模块，将Neb功能模块和amtR功能模块转入野生型菌株中，获得泌铵固氮工程菌。
2、分别构建三种植物底盘的氮高效利用模块
1)在玉米中构建氮高效玉米品系
将玉米作为模式植物，用人工合成的目的基因构建氮高效利用玉米底盘。
方法是：将目的基因构建到pCAMBIA 3300-bar表达载体(购买自中国质粒载体菌株细胞株基因保藏中心Biovector Science Lab，Inc)上，以玉米HiII为受体，通过农杆菌介导的玉米幼胚转化法获得。除草剂筛选、PCR、RT-PCR、Southern blot等技术验证目的基因插入玉米基因组中，稳定表达，且多世代稳定遗传。以此获得氮高效玉米品系。
氮高效玉米品系所用的目的基因是：通过人工合成的铵高亲蛋白的目的基因Ham和抗除草剂草铵膦筛选基因bar。
2)在水稻中构建氮高效水稻品系
将水稻作为模式植物，用人工合成的目的基因构建氮高效利用水稻底盘。
方法是：将目的基因构建到pCXK1301-hyg表达载体(购买自中国质粒载体菌株细胞株基因保藏中心Biovector Science Lab，Inc)上，以水稻IR8为受体，通过农杆菌介导的水稻幼胚转化法获得。抗生素筛选、PCR、RT-PCR、Southern blot等技术验证目的基因插入水稻基因组中，稳定表达，且多世代稳定遗传。以此获得氮高效水稻品系。
氮高效水稻品系所用的目的基因是：通过人工合成的铵高亲蛋白的目的基因Ham和抗潮霉素筛选基因hyg。
3)在小麦中构建氮高效小麦品系
将小麦作为模式植物，用人工合成的目的基因构建氮高效利用小麦底盘。
方法是：将目的基因构建到pCAMBIA1301-35S-NPT II表达载体(购买自中国质粒载体菌株细胞株基因保藏中心Biovector Science Lab，Inc)上，以小麦京411为受体，通过农杆菌介导的小麦幼胚转化法获得。抗生素筛选、PCR、RT-PCR、Southern blot等技术验证目的基因插入小麦基因组中，稳定表达，且多世代稳定遗传。以此获得氮高效小麦品系。
氮高效小麦品系所用的目的基因是：通过人工合成的铵高亲蛋白的目的基因Ham和抗卡那霉素筛选基因NPT II。
3、微生物和植物底盘的功能适配与田间应用考察
将上述泌铵工程菌分别和氮高效玉米、水稻和小麦进行组合，形成以下三个人工根际联合固氮体系(图1)，分别是
neb+amtR泌铵工程菌与Ham+bar玉米品系
neb+amtR泌铵工程菌与Ham+hyg水稻品系
neb+amtR泌铵工程菌与Ham+NPT II小麦品系
考察评估三个人工根际联合固氮体系的根际固氮效率和植物促生效果，方法是在温室条件下分别对这三个人工联合固氮体系的植物生长量和生物固氮量进行测定。
结果表明：三个人工根际联合固氮体系的根际固氮效率和促进植物生长量都显著高于对照体系。
本发明采用合成生物学技术，分别构建了微生物的固氮泌铵模块和植物的氮高效利用模块，并分别在固氮菌底盘和玉米、水稻和小麦底盘中完成了功能适配，将固氮微生物泌铵基因线路与植物底盘的氮高效利用模块进行功能偶联，形成一个全新的人工根际联合固氮体系，相比对照体系，具有更好的固氮效率和促生效果，具有减肥增产的潜力，为解决农业生产系统中氮素限制和利用率低下等难题提供了一种新方法。该方法也克服了传统方法研究周期长、无法对植物和微生物同时进行基因改造和实践等缺陷。
序列表信息
SEQ ID NO.1：neb基因的DNA序列。
SEQ ID NO.2：amtR基因的DNA序列。
SEQ ID NO.3：ham基因的DNA序列。
附图说明：
图1人工根际联合固氮体系示意图。高效泌铵固氮微生物底盘包括Neb+amtR泌铵工程菌。氮高效利用作物底盘包括Ham+bar玉米底盘、Ham+hyg水稻底盘和Ham+NPY II小麦底盘。
图2高效泌铵固氮微生物底盘的生长曲线；
