WO2022227102A1 - γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法 - Google Patents

γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法 Download PDF

Info

Publication number
WO2022227102A1
WO2022227102A1 PCT/CN2021/091993 CN2021091993W WO2022227102A1 WO 2022227102 A1 WO2022227102 A1 WO 2022227102A1 CN 2021091993 W CN2021091993 W CN 2021091993W WO 2022227102 A1 WO2022227102 A1 WO 2022227102A1
Authority
WO
WIPO (PCT)
Prior art keywords
maize
transgenic
plant
callus
yield
Prior art date
Application number
PCT/CN2021/091993
Other languages
English (en)
French (fr)
Inventor
夏涛
马海珍
李�灿
Original Assignee
齐鲁工业大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 齐鲁工业大学 filed Critical 齐鲁工业大学
Publication of WO2022227102A1 publication Critical patent/WO2022227102A1/zh

Links

Images

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001—Culture apparatus for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • 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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • 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)
    • 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
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C12Y104/01013—Glutamate synthase (NADPH) (1.4.1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C12Y104/01014—Glutamate synthase (NADH) (1.4.1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/07—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with an iron-sulfur protein as acceptor (1.4.7)
    • C12Y104/07001—Glutamate synthase (ferredoxin) (1.4.7.1)

Definitions

  • the invention relates to a method for improving plant stress resistance and yield, in particular to a method for heterologous synthesis of ⁇ -polyglutamic acid in plants to improve plant stress resistance and yield, belonging to genetic engineering, genetic breeding, synthetic biology field of study technology.
  • Abiotic stresses such as drought and soil salinization have become the main limiting factors for crop production in many countries and regions in the world. About half of China's land area is in arid and semi-arid regions. As the global temperature rises, my country's water resources are becoming increasingly scarce, and drought has an increasing impact on crop yields. In addition, soil salinization is also a major environmental factor affecting crop yield. At present, about 20% of the cultivated land and nearly 50% of the irrigated land in the world are seriously damaged by salinization, and the salinized land in China accounts for about 10% of the global saline soil area.
  • Plant transgenic technology is widely used, mainly in insect resistance, herbicide resistance, stress resistance and yield enhancement.
  • GM insect-resistant corn was approved for commercial planting in the United States in 1995, more and more GM crops have been widely promoted in the United States, Brazil and other countries.
  • the enzymes GSMT2, DMT2 and betA in the osmotic regulation substance betaine synthesis pathway from bacteria were transferred into maize to improve the drought resistance of maize; the introduction of the HAV1 gene of the LEA protein family from barley into maize significantly improved the drought resistance of maize; As a protein kinase in the signaling pathway in response to stress, ZmCIPK2 can significantly improve the drought resistance of maize after overexpression in maize; heterologous expression of Arabidopsis LOS5 gene in maize can significantly improve the ABA content and drought resistance in maize .
  • salt stress signaling pathways can be mainly divided into osmotic and ion homeostasis signaling pathways, cell damage and repair, and growth regulation processes. So far, many key stress tolerance genes involved in the synthesis of osmoprotective substances, ion balance, reactive oxygen species scavenging and transcription factors have been cloned, and these genes have improved the stress tolerance of transgenic plants to varying degrees.
  • transfection of the CMO (choline monooxidase) gene for betaine synthesis into tobacco can significantly improve the salt tolerance of tobacco (Wu et al., 2010); overexpression of Arabidopsis AtSOS1 significantly improved the salt tolerance of transgenic plants (Qiu et al., 2002; Shi et al., 2003); overexpression of the Arabidopsis tonoplast Na + /H + antiporter (AtNHX1) gene can significantly improve the salt tolerance of transgenic Arabidopsis and tomato (Zhang and Blumwald, 2001; Zhang et al., 2001); Overexpression of Arabidopsis ANAC019, ANAC055 and ANAC072/RD26 can significantly improve the salt tolerance of transgenic plants; Overexpression of Arabidopsis H + -PPase gene in cotton AtAVP1 increases the salinity and drought resistance of transgenic cotton (Pasapula et al., 2011); transgenic maize can significantly improve the drought and salinity resistance of maize
  • transgenic stress-resistant maize is a single functional gene. Although some stress resistance of maize has been improved, there is little or no report of simultaneously improving maize yield under normal growth environment. Therefore, screening for genes that can both enhance crop stress resistance and maintain high yields under normal and stress conditions has become a focus.
  • ⁇ -polyglutamic acid a microbial fermentation product
  • ⁇ -PGA ⁇ -polyglutamic acid
  • ⁇ -PGA ⁇ -polyglutamic acid
  • ⁇ -PGA ⁇ -polyglutamic acid
  • Its molecule is straight-chain and contains a large number of amide bonds and free carboxyl groups. It is an anionic polymer.
  • the finished product is white and odorless. powdered solid.
  • ⁇ -PGA has the characteristics of good ductility, flexibility, biocompatibility, adhesion, stability, moisture retention, water absorption, oxygen barrier, film formation and biodegradability, it is a very Potential biodegradable polymer materials are widely used in medicine, food, cosmetics, feed, agriculture, environmental protection and other fields. It is a recognized green chemical product with great development potential.
  • ⁇ -PGA can be used as a synergist for pesticides and fertilizers to promote crop growth under low nutrient conditions, which is beneficial to improve seed vigor and germination rate, and promote the growth and development of germ and radicle; ⁇ - PGA can also improve the physicochemical and adsorption characteristics of soil, thereby promoting the absorption of nutrients by plants; because ⁇ -PGA has strong adsorption characteristics, it can be used as an adsorbent or chelating agent, and has obvious chelating effect on toxic heavy metal ions in soil.
  • ⁇ -PGA can also increase the biomass of plant roots, thereby enhancing the absorption of nitrogen, phosphorus and potassium by plants, and promoting plant growth.
  • ⁇ -PGA as a water-retaining agent can significantly improve the drought resistance of crops. Because ⁇ -PGA contains a large number of hydrophilic groups in its molecule, it has strong water absorption and water retention, which determines its important value in the field of agricultural water saving.
  • the applicant cloned the key genes in the process of synthesizing ⁇ -polyglutamic acid from microorganisms, using synthetic biology and genetic engineering technology, taking corn as an example, and transferring it into plants to evaluate heterologous ⁇ -polyglutamic acid.
  • the obtained ⁇ -polyglutamic acid-producing transgenic maize not only significantly improves the drought resistance of maize, but also significantly improves the salt resistance of maize, and under drought and normal growth conditions It can also significantly increase the biomass of corn.
  • the invention also measures the quality of the transgenic corn grains, and finds that synthesizing ⁇ -PGA in the corn can also increase the starch content and the amylose content in the corn grains. After searching, there is no report on the heterologous synthesis of ⁇ -polyglutamic acid in crops and its effects on drought resistance, salt tolerance, yield and quality of crops.