图3高效泌铵固氮微生物底盘的固氮酶活特征；
图4高效泌铵固氮微生物底盘的泌铵能力。
图2—图4中：
野生型菌株，野生型Pseudomonas stutzeri A1501；
固氮基因突变株，nifH基因缺失突变株；
泌铵工程菌株，Neb+amtR泌铵工程菌；
n.d.，未检出。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于举例说明本发明的方法，而不用于限制本发明的范围。凡未注明具体实验条件的，均为按照本领域技术人员熟知的常规条件。
实施例1 Neb+amtR泌铵工程菌的获得
一、固氮施氏假单胞菌A1501(P.stutzeri A1501)中amt基因缺失突变株的构建：首先利用融合PCR技术将目的基因的上游同源片段、氯霉素抗性盒基因以及目的基因的下游同源片段融合成一大小约为4.1kb的融合片段，然后将克隆片段进行BamH I和Hind III双酶切，连接到自杀载体pk18mobsacB上。将构建好的自杀重组质粒通过三亲结合的方法导入到野生型A1501菌中，通过与染色体上基因的同源重组将自杀质粒整合到了染色体上，利用抗性筛选和PCR验证得到单交换菌株，然后根据amt基因在10％蔗糖选择压下致死的特性，将经过PCR验证的单交换克隆按照10 -3、10 -4和10 -5的稀释梯度分别涂布在含有10％蔗糖的氯霉素抗性的LB平板，进行双交换的筛选，经PCR验证得到目的基因amt的缺失突变菌株Δamt。
二、固氮施氏假单胞菌A1501(P.stutzeri A1501)中Neb和amtR基因转化重组菌株的构建：首先PCR扩增获得完整的Neb和amtR DNA片段，然后将克隆片段进行BamHI和HindIII酶切，插入到广宿主表达载体pLAFR3的多克隆位点处，最后将neb和amtR表达载体转入到大肠杆菌感受态细胞Trans109中获得具有四环素抗性的重组表达菌株E.coli Trans109(pneb+amtR)。将构建好的E.coli Trans109(pneb+amtR)供体质粒，同样通过三亲结合的方法导入到amt的缺失突变菌株中。
实施例2 Neb+amtR泌铵工程菌的固氮效果测定
一、实验目的：考察工程菌的固氮酶活和泌铵能力(固氮菌株的生长曲线、固氮酶活和泌铵特性)
二、实验对象：
对照菌株：野生型Pseudomonas stutzeri A1501；
固氮基因突变株：nifH基因缺失突变株；
泌铵工程菌株：Neb+amtR泌铵工程菌；
三、实验方法：
固氮菌株酶活测定，方法如下：
挑取待测菌株野生型、泌铵工程菌和nifH基因缺失突变株接种于含对应抗生素的LB液体培养基中，30℃、220r/min振荡培养过夜。4℃、4000r/min离心10min收集培养过夜的菌株，经0.85％的NaCl溶液洗涤菌体2次，转接至A15培养基中，调整初始OD600至0.1，30℃、200r/min振荡培养，每隔2h取样测定菌株的OD600。以时间(h)为横坐标轴，OD600为纵坐标轴绘制生长曲线。
采用乙炔还原法测定菌株的固氮酶活性。具体方法如下：
取10mL过夜培养的菌液，6000rpm/min离心10min，再使用0.85％的NaCl溶液悬浮洗涤菌体沉淀2次，取1mL OD600为1.0的菌液至转接至9mL装有A15无氮源培养基的三角瓶中，初始OD600为0.1，体积为10mL。盖上胶塞，密封瓶口，充氩气4min用以排出三角瓶中的空气。向瓶中注入0.5％的氧气和10％的乙炔。实验设置4个重复，30℃、220r/min振荡培养。每隔2h用微量进样器从瓶中抽取250μL气体注入气相色谱中(SP-2305型气相色谱仪)检测乙烯含量，记录乙烯峰面积。利用公式计算固氮酶的活性：固氮酶活性[nmol C2H4/(mg protein·h)]＝所测菌株乙烯峰面积×(三角瓶中气相体积/进样量)/1nmol标准乙烯峰的面积×菌体蛋白浓度×反应时间。
固氮菌株泌铵能力测定，方法如下：
挑取待测菌株接种于含对应抗生素的LB液体培养基中过夜培养，菌株离心收集后转至10mL A15液体培养基中，调整初始OD600为0.1。菌液在固氮条件下培养72h后收集上清液利用酶标仪测定铵离子浓度。实验设置4个重复。
四、实验结果：
固氮施氏假单胞菌野生型、泌铵工程菌和nifH基因缺失突变株在LB培养基中长势基本一致，均在2h后进入对数生长期，在8-10h时到达平台期(图2)。固氮条件下，泌铵工程菌的固氮酶活达到1477nmol acetylene/(mg protein h)-1，略高于野生型的固氮酶活(图3)。同样在固氮条件下，培养72h后泌铵工程菌的胞外铵离子浓度达到18μM(图4)。
五、实验结论：
泌铵工程菌株的生理活性正常，其中固氮酶活和泌铵能力最佳，可用于后续高效联合固氮体系的构建。
实施例3泌铵工程菌株与玉米构建的高效联合固氮体系的田间应用效果
实验方法
(1)植物生长量测定
实验在智能温室进行，可以全时控制温度和湿度。实验接种的固氮菌包括5个处理(泌铵工程菌和野生型菌株、nifH基因缺失突变菌株以及50mg N每千克土施氮处理和不施肥不接种处理对照)；玉米品种包括2个处理(氮高效利用品系及对照品系)；实验设置8个重复。本实验总设置5×2×8＝80个样品；
将玉米种子在无菌水中浸泡清洗30min后转移至5％NaClO溶液浸泡1min，接着在75％乙醇中处理2min，最后用无菌水清洗5次。表面消毒后的种子分别放入准备好的各种菌悬液以及灭菌的野生型悬浮液(不接种对照)浸泡30min。将浸泡后的玉米种子放入装有2.5千克土壤基质(Klasmann-Deilmann)的塑料盆中(内径20cm、高20cm)，每盆4颗种子，每组8个重复。试验设置8个重复。玉米出苗后间苗至2颗/盆，玉米生长周期内按照正常管理。种植60天后分别测定玉米地上和地下部生长量。
实验结果：
玉米生长期第60天采集地上和地下部进行植物生物量测定，包括株高、株重和根系重量(表1)。数据分析表明相比施肥和不接种对照体系，由Neb+amtR泌铵工程菌与Ham+bar玉米品系构建的联合固氮体系的植物生物量均显著增加。
在这个高效固氮体系中，植物株高、地上和地下部及总生物量显著高于对照(表1)，接种泌铵工程菌株的植株总生物量较施肥处理提高7.2％，较不接种对照提高14.9％。其中地上部生物量较施肥处理提高13.5％，较不接种对照提高19.0％；根系重量较施肥处理提高1.2％，较不接种对照提高3.6％。
人工设计高效固氮体系的平均株高显著高于对照体系(表1)，接种泌铵工程菌后玉米平均株高较施肥处理增加1.9％，较不接种对照增加4.7％。
实验结论：
高效泌铵固氮微生物底盘与氮高效利用植物底盘构建的人工联合固氮系统可以显著提高植株生长量，对玉米的促生效果最佳。
(2)15N稳定性同位素稀释法测定生物固氮量
实验结果：
利用15N稀释法对人工设计根际联合固氮体系中的固氮菌固氮能力进行评价(表2)。对Neb+amtR泌铵工程菌底盘与Ham+bar玉米底盘构建的人工联合固氮体系的数据分析表明，与对照体系相比，泌铵工程菌显著提高了玉米植株的全氮含量、从空气 中固定的氮素占植物总氮量的百分比(生物固氮效率)和生物固氮量。
在这个高效固氮体系中，接种泌铵工程菌株后植株全氮含量相对于对照体系增加40.6％，生物固氮贡献率为21.5％，经换算后生物固氮量为0.8g/株，假设玉米每公顷产6万株，估算每公顷的生物固氮量为48kg，按照玉米的正常施肥量是187.5kg N/公顷估算节肥率约为25.6％(表2)。