  • the problem to be solved by the present invention is to provide a method for heterologous synthesis of ⁇ -polyglutamic acid in plants to improve plant stress resistance and yield.
  • the method for heterologous synthesis of ⁇ -polyglutamic acid of the present invention to improve plant stress resistance and yield in plants the steps are:
  • Gene for cloning and synthesizing ⁇ -polyglutamic acid clone and synthesize 3 key enzyme genes PgsA, PgsB and PgsC of ⁇ -PGA from the strain producing ⁇ -polyglutamic acid; wherein, the GenBank ID of gene PgsA: AIA08848.1, whose amino acid sequence is shown in SEQ ID NO.1; Genebank ID of gene PgsB: AIA08846.1, whose amino acid sequence is shown in SEQ ID NO.2; Genebank ID of gene PgsC: AIA08847.1, whose amino acid The sequence is shown in SEQ ID NO.3;
  • Codon optimization According to the amino acid sequences of PgsA, PgsB, and PgsC genes, analyze the codon preference in maize or other plants to be transgenic and combine CpG dinucleotides content, GC content, mRNA secondary structure, Cryptic splicing sites, Analysis of Premature PolyA sites, Internal chi sites and ribosomal binding sites, Negative CpG islands, RNA instability motif(ARE), Repeat sequences(direct repeat, reverse repeat, and Dyad repeat), Restriction sites that may interfere with cloning, etc. Sequence, and artificially de novo synthesize optimized PgsA, PgsB, PgsC genes according to the sequence;
  • the ⁇ -polyglutamic acid-producing strain in step (1) is Bacillus licheniformis or Bacillus amyloliquefaciens;
  • the nucleotide sequence of the PgsA gene after the codon optimization in step (2) is shown in SEQ ID NO.4; the nucleotide sequence of the PgsB gene after the codon optimization is shown in SEQ ID NO.5; the The nucleotide sequence of the PgsC gene after codon optimization is shown in SEQ ID NO.6;
  • nucleotide sequence of the plant expression vector PGA001 in step (3) is shown in SEQ ID NO.7; each functional element of the expression vector is described as:
  • transgenic maize The transgenic plant that the described plant stress resistance and yield of step (4) improve is transgenic maize, and the method that obtains this transgenic maize is:
  • Select Agrobacterium EH105 as the transformed strain add 3 ⁇ l of plasmid containing PGA001 expression vector to 50 ⁇ l of the Agrobacterium competent cells, ice bath for 30 minutes, quick-freeze in liquid nitrogen for 5 minutes, and water bath at 37°C for 5 minutes; then add 800-1000 ⁇ l YEP liquid Culture medium, 25-28°C, 180-250rpm shaking culture for 3 hours; take out the bacterial liquid and spread it on solid YEP medium containing rifampicin and kanamycin, and place it in the dark at 25-28°C and invert for 3-4 days , take the colonies for colony PCR verification, and sequence them, and save the correctly sequenced Agrobacterium for transformation;
  • the transformed callus was transferred to the screening medium supplemented with glufosinate-ammonium, screened and cultured for two weeks, then replaced with a new screening medium, cultivated in the dark at 25-28°C for 15-20 days, and the callus was cut.
  • the powder was then transferred to a new screening medium to continue screening, a total of 2 rounds of screening, and cultured for 30-40 days;
  • the seedlings are hardened for 2-3 days, the root medium is washed and then transplanted into sterilized nutrient soil, and the seedlings are transplanted to the field after 7 days of indoor hardening; during the period, the young leaves are taken for PAT/bar Protein rapid detection test strips are used to detect the transgenic positive corn, and the seeds are harvested by selfing; after the T1 generation transgenic maize plants are obtained, after two generations of strict selfing, the transgenic maize homozygous line is finally obtained.
  • the culture medium involved in the above-mentioned transgenic corn method is as follows:
  • the present invention also provides a plant expression vector capable of heterologously synthesizing ⁇ -polyglutamic acid in plants and improving plant stress resistance and yield, characterized in that: the plant expression vector is named as plant expression vector PGA001, Its nucleotide sequence is shown in SEQ ID NO.7.
  • the invention also discloses a method for improving plant stress resistance and yield by utilizing ⁇ -polyglutamic acid to synthesize heterologously in plants, and the obtained plant transgenic line.
  • the plant transgenic line is preferably a transgenic maize homozygous line
  • the stress resistance refers to drought resistance and salt resistance
  • the detection method includes PAT/bar protein rapid detection test strip (Artron, see the specification for detailed steps) detection, PCR detection, RT-PCR detection, and detection of product ⁇ -PGA content. Among them, the detection of ⁇ -PGA content was carried out with reference to the method of NY/T 3039-2016.
  • Transgenic-positive maize lines were selected to characterize their phenotypes throughout their developmental stages, and yield assays, as well as their seed quality (starch and amylose content).
  • Drought resistance test of transgenic maize Transgenic positive maize and negative control maize were respectively subjected to drought stress treatment, and their phenotypes were studied and analyzed. Drought treatment was divided into PEG simulated drought treatment and water cut-off drought stress treatment.
  • the surface of the corn seeds was first sterilized and then germinated, and then the sprouted seedlings were inserted into the nutrient solution (Hogland corn nutrient solution) for cultivation. Or 18% PEG nutrient solution for treatment, observation of phenotypic changes, and determination of biomass.
  • nutrient solution Hogland corn nutrient solution
  • Drought test The seeds of the T3 generation of transgenic maize and the wild type KN5585 seeds of uniform size were selected and sown in small plastic flowerpots of the same size, and the plate was filled with the same amount of fertile soil with uniform texture. Half of each plate was sown with transgenic material and half of wild-type material, 2 grains each, 3 plates per line. Water normally until the seedlings grow to the 3-leaf stage, and then carry out drought stress treatment, that is, stop watering after pouring enough water at one time, and then resume watering until the wild-type plants die, and observe the growth of the transgenic lines under drought conditions. condition and survival and recovery rates after re-watering.
  • Field drought treatment chooses the method of cutting off water before flowering.
  • the specific implementation method is: cut off water for 15 days, rewater once, and cut off water for 15 days until harvest, and measure against adverse phenotype and yield .
  • the salt resistance study of transgenic maize includes germination stage and seedling stage: different concentrations of NaCl solution were selected for salt stress treatment for maize in germination stage. Select the seeds of the transgenic positive material and the negative control material, germinate on filter paper containing 150 mM NaCl and 200 mM NaCl solution after surface disinfection, and observe the germination situation; the salt resistance test at the seedling stage adopts the method of solution stress treatment: the surface of the corn seeds is sterilized After germination, it was transferred to a nutrient solution (Hogland corn nutrient solution) for cultivation, and the corn cultured to the three-leaf stage was transferred to a nutrient solution containing 200 mM NaCl for cultivation, and the phenotypic changes were counted.