而在对照体系中，生物固氮量贡献率为10.1％，经换算后生物固氮量约为0.23g/株，估算每公顷的生物固氮量为13.8kg，节肥率约为7.3％(表2)。
实验结论：
高效泌铵固氮微生物底盘与氮高效利用植物底盘构建的人工联合固氮体系可以使植物获得具有更高的生物固氮效率和固氮量，预测节肥效果最佳，据估算每公顷可节省化肥约25.6％，而对照体系为7.3％。
实施例4泌铵工程菌株与水稻构建的高效联合固氮体系的田间应用效果
实验方法
(1)植物生长量测定
实验在智能温室进行，可以全时控制温度和湿度。实验接种的固氮菌包括5个处理(泌铵工程菌和野生型菌株、nifH基因缺失突变菌株以及50mg N每千克土施氮处理和不施肥不接种处理对照)；水稻品种包括2个处理(氮高效利用品系及对照品系)；实验设置8个重复。本实验总设置5×2×8＝80个样品；
将水稻种子在无菌水中浸泡清洗30min后转移至5％NaClO溶液浸泡1min，接着在75％乙醇中处理2min，最后用无菌水清洗5次。表面消毒后的种子分别放入准备好的各种菌悬液以及灭菌的野生型悬浮液(不接种对照)浸泡30min。将浸泡后的水稻种子放入装有2千克土壤基质(Klasmann-Deilmann)的塑料盆中(内径20cm、高20cm)，每盆4颗种子，每组8个重复。试验设置8个重复。水稻出苗后间苗至2颗/盆，水稻生长周期内按照正常管理。种植60天后分别测定水稻地上和地下部生长量。
实验结果：
水稻生长期第60天采集地上和地下部进行植物生物量测定，包括株高、株重和根系重量(表1)。数据分析表明相比施肥和不接种对照体系，由Neb+amtR泌铵工程菌与Ham+hyg水稻品系构建的联合固氮体系的植物生物量均显著增加。
在这个高效固氮体系中，植物株高、地上和地下部及总生物量显著高于对照(表1)，接种泌铵工程菌株的植株总生物量较施肥处理提高6.6％，较不接种对照提高 10.9％。其中地上部生物量较施肥处理提高10.4％，较不接种对照提高20.6％；根系重量较施肥处理提高2.4％，较不接种对照提高2.6％。
人工设计高效固氮体系的平均株高显著高于对照体系(表1)，接种泌铵工程菌后水稻平均株高较施肥处理增加4.3％，较不接种对照增加6.8％。
实验结论：
高效泌铵固氮微生物底盘与氮高效利用植物底盘构建的人工联合固氮系统可以显著提高植株生长量，对水稻的促生效果最佳。
(2)15N稳定性同位素稀释法测定生物固氮量
实验结果：
利用15N稀释法对人工设计根际联合固氮体系中的固氮菌固氮能力进行评价(表2)。对Neb+amtR泌铵工程菌底盘与Ham+hyg水稻底盘构建的人工联合固氮体系的数据分析表明，与对照体系相比，泌铵工程菌显著提高了水稻植株的全氮含量、从空气中固定的氮素占植物总氮量的百分比(生物固氮效率)和生物固氮量。
在这个高效固氮体系中，接种泌铵工程菌株后植株全氮含量相对于对照体系增加48.2％，生物固氮贡献率为18.5％，经换算后生物固氮量为0.14g/株，假设玉米每公顷产21万株，估算每公顷的生物固氮量为29.4kg，按照水稻的正常施肥量是120kg N/公顷估算节肥率约为24.5％(表2)。
而在对照体系中，生物固氮量贡献率为9.0％，经换算后生物固氮量约为0.06g/株，估算每公顷的生物固氮量为12.6kg，节肥率约为10.5％(表2)。
实验结论：
高效泌铵固氮微生物底盘与氮高效利用植物底盘构建的人工联合固氮体系可以使植物获得具有更高的生物固氮效率和固氮量，预测节肥效果最佳，据估算每公顷可节省化肥约24.5％，而对照体系为10.5％。
实施例5泌铵工程菌株与小麦构建的高效联合固氮体系的田间应用效果
实验方法
(1)植物生长量测定
实验在智能温室进行，可以全时控制温度和湿度。实验接种的固氮菌包括5个处理(泌铵工程菌和野生型菌株、nifH基因缺失突变菌株以及50mg N每千克土施氮处理和不施肥不接种处理对照)；小麦品种包括2个处理(氮高效利用品系及对照品系)；实验设置8个重复。本实验总设置5×2×8＝80个样品；
将小麦种子在无菌水中浸泡清洗30min后转移至5％NaClO溶液浸泡1min，接着在75％乙醇中处理2min，最后用无菌水清洗5次。表面消毒后的种子分别放入准备好的各种菌悬液以及灭菌的野生型悬浮液(不接种对照)浸泡30min。将浸泡后的小麦种子放入装有2.5千克土壤基质(Klasmann-Deilmann)的塑料盆中(内径20cm、高20cm)，每盆4颗种子，每组8个重复。试验设置8个重复。小麦生长周期内按照正常管理。种植60天后分别测定小麦地上和地下部生长量。
实验结果：
小麦生长期第60天采集地上和地下部进行植物生物量测定，包括株高、株重和根系重量(表1)。数据分析表明相比施肥和不接种对照体系，由Neb+amtR泌铵工程菌与Ham+NPT II小麦品系构建的联合固氮体系的植物生物量均显著增加。
在这个高效固氮体系中，植物株高、地上和地下部及总生物量显著高于对照(表1)，接种泌铵工程菌株的植株总生物量较施肥处理提高7.2％，较不接种对照提高14.9％。其中地上部生物量较施肥处理提高13.5％，较不接种对照提高19.0％；根系重量较施肥处理提高1.2％，较不接种对照提高3.6％。
人工设计高效固氮体系的平均株高显著高于对照体系(表1)，接种泌铵工程菌后小麦平均株高较施肥处理增加1.9％，较不接种对照增加4.7％。
实验结论：
高效泌铵固氮微生物底盘与氮高效利用植物底盘构建的人工联合固氮系统可以显著提高植株生长量，对小麦的促生效果最佳。
(2)15N稳定性同位素稀释法测定生物固氮量
实验结果：
利用15N稀释法对人工设计根际联合固氮体系中的固氮菌固氮能力进行评价(表2)。对Neb+amtR泌铵工程菌底盘与Ham+NPT II小麦底盘构建的人工联合固氮体系的数据分析表明，与对照体系相比，泌铵工程菌显著提高了小麦植株的全氮含量、从空气中固定的氮素占植物总氮量的百分比(生物固氮效率)和生物固氮量。
在这个高效固氮体系中，接种泌铵工程菌株后植株全氮含量相对于对照体系增加42.6％，生物固氮贡献率为14.3％，经换算后生物固氮量为0.02g/株，估算每公顷的生物固氮量为80kg，节肥率约为66.6％(表2)。
而在对照体系中，生物固氮量贡献率为8.5％，经换算后生物固氮量约为0.01g/株，假定小麦每公顷产400万株，估算每公顷的生物固氮量为40kg，按照小麦的正常施肥量是120kg N/公顷估算节肥率约为33.3％(表2)。
实验结论：
高效泌铵固氮微生物底盘与氮高效利用植物底盘构建的人工联合固氮体系可以使植物获得具有更高的生物固氮效率和固氮量，预测节肥效果最佳，据估算每公顷可节省化肥约66.6％，而对照体系为33.3％。
实施例3—5中，实验结果的测定指标包括植物生长量和生物固氮量。其中：
1、植物生长量包括株高、地上部和地下部重量的相对增长率结果见表1。
表1人工根际联合固氮体系作物植株性状及生物量相对于对照体系的增长率
Figure PCTCN2020100411-appb-000001
注：施肥对照系统中氮肥的施加量为50毫克氮每千克土壤。
2、生物固氮量测定，用15N稳定性同位素稀释法进行
15N稳定性同位素稀释法，是一种测定生物固氮量的通用方法。本发明中，玉米、水稻、小麦的生长量评价和15N测定生物固氮量的方法基本一致。具体操作和计算方法为：
1、实验开始前两周将15N稳定性同位素标记的硫酸铵(浓度为50mg N每千克土壤)加入土壤中并充分混合待用。
2、经菌液浸泡后的玉米(或水稻，或小麦)种子放入盆装土壤中。栽培60天后取茎叶烘干至恒重，研磨过100目筛后，经稳定性同位素质谱检测。
实验设置4个重复。
3、计算