  • a nutrient solution Hogland corn nutrient solution
  • Yield analysis was carried out under field conditions, divided into normal irrigation conditions and drought and water cutoff conditions.
  • the normal irrigation condition is to keep the transgenic maize and the control inbred line growing under normal natural growth conditions; the drought test mainly adopts the method of cutting off water before flowering.
  • yield and yield-related traits ear shape, ear length, ear weight, number of grains per ear, grain weight per ear, 100-grain weight, etc.
  • the invention discloses a method for heterologous synthesis of ⁇ -polyglutamic acid in plants to improve plant stress resistance and yield, and at the same time discloses the use of ⁇ -polyglutamic acid in plants for heterologous synthesis to improve plant stress resistance and yield.
  • the plant transgenic line obtained by the method of yield is preferably a transgenic maize homozygous line.
  • the obtained transgenic maize producing ⁇ -polyglutamic acid not only significantly improved the drought resistance of maize, but also significantly improved the salt resistance of maize, and also significantly increased the biomass of maize under normal growth conditions. .
  • the beneficial effects of the present invention are: (1) The synthesis of ⁇ -polyglutamic acid in plants is creatively realized, and it is proved that ⁇ -polyglutamic acid can be produced in the obtained transgenic corn. (2) Evaluating the expression of heterologous ⁇ -polyglutamic acid synthesis gene in maize and the effect on maize drought resistance, salt tolerance, yield and improvement effect on other traits.
  • the transgenic plants obtained by the method of the present invention show that It shows obvious drought resistance, salt tolerance and the effect of improving yield, which proves that the method of the present invention is a green, efficient and sustainable solution to stabilize or reduce yield of crops under drought, water shortage and saline-alkali stress. It can be widely used in the drought-resistant and salt-tolerant molecular breeding of plants and crops and the cultivation of new varieties, and has broad application prospects.
  • Figure 1 shows the schematic diagram of the plant expression vector map and the detection of transgenic maize.
  • A Schematic diagram of plant expression vector PGA001; B: rapid detection of PAT/bar protein in transgenic maize, CK: negative control inbred line KN5585; 1-9: transgenic positive maize lines; C: PCR of transgenic maize bar gene Test results; M: DNA marker; CK: negative control inbred line KN5585; 1-9, 12, 13, 15: transgenic positive maize lines; D: RT-PCR detection results of PgsA, PgsB, and PgsC genes in transgenic maize ; T1, T2, T5: transgenic positive maize lines.
  • Figure 2 shows the phenotype of transgenic positive maize and negative control maize inbred lines under normal growth conditions.
  • A the inbred line of transgenic positive maize and negative control maize after 30h germination under normal conditions
  • CK negative control inbred line KN5585, T: transgenic positive maize line
  • B transgenic positive maize and negative control maize line 48h after germination of control maize inbred line under normal conditions
  • CK negative control inbred line KN5585, T: transgenic positive maize line
  • C root development of transgenic positive maize and negative control maize
  • CK Negative control inbred line KN5585, T1, T2, T5: transgenic positive maize line
  • D growth condition of transgenic positive maize and negative control maize in potted condition at seedling stage
  • CK negative control inbred line KN5585, T: transgenic Positive maize lines
  • E Growth status of transgenic positive maize and negative control maize in pots
  • F Fruit phenotype of transgenic positive maize and negative control maize
  • CK Negative control inbred line KN5585
  • Figure 3 shows the detection of drought resistance at the germination stage of transgenic maize and wild-type control maize inbred lines.
  • Figure 4 shows transgenic positive plants and negative control maize PEG simulated drought experiments.
  • A transgenic positive (endogenous synthesis of ⁇ -PGA) corn, ⁇ -PGA exogenously treated and untreated negative control corn PEG simulated drought comparison test
  • B transgenic positive (endogenous synthesis of ⁇ -PGA) corn, Comparison of fresh weight of aerial parts of ⁇ -PGA-treated and untreated negative control maize under normal conditions and PEG-treated conditions
  • C Transgenic positive (endogenously synthesized ⁇ -PGA) maize, exogenously treated with ⁇ -PGA Comparison of fresh weight of belowground part of normal and untreated negative control maize under normal and PEG-treated conditions.
  • Fig. 5 shows the potted drought and water cut-off test of transgenic maize.
  • A the test results of the bar gene test strips of the transgenic positive plants and the negative control inbred lines
  • B the results of the drought and water loss test of the transgenic positive corn pot plants.
  • Figure 6 shows the drought and water cutoff test and yield analysis of transgenic positive maize under field conditions.
  • A phenotype of transgenic positive maize and negative control maize after water cut off for 8 days at seedling stage
  • B phenotype of transgenic positive maize and negative control maize after drought and water cut off for 8 days before flowering
  • C transgenic positive maize and negative control maize under drought conditions Yield analysis of negative control maize
  • CK negative control inbred line KN5585; T1, T2, T5, T6, T8, T9, T12, T13: transgenic positive maize lines.
  • Figure 7 shows the detection of salt tolerance at the germination stage of transgenic maize and wild-type control maize inbred lines.
  • Figure 8 shows the salt resistance test of the transgenic positive lines and negative control plants at the seedling stage.
  • CK negative control inbred line KN5585
  • T1, T2, T5 transgenic positive inbred line KN5585.
  • Figure 9 shows a comparison test of drought and salt resistance of transgene-positive (endogenously synthesized ⁇ -PGA) maize and wild-type control maize exogenously treated with ⁇ -PGA.
  • A salt resistance comparison test of transgenic positive (endogenously synthesized ⁇ -PGA) maize and wild-type control maize exogenously treated with ⁇ -PGA
  • B transgenic positive (endogenous synthesized ⁇ -PGA) maize, ⁇ -PGA The fresh weight comparison of exogenous-treated wild-type control maize and wild-type control maize under normal conditions and under salt stress treatment conditions
  • C Transgenic positive (endogenously synthesized ⁇ -PGA) maize, exogenously treated with ⁇ -PGA The fresh weight comparison of wild-type control maize and wild-type control maize under normal conditions and under salt stress treatment conditions
  • CK negative control inbred line KN5585
  • T1, T2 transgenic positive inbred lines.
  • Figure 10 is a schematic diagram of the map of the plant expression vector pU130-bar.
  • Example 1 Acquisition of pgsA, pgsB, pgsC amino acid sequences
  • PgsA, PgsB and PgsC for synthesizing ⁇ -PGA were cloned from the ⁇ -polyglutamic acid-producing strain Bacillus licheniformis by a known method; among them, the GenBank ID of gene PgsA: AIA08848.1 , its amino acid sequence is shown in SEQ ID NO.1; the Genebank ID of gene PgsB: AIA08846.1, its amino acid sequence is shown in SEQ ID NO.2; the Genebank ID of gene PgsC: AIA08847.1, its amino acid sequence is as shown in SEQ ID NO.2 ID NO.3 is shown.
  • the codon bias analysis in maize or other plants to be transgenic is combined with CpG dinucleotides content, GC content, mRNA secondary structure, Cryptic splicing sites, Premature PolyA sites, Internal chi sites and ribosomal binding sites, Negative CpG islands, RNA instability motif(ARE), Repeat sequences (direct repeat, reverse repeat, and Dyad repeat), Restriction sites that may interfere with cloning and other analyses finally determined the optimized sequence, and determined the optimized sequence according to The PgsA, PgsB, PgsC genes after the artificial de novo synthesis of the sequence optimization; wherein, the PgsA gene nucleotide sequence after the codon optimization is as shown in SEQ ID NO.4; the PgsB gene nucleotide sequence after the codon optimization The sequence is shown in SEQ ID NO.5; the nucleotide sequence of the PgsC
  • the codon-optimized PgsA, PgsB, and PgsC genes are linked into the plant expression vector pU130-bar (the vector map is shown in Figure 10) containing the bar gene selection marker by a known method, and the plant expression vector containing the target gene is obtained, named It is a plant expression vector PGA001, and the vector map is shown in Figure 1; the nucleotide sequence of the plant expression vector PGA001 is shown in SEQ ID NO.7; each functional element of the expression vector is described as:
  • the above-mentioned plant expression vector PGA001 is constructed using the multi-fragment recombinase method, and the specific construction steps are:
  • PCR products 35S+pgsA+Tnos, 35S+pgsB+Tnos, 35S+pgsC+Tnos of the codon-optimized pgsA, pgsB, and pgsC PCR products in Example 2 were directly synthesized by a bio company.
  • the recombination ligation system was as follows: the synthetic PCR products pgsA+pgsB+pgsC were 3 ⁇ l respectively; the enzyme digested vector product, 3 ⁇ l; the recombinase, 2 ⁇ l; the recombination buffer, 4 ⁇ l; H 2 O, 2 ⁇ l. Store on ice or 4°C after 30min at 37°C;
  • Plasmids with correct sequencing results were used to transform Agrobacterium.
  • the embryogenic callus After 2-3 weeks, from the induced callus, select the embryogenic callus with fast growth rate, soft texture, loose and brittle, bright color and transfer it to the subculture medium (MS+1mg/L2,4 -D+0.69g/L L-proline+0.5g/L hydrolyzed casein+30g/L sucrose+7g/L agar, pH5.8) subculture, subculture once every 2 weeks, for Agrobacterium infection .
  • the subculture medium MS+1mg/L2,4 -D+0.69g/L L-proline+0.5g/L hydrolyzed casein+30g/L sucrose+7g/L agar, pH5.8 subculture, subculture once every 2 weeks, for Agrobacterium infection .
  • EH105 Agrobacterium as the transformed strain, add 3 ⁇ l of plasmid containing PGA001 expression vector to 50 ⁇ l of EHA105 commercial Agrobacterium competent cells (Bomed), ice bath for 30 minutes, quick-freeze in liquid nitrogen for 5 minutes, and water bath at 37°C for 5 minutes; Then add 800 ⁇ l of YEP liquid medium, and shake at 28°C and 250rpm for 3 hours; take out the bacterial liquid and spread it on the solid YEP medium containing rifampicin and kanamycin, and place it at 28°C in the dark for 3-4 days. Colonies were verified by colony PCR and sequenced, and the correctly sequenced Agrobacterium was preserved for transformation;
  • the transformed callus was transferred to the screening medium supplemented with glufosinate-ammonium (PPT) (MS+1mg/L2,4-D+0.69g/L L-proline+0.5g/L hydrolyzed casein). +20g/L sucrose+250mg/L cephalosporin+10mg/L PPT+7.5g/L agar, pH5.8), start screening and culture for two weeks, then change to a new screening medium, 25-28 °C dark culture On the 15th day, the callus was broken up and transferred to a new screening medium to continue screening, for a total of 2 rounds of screening.
  • PPT glufosinate-ammonium
  • the seedlings are hardened for 2-3 days, and the root culture medium is washed and transplanted in the sterilized nutrient soil, and the indoor hardening is transplanted to the field after 7 days (cover the flowerpot with a film during the hardening period. increase humidity).
  • Transgenic detection methods include PAT/bar protein rapid detection test strip detection, PCR detection, RT-PCR detection, and detection of product ⁇ -PGA content.
  • PAT/bar protein rapid detection test strip (Artron) detection was performed according to the kit instructions; PCR, RT-PCR detection reference (Xie Guangning. Maize ubiquitin receptor ZmDA1, the effect of ZmDAR1 on grain development [D]. Shandong University, 2017 .) described method. The detection of product ⁇ -PGA content was carried out with reference to the method of NY/T 3039-2016.
  • Transgenic-positive maize lines were selected to identify their entire developmental stages (germination stage, seedling stage, jointing stage, etc.) and seed phenotypes.
  • test results are shown in Figure 2 and Table 3.
  • the phenotypes of the transgenic positive maize and the negative control maize under normal growth conditions were observed to determine the effect of heterologous synthesis of ⁇ -PGA in maize on the growth and development of maize.
  • the results are shown in Figure 2. Show.
  • the transgenic positive maize and negative control maize were treated with drought stress respectively, and their phenotypes were studied. Drought treatment was divided into PEG simulated drought treatment and water cut-off drought stress treatment.
  • the germination stage and the three-leaf stage were mainly selected for PEG simulated drought treatment.
  • Hogland nutrient solution Hibo Bio
  • the concentration of PEG6000 selected for drought stress treatment in the germination stage was 14% and 18%, respectively.
  • the seeds of the transgenic material and the control material were selected for surface disinfection, germinated on filter paper containing 0 mmol/L, 12% PEG and 14% PEG solution respectively, the germination rate was counted, and the growth potential was observed.
  • the concentration of PEG6000 selected for simulated drought treatment at seedling stage was 18%; for seedling stage drought treatment, potted plants were cut off water, and physiological and biochemical indicators such as free proline, soluble sugar, chlorophyll content and ABA content were measured.
  • the site of field drought treatment was Sanya Nanfan Base, and the method of cutting off water before flowering was selected. The specific implementation method was: cutting off water for 15 days, rewatering once, and cutting off water for 15 days until harvest, and measuring the anti-adverse phenotype and yield.
  • the inventors conducted fresh weight statistics of the aboveground and underground parts of the transgenic positive maize lines treated with 18% PEG for 5 days, the negative maize plants exogenously applied with ⁇ -PGA, and the negative control lines, and found that the transgenic positive maize lines were in The biomass under the PEG treatment condition was significantly higher than that of the negative control maize, and even slightly higher than that of the exogenously applied ⁇ -PGA maize group (Fig. 4B-4C). It shows that the heterologous synthesis of ⁇ -PGA in maize can achieve the effect of exogenous application of ⁇ -PGA to improve the drought resistance of maize. This suggests that the increased stress resistance of transgenic maize is due to the production of ⁇ -PGA.
  • the inventors also conducted a potted water cut-off treatment test on the transgenic positive corn and negative control corn at the three-leaf stage. It was found that in the early stage of drought stress, the transgenic positive maize showed a better growth state than the transgenic negative control maize. After 8 days of water cut-off treatment, the negative control maize almost wilted to death, and when watering was resumed, the transgenic positive maize could still recover rapidly, while the control maize almost died (Fig. 5). The contents of ABA, soluble sugar, proline and chlorophyll in leaves treated without water for 6 days were measured. The results showed that under drought stress conditions, heterologous synthesis of ⁇ -PGA in maize could significantly increase the levels of ABA, soluble sugar in maize leaves. , proline and chlorophyll content, thereby improving the drought resistance of maize (Table 2).
  • the salt resistance study of transgenic maize includes germination stage and seedling stage: different concentrations of NaCl solution were selected for salt stress treatment for maize in germination stage. After the seeds of transgenic positive materials and negative control materials were selected for surface disinfection, they were germinated on filter paper containing 150 mM NaCl and 200 mM NaCl solutions respectively, and the germination was observed; the salt resistance test at seedling stage was treated with solution stress: the surface of corn seeds was sterilized to make it After germination, it was transferred to a nutrient solution (Hogland corn nutrient solution) for cultivation, and the corn cultured to the three-leaf stage was transferred to a nutrient solution containing 200 mM NaCl for cultivation, and the phenotypic changes were counted.
  • a nutrient solution Hogland corn nutrient solution
  • Comparison of salt resistance and drought resistance of transgene-positive (endogenously synthesized ⁇ -PGA) maize and wild-type control maize treated with exogenous ⁇ -PGA Transgene-positive (endogenously synthesized ⁇ -PGA) maize, wild-type treated with exogenous ⁇ -PGA
  • the comparison test of drought resistance and salt resistance of the inbred lines was carried out by solution stress treatment. First, the surface of the corn seeds was sterilized and germinated, and then the sprouted seedlings were inserted into the nutrient solution (Hogland corn nutrient solution) for cultivation. When the plants grew to the three-leaf stage, the treatment was started, and the transgenic corn lines were transferred to 18% PEG.
  • Example 8 Yield analysis of transgenic maize under normal and drought conditions
  • transgenic positive maize plants and transgenic negative control maize plants planted in the field were subjected to drought treatment before flowering.
  • the specific implementation method was watering off for 15 days, then watering again for 15 days, and then rewatering. Finally, the output is counted.
  • Example 9 Content detection of starch and amylose in transgenic positive corn and negative control corn seeds
  • transgenic maize was compared, and it was found that transgenic maize could significantly increase the content of starch and amylose in maize seeds.
  • the present invention provides a method for synthesizing ⁇ -PGA in plants for the first time by using synthetic biology and genetic engineering technology, and successfully detected the synthesis of ⁇ -PGA in transgenic maize, which proves that ⁇ -PGA is synthesized.
  • PGA can be heterologously synthesized in plants; and the present invention selects the main food crop maize as the test material, and the obtained transgenic maize can be confirmed to significantly improve its drought resistance and salt resistance, and its yield can be significantly improved under drought conditions; The yield under normal irrigation conditions of stress also does not decrease, and even increases slightly; the present invention also tests the quality of transgenic corn grains, and it is found that synthesizing ⁇ -PGA in corn can also improve the starch content and linear chain in corn grains. starch content.
  • the above research results suggest that the method of the present invention can be widely used in crop stress-resistant molecular breeding, crop biomass improvement, etc., and has important theoretical significance and huge economic value.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Environmental Sciences (AREA)
  • Botany (AREA)
  • Medicinal Chemistry (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

本发明公开了一种γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法，是从产γ-PGA的菌株中克隆合成γ-PGA的3个关键酶基因PgsA、PgsB、PgsC，并根据植物中的密码子偏好性重新合成密码子优化后的PgsA、PgsB、PgsC基因序列，并连入载体pU130-bar，获得植物表达载体PGA001，通过农杆菌介导转入到植物中，获得产γ-聚谷氨酸的转基因植物新品系。实验证实本发明方法获得的新品系转基因玉米不仅可以提高抗旱性同时还可以提高抗盐性，并且在干旱胁迫条件下、正常生长条件下还可以显著提高玉米的生物量。上述结果预示本发明方法是一种绿色、高效、可持续的解决作物在干旱、缺水、盐碱胁迫下稳产或少减产的有效途径，可广泛应用于植物的抗旱、耐盐分子育种与新品种培育。

Description

γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法 技术领域
本发明涉及一种提高植物抗逆性和产量的方法，尤其涉及一种γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法，属于基因工程、遗传育种、合成生物学技术领域。
背景技术
干旱、土壤盐渍化等非生物胁迫已经成为世界上许多国家、地区农作物生产的主要限制因素。在中国，约一半的国土面积处于干旱和半干旱区。随着全球气温升高，我国水资源愈发短缺，干旱对农作物产量的影响越来越大。此外，土壤盐渍化也是影响作物产量的主要环境因素。目前世界上约20％耕地和近50％灌溉用地受到盐渍的严重危害，中国盐渍化土地约占全球盐渍土面积的10％。利用现代生物学技术培育抗旱、抗盐的优质高产作物新品种具有重要的战略意义，是提高作物产量、保障粮食安全和农业可持续发展的重要选择。而开发抗逆且高产的优质基因并通过转基因工程改造农作物是避免其在逆境条件下减产的有效途径。
植物转基因技术应用十分广泛，主要应用在抗虫、抗除草剂、抗逆和提高产量等方面。随着1995年世界第一例转基因抗虫玉米在美国获得商业化种植批准，越来越多的转基因作物在美国、巴西等国家开始大面积推广种植。
目前，国际上转基因抗旱作物的研究大部分来自于一些渗透调节物质合成相关的基因、转录因子、抗逆信号传导途径中的相关基因等，还有一些水通道蛋白基因、伴侣蛋白基因、ABA合成途径中的相关酶基因等。例如，2007年Qin等在玉米中过表达转录因子ZmDREB2A显著提高了玉米的抵抗干旱和高温的能力；2010年Zhang等将盐芥中的转录因子TsCBF1转入玉米中从而提高了玉米的抗旱性；将来自细菌的渗透调节物质甜菜碱合成途径中的酶GSMT2、DMT2、betA转入玉米中提高了玉米的抗旱性；将来自大麦的LEA蛋白家族的HAV1基因导入玉米显著提高了玉米的抗旱性；ZmCIPK2作为响应逆境的信号途径中的一个蛋白激酶，在玉米中过表达后能明显改善玉米的抗旱性；在玉米中异源表达拟南芥LOS5基因，可以显著提高玉米中的ABA含量和抗旱性。
研究报道表明，盐胁迫信号途径主要可以分为渗透和离子平衡信号途径、细胞损伤与修复以及生长调节过程。目前为止，许多参与渗透保护物质合成、离子平衡、活性氧清除以及转录因子等过程的关键耐逆基因已被克隆，并且这些基因都不同程度的提高了转基因植物的耐逆性。例如，将合成甜菜碱的CMO(胆碱单氧化酶)基因转入烟草后，可以显著提高烟草的抗盐性(Wu等，2010)；过表达拟南芥AtSOS1显著提高了转基因植物的耐盐性(Qiu et al.,2002；Shi et al.,2003)；过表达拟南芥液泡膜Na +/H +反向转运蛋白(AtNHX1)基因可以显著提高转基因拟南芥、番茄的抗盐性(Zhang and Blumwald,2001；Zhang et al.,2001)；过表达拟南芥ANAC019、ANAC055和ANAC072/RD26能显著提高转基因植物的抗盐性；在棉花中过量表达拟南芥H +-PPase基因AtAVP1增加了转基因棉花的耐盐性和抗旱能力 (Pasapula et al.,2011)；转TsVP基因玉米可以显著提高玉米的抗旱和抗盐性(wei等，2008)。上述研究结果表明，虽然已经有许多植物耐盐基因被克隆和表达，但是仍主要集中在拟南芥、烟草和番茄等模式植物中，在作物中并未广泛应用。
已报道的一些转基因抗逆玉米大都是转单个功能基因，虽然提高了玉米的某些抗逆性，但是在正常生长环境下极少或并未有同时提高玉米产量的报道。因此，筛选既能增强作物抗逆性又能在正常条件下和胁迫条件下都能保持高产的基因成为焦点。
γ-聚谷氨酸(γ-poly glutamic acid，γ-PGA)是一种微生物发酵产物，在纳豆中首次被发现。γ-PGA由L-谷氨酸和D-谷氨酸通过脱水缩合而成的，其分子呈直链状，含有大量酰胺键和可游离的羧基，是一种阴离子聚合物，成品为白色无味的粉末状固体。由于γ-PGA具有良好的延展性、柔韧性、生物相容性、黏着性、稳定性、保湿性、吸水性、阻氧性、成膜性及生物可降解性等特点，是一种极具开发潜力的生物可降解高分子材料，广泛应用于医药、食品、化妆品、饲料、农业、环保等多个领域，是一种公认的极具发展潜力的绿色化学产品。
在农业领域，大量的研究发现，γ-PGA可以作为农药和肥料的增效剂，在低养分条件下促进作物生长，有利于提高种子活力和发芽率，促进胚芽和胚根生长发育；γ-PGA还可以改善土壤的理化和吸附特性，从而促进植物对养分的吸收；由于γ-PGA具有较强的吸附特性，可以作为吸附剂或螯合剂，对土壤中的有毒重金属离子具有明显的螯合效果，避免作物从土壤中吸收过多的有毒重金属；γ-PGA还可以提高植物根系的生物量，从而增强植物对氮、磷、钾的吸收，促进植物的生长。此外，γ-PGA作为一种保水剂可以显著提高作物的抗旱性。γ-PGA因其分子中含有大量的亲水基团，具有很强的吸水性与保水性，决定了其在农业节水领域的重要价值。
作为一种新型的绿色高分子生物材料，虽然γ-PGA具有广泛的应用前景，但是由于利用微生物发酵生产γ-PGA相对成本较高，也限制了其在农业生产中的大规模应用。同时，作为一种高分子生物材料，其长期在土壤中使用的生态、环境效应也缺乏系统、全面的研究与评估。
基于现有技术，申请人克隆了来自于微生物的、合成γ-聚谷氨酸过程中的关键基因，利用合成生物学和基因工程技术，以玉米为实施案例转入植物中，评估异源γ-聚谷氨酸合成基因在玉米中的表达以及对玉米抗旱性、耐盐性、产量的影响及对其他性状的改良效果，并建立了一种γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法，获得的产γ-聚谷氨酸的转基因玉米不仅显著的提高了玉米的抗旱性，同时也显著提高了玉米的抗盐性，并且在干旱和正常生长条件下还可以显著提高玉米的生物量。本发明还对转基因玉米籽粒的品质进行了测定，发现在玉米中合成γ-PGA还可以提高玉米籽粒中的淀粉含量和直链淀粉含量。经检索，目前尚未有在作物体内异源合成γ-聚谷氨酸并研究其对作物抗旱性、耐盐性、产量、品质影响的报道。
发明内容
针对现有技术的不足，本发明要解决的问题是提供一种γ-聚谷氨酸在植物中异源合成提 高植物抗逆性和产量的方法。
本发明所述的γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法，步骤是：
(1)克隆合成γ-聚谷氨酸的基因：从产γ-聚谷氨酸的菌株中克隆合成γ-PGA的3个关键酶基因PgsA、PgsB、PgsC；其中，基因PgsA的GenBank ID:AIA08848.1，其氨基酸序列如SEQ ID NO.1所示；基因PgsB的Genebank ID:AIA08846.1，其氨基酸序列如SEQ ID NO.2所示；基因PgsC的Genebank ID:AIA08847.1，其氨基酸序列如SEQ ID NO.3所示；
(2)密码子优化：按照PgsA、PgsB、PgsC基因的氨基酸序列，通过玉米或待转基因的其他植物中的密码子偏好性分析并结合CpG dinucleotides content、GC content、mRNA secondary structure、Cryptic splicing sites、Premature PolyA sites、Internal chi sites and ribosomal binding sites、Negative CpG islands、RNA instability motif(ARE)、Repeat sequences(direct repeat,reverse repeat,and Dyad repeat)、Restriction sites that may interfere with cloning等分析最终确定优化的序列，并根据序列人工从头合成优化后的PgsA、PgsB、PgsC基因；
(3)构建植物表达载体：将密码子优化后的PgsA、PgsB、PgsC基因连入含有bar基因筛选标记的植物表达载体pU130-bar，获得含有目的基因的植物表达载体，命名为植物表达载体PGA001；
(4)植物抗逆性和产量提高的转基因植物的获得；
其特征在于：
步骤(1)所述产γ-聚谷氨酸的菌株是地衣芽孢杆菌(Bacillus licheniformis)或解淀粉芽孢杆菌(Bacillus amyloliquefaciens)；
步骤(2)所述密码子优化后的PgsA基因核苷酸序列如SEQ ID NO.4所示；所述密码子优化后的PgsB基因核苷酸序列如SEQ ID NO.5所示；所述密码子优化后的PgsC基因核苷酸序列如SEQ ID NO.6所示；
步骤(3)所述植物表达载体PGA001的核苷酸序列如SEQ ID NO.7所示；该表达载体各功能元件描述为：
Figure PCTCN2021091993-appb-000001
Figure PCTCN2021091993-appb-000002
步骤(4)所述植物抗逆性和产量提高的转基因植物是转基因玉米，获得该转基因玉米的方法是：
1)含植物表达载体PGA001的农杆菌菌株制备
选择EH105农杆菌为转化菌株，在50μl该农杆菌感受态细胞中加入含PGA001表达载体的质粒3μl，冰浴30分钟，液氮速冻5分钟，37℃水浴5分钟；再加入800-1000μl YEP液体培养基，25-28℃、180-250rpm振荡培养3小时；取出菌液涂布于含利福平及卡那霉素的固体YEP培养基上，置25-28℃黑暗倒置培养3-4天，取菌落进行菌落PCR验证，并进行测序，对测序正确的农杆菌保存，用于转化；
2)愈伤组织的准备
选取玉米自交系KN5585作为受体自交系，取其授粉后10-12d的果穗，去苞叶，在无菌工作台中，先用70％酒精处理5-6min，再用无菌水冲洗4-5遍，剥取1.5-2mm的幼胚，盾片向上放置在幼胚诱导培养基上，28℃、黑暗培养诱导愈伤组织；2-3周后从诱导出的愈伤组 织中，选取生长速度较快、质地松软、松散易碎、颜色鲜艳的胚性愈伤组织转移到继代培养基上继代培养，每2周继代一次，用于农杆菌侵染；
3)农杆菌侵染
挑取含有PGA001表达载体的农杆菌单菌落，加入5-6mL的YEP(Kan)培养基中，培养过夜；室温下，5000-6000rpm，离心5-10min，收集菌体；将菌体悬浮于含有乙酰丁香酮(AS)的侵染液中，并使OD 600＝0.6-0.8，混匀，待用；将制得的侵染液在25-28℃，摇床180rpm活化1-2小时，用于侵染；将玉米的胚性愈伤组织收集在一起放入一个无菌的三角瓶中，然后倒入侵染液，将大的愈伤组织块打散摇匀，使愈伤组织充分接触农杆菌，侵染15-20min；将愈伤组织取出，在滤纸上吸干多余的菌液，然后接种到共培养培养基上，19-22℃黑暗培养3天；
4)恢复培养
使用含有抗生素的无菌水冲洗愈伤组织表面3-5次，待加入的水不再浑浊时倒掉液体，将愈伤组织转入铺有滤纸的平皿中，在超净台中吹干愈伤表面水分，转入恢复培养基，28℃暗培养7-10天；
5)筛选培养
恢复培养后将转化愈伤组织转入添加有草铵膦的筛选培养基上，筛选培养两周，然后更换新的筛选培养基，25-28℃暗培养15-20天，将愈伤组织打散再转入新的筛选培养基继续筛选，总共筛选2轮，培养30-40天；
6)分化生根
将抗性愈伤组织转入分化培养基，25-28℃暗培养7-10天，然后转入光照培养箱25-28℃培养，直至再生芽长至3-5cm时转入生根培养基；
7)炼苗移栽后自交收种
待长出大量健壮根后，炼苗2-3天，洗净根部培养基后移栽在灭菌的营养土中，室内炼苗7天后移栽到大田；期间取其幼叶进行PAT/bar蛋白快速检测试纸条检测，转基因阳性玉米自交收获种子；获得T1代转基因玉米植株后，经过两代严格自交，最终获得转基因玉米纯合株系。
上述转基因玉米方法涉及的培养基如下：
Figure PCTCN2021091993-appb-000003
Figure PCTCN2021091993-appb-000004
本发明还提供了一种能使γ-聚谷氨酸在植物中异源合成并提高植物抗逆性和产量的植物表达载体，其特征在于：所述植物表达载体命名为植物表达载体PGA001，其核苷酸序列如SEQ ID NO.7所示。
本发明还公开了利用γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法及获得的植物转基因品系。
其中：所述植物转基因品系优选是转基因玉米纯合株系；所述抗逆性是指抗旱、抗盐性。
(5)转基因玉米的检测：
检测方法包括PAT/bar蛋白快速检测试纸条(Artron，详细步骤见说明书)检测、PCR检测、RT-PCR检测，产物γ-PGA含量的检测。其中，γ-PGA含量的检测参考NY/T 3039-2016方法进行。
将玉米植株在60℃真空烘干后，研碎，称取10g混合均匀的试样，分别测定试样中经盐酸水解后的和未经水解的游离谷氨酸的含量，两者之差即为聚谷氨酸的含量。
结果显示PgsA、PgsB、PgsC三个基因已经成功转入到玉米中(图1)，并成功在玉米中检测到了γ-PGA(表1)。
(6)转基因玉米表型鉴定：
选择转基因阳性玉米株系对其整个发育期的表型进行鉴定，并进行产量测定，并对其种子品质(淀粉含量和直链淀粉含量)进行测定。
结果显示，转基因阳性玉米株系具有更好的萌发率，更高的株高，更发达的根系，说明在玉米中合成γ-PGA可以促进玉米的生长，同时可以提高正常条件下玉米的产量，主要是提高了穗粒数、穗粒重、百粒重(表3)，还可以提高玉米籽粒的品质，主要是淀粉含量和直链淀粉含量(表4)。
(7)转基因玉米萌发期、苗期抗旱性和抗盐性的检测：
转基因玉米的抗旱性检测：对转基因阳性玉米和阴性对照玉米分别进行干旱胁迫处理，并对其表型进行研究分析，干旱处理分PEG模拟干旱处理和断水干旱胁迫处理。
PEG模拟干旱处理主要选萌发期和三叶期这两个时期，玉米培养液选择Hogland营养液(海博生物)，萌发期干旱胁迫处理选择的PEG6000的浓度分别为14％、18％。苗期模拟干旱处理选择的PEG6000的浓度为18％；苗期干旱处理采用盆栽断水处理，并测定游离脯氨酸、可溶性糖、叶绿素和ABA含量等生理生化指标。
在苗期抗性检测中，首先将玉米种子表面消毒后萌发，再将萌芽小苗插入到营养液(Hogland玉米营养液)中培养，待植株长至三叶期时分别转移到含有200mmol/L NaCl或18％PEG的营养液中进行处理，观察表型变化，并进行生物量测定。
干旱试验：选取大小均匀的转基因玉米T3代种子和野生型KN5585种子播种于大小一致塑料小花盆中，盘中装有等量的质地均匀的肥沃土壤。每盘一半播种转基因材料，一半播种野生型材料，各2粒，每株系种3盘。正常浇水至小苗长至3叶期，然后进行干旱胁迫处理，即一次性浇足水分后停止浇水，一直到野生型植株死亡后再恢复浇水，观察转基因株系在干旱条件下的生长状况及恢复浇水后的成活率和恢复速率。田间干旱处理(实施地点为三亚南繁基地)选择开花前断水处理的方法，具体实施办法为：断水15天，复水一次，再断水15天，一直到收获，对抗逆表型和产量进行测定。
结果表明，转基因阳性玉米在PEG处理下仍可以较好的萌发，而阴性对照玉米的萌发却受到了明显的抑制(图3)。大田干旱断水试验结果表明，在大田干旱条件下，转基因阳性玉米也表现出良好的抗旱性。
转基因玉米抗盐性研究包括萌发期和苗期：对萌发期玉米选择不同浓度的NaCl溶液进行盐胁迫处理。选取转基因阳性材料及阴性对照材料的种子，表面消毒后分别在含150mM NaCl、200mM NaCl溶液的滤纸上萌发，并观察萌发情况；苗期抗盐试验采取溶液胁迫处理的方法：将玉米种子表面消毒使其萌发后转移至营养液(Hogland玉米营养液)中培养，将培养至三叶期的玉米分别移至含200mM NaCl的营养液中培养，并统计表型变化。
结果表明，无论在较低浓度还是高浓度NaCl胁迫条件下，转基因玉米种子的萌发情况都比对照阴性玉米好(图7-图8)，说明转基因玉米在萌发期具有更好的抗盐性，苗期水培抗盐试验结果也显示转基因阳性玉米具有明显的抗盐性。
(8)转基因玉米在正常条件下和干旱条件下产量分析
在田间条件下进行了产量分析，分正常灌溉条件和干旱断水条件。正常灌溉条件是保持转基因玉米和对照自交系在正常自然生长条件下生长；干旱试验主要采取开花前断水处理的方法，具体实施办法为：断水15天，复水一次，再断水15天，一直到收获，对产量和产量相关性状进行测定(果穗形态、穗长、穗重、穗粒数、穗粒重、百粒重等)。
结果显示，在干旱条件下，转基因阳性玉米的产量显著提高。与正常灌溉条件下产量相比，转基因玉米受干旱的影响较小，而阴性对照玉米在干旱条件下产量显著下降。
本发明公开了一种γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法，同时公开了利用γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法所获得的植物转基因品系，优选是转基因玉米纯合株系。实验证实：获得的产γ-聚谷氨酸的转基因玉米不仅显著的提高了玉米的抗旱性，同时也显著提高了玉米的抗盐性，并且在正常生长条件下还显著提高了玉米的生物量。本发明的有益效果是：(1)创造性的实现了在植物中合成γ-聚谷氨酸，并证明在获得的转基因玉米中可以产生γ-聚谷氨酸。(2)评估异源γ-聚谷氨酸合成基因在玉米中的表达以及对玉米抗旱性、耐盐性、产量的影响及对其他性状的改良效果显示，通过本发 明方法得到的转基因植物表现出明显的抗旱性、耐盐性和对产量的提升作用，证实本发明方法是一种绿色、高效、可持续的解决作物在干旱、缺水、盐碱胁迫下稳产或少减产的有效途径，可广泛应用于植物、作物的抗旱、耐盐分子育种与新品种培育，具有广阔的应用前景。
附图说明
图1示植物表达载体图谱示意图和转基因玉米的检测。
其中，A：植物表达载体PGA001图谱示意图；B：转基因玉米的PAT/bar蛋白快速检测，CK：阴性对照自交系KN5585；1-9：转基因阳性玉米株系；C：转基因玉米bar基因的PCR检测结果；M：DNA marker；CK：阴性对照自交系KN5585；1-9、12、13、15：转基因阳性玉米株系；D：转基因玉米中PgsA、PgsB、PgsC基因的RT-PCR检测结果；T1、T2、T5：转基因阳性玉米株系。
图2示转基因阳性玉米与阴性对照玉米自交系在正常生长条件下的表型。
其中，A：转基因阳性玉米与阴性对照玉米自交系的在正常条件下萌发30h后的情况；其中CK：阴性对照自交系KN5585，T：转基因阳性玉米株系；B：转基因阳性玉米与阴性对照玉米自交系在正常条件下萌发48h后的情况；其中CK：阴性对照自交系KN5585，T：转基因阳性玉米株系；C：转基因阳性玉米和阴性对照玉米的根系发育情况；其中CK：阴性对照自交系KN5585，T1、T2、T5：转基因阳性玉米株系；D：转基因阳性玉米和阴性对照玉米盆栽条件下苗期的长势情况；其中CK：阴性对照自交系KN5585，T：转基因阳性玉米株系；E：转基因阳性玉米和阴性对照玉米盆栽条件下苗期的长势情况；F：转基因阳性玉米与阴性对照玉米的果实表型；其中CK：阴性对照自交系KN5585，T1、T2、T5、T6：转基因阳性玉米株系。
图3示转基因玉米和野生型对照玉米自交系萌发期抗旱性检测。
图4示转基因阳性植株和阴性对照玉米PEG模拟干旱试验。
其中，A：转基因阳性(内源合成γ-PGA)玉米、γ-PGA外源处理的及未处理的阴性对照玉米PEG模拟干旱对比试验；B：转基因阳性(内源合成γ-PGA)玉米、γ-PGA外源处理的及未处理的阴性对照玉米在正常条件下和PEG处理条件下地上部分的鲜重对比；C：转基因阳性(内源合成γ-PGA)玉米、γ-PGA外源处理的及未处理的阴性对照玉米在正常条件下和PEG处理条件下地下部分的鲜重对比。
图5示转基因玉米的盆栽干旱断水试验。
其中，A：转基因阳性植株与阴性对照自交系bar基因试纸条检测结果；B：转基因阳性玉米盆栽干旱断水试验结果。
图6示转基因阳性玉米在田间条件下的干旱断水试验及产量分析。
其中，A：转基因阳性玉米及阴性对照玉米苗期断水8d后的表型；B：转基因阳性玉米及阴性对照玉米在开花前干旱断水8d后的表型；C：干旱条件下的转基因阳性玉米和阴性对照玉米的产量分析；其中CK：阴性对照自交系KN5585；T1、T2、T5、T6、T8、T9、T12、T13：转基因阳性玉米株系。
图7示转基因玉米和野生型对照玉米自交系萌发期抗盐性检测。
图8示转基因阳性株系和阴性对照植株的苗期抗盐试验。
其中，CK：阴性对照自交系KN5585；T1、T2、T5：转基因阳性自交系KN5585。
图9示转基因阳性(内源合成γ-PGA)玉米与γ-PGA外源处理的野生型对照玉米抗旱和抗盐性对比试验。
其中，A：转基因阳性(内源合成γ-PGA)玉米与γ-PGA外源处理的野生型对照玉米抗盐性对比试验；B：转基因阳性(内源合成γ-PGA)玉米、γ-PGA外源处理的野生型对照玉米以及野生型对照玉米在正常条件下和盐胁迫处理条件下地上部分的鲜重对比；C：转基因阳性(内源合成γ-PGA