1)生物固氮对植物氮素营养的贡献率，以从空气中固定的氮素占植物总氮量的百分比(％Ndfa)计，计算方法如下：
％Ndfa＝(1-atom％15NF excess/％15NNF excess)×100％
式中：
atom％15NF excess—接种固氮菌玉米(或水稻、或小麦)植株15N原子百分超；
atom％15NNF excess—接种niH基因突变株玉米(或水稻、或小麦)植株15N原子百分超；
计算结果以重复性条件下获得的3次独立测定结果的算术平均值表示。
2)植物体内总的固氮量(N fixed)以克计，按以下公式计算：
Nfixed＝Nt×％Ndfa×Biomass
式中：
Nt—植物总氮含量(％)，单位％；
Biomass—植株地上和地下部的生物量总和，单位克每株植物；
计算结果以重复性条件下获得的3次独立测定结果的算术平均值表示。
结果见表2。
表2人工设计根际联合固氮体系和对照体系的作物植株氮含量及生物固氮量计算
Figure PCTCN2020100411-appb-000002

Claims (10)

  1. 一种人工根际联合固氮体系，由固氮微生物底盘构建的固氮泌铵基因模块和非豆科植物底盘的氮高效利用模块组成；
    所述固氮泌铵基因模块，是含有人工设计的固氮激活蛋白Neb功能模块和amtR铵转运模块的重组工程菌；所述固氮激活蛋白Neb，由SEQ ID NO.1所示的DNA序列编码；所述amtR铵转运模块，是SEQ ID NO.2所示DNA序列的基因；
    所述植物底盘的氮高效利用模块，是含有人工合成的铵高亲蛋白Ham的重组植物；所述铵高亲蛋白Ham由SEQ ID NO.3所示的DNA序列编码。
  2. 权利要求1所述的人工根际联合固氮体系，所述重组植物选自以下之一：
    含有基因Ham和基因bar的重组玉米；或
    含有基因Ham和基因hyg的重组水稻；或
    含有基因Ham和基因NPT II的重组小麦。
  3. 权利要求1所述的人工根际联合固氮体系的构建方法，包括：
    1)将所述固氮泌铵模块导入固氮微生物中，获得含所述固氮泌铵模块的重组微生物；
    2)将所述氮高效利用模块分别导入不同目标植物中，分别获得含所述氮高效利用模块的不同重组植物。
  4. 权利要求1所述的人工根际联合固氮体系的应用方法，是将所述重组工程菌接种于所述重组植物中。
  5. 权利要求4所述的应用方法，所述接种是在作物根际使用种子包衣接种技术进行。
  6. SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示序列的基因。
  7. 含SEQ ID NO.1和/或SEQ ID NO.2所示序列基因的质粒、载体或工程菌。
  8. 含SEQ ID NO.3所示序列基因的质粒、载体或工程菌。
  9. SEQ ID NO.1和SEQ ID NO.2所示序列的基因在构建固氮泌铵重组工程菌中的应用。
  10. SEQ ID NO.3所示序列的基因在构建氮高效利用的重组植物中的应用。
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Publication number Priority date Publication date Assignee Title
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101298600A (zh) * 2008-06-23 2008-11-05 中国科学院南海海洋研究所 红树植物根际促生固氮菌(dzy-hs14)及其应用
CN107119000A (zh) * 2017-04-19 2017-09-01 山东大学 荧光假单胞菌突变菌株的筛选方法及其在生物防治中的应用
CN108602729A (zh) * 2015-07-13 2018-09-28 皮沃特生物公司 改良植物性状的方法及组合物
WO2019213939A1 (zh) * 2018-05-11 2019-11-14 北京大学 基于多聚蛋白对复杂生物系统的重构方法，及其在高活性超简化固氮体系构建中的应用
CN110573623A (zh) * 2017-02-09 2019-12-13 福建农林大学 用于提高植物产量的磷酸盐转运蛋白的表达

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
UY37566A (es) * 2017-01-12 2018-07-31 Pivot Bio Inc Métodos y composiciones para mejorar características en plantas

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101298600A (zh) * 2008-06-23 2008-11-05 中国科学院南海海洋研究所 红树植物根际促生固氮菌(dzy-hs14)及其应用
CN108602729A (zh) * 2015-07-13 2018-09-28 皮沃特生物公司 改良植物性状的方法及组合物
CN110573623A (zh) * 2017-02-09 2019-12-13 福建农林大学 用于提高植物产量的磷酸盐转运蛋白的表达
CN107119000A (zh) * 2017-04-19 2017-09-01 山东大学 荧光假单胞菌突变菌株的筛选方法及其在生物防治中的应用
WO2019213939A1 (zh) * 2018-05-11 2019-11-14 北京大学 基于多聚蛋白对复杂生物系统的重构方法，及其在高活性超简化固氮体系构建中的应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PERRONE SHARON; GROSSMAN JULIE; LIEBMAN ALEX; SOOKSA-NGUAN THANWALEE; GUTKNECHT JESSICA: "Nitrogen fixation and productivity of winter annual legume cover crops in Upper Midwest organic cropping systems", NUTRIENT CYCLING IN AGROECOSYSTEMS, vol. 117, no. 1, 6 March 2020 (2020-03-06), pages 61 - 76, XP037090441, ISSN: 1385-1314, DOI: 10.1007/s10705-020-10055-z *
SUN SHUAI-XIN, CHENG JIE-JIE;CHEN YUN-PENG: "Generation of Tn5 Insertion Mutations in Nitrogen-Fixing Bacterium Kosakonia Radicincitans GXGL-4A Associated with Maize", MICROBIOLOGY CHINA, vol. 45, no. 8, 20 December 2017 (2017-12-20), CN, pages 1711 - 1718, XP055869968, ISSN: 0253-2654, DOI: 10.13344/j.microbiol.china.170916 *

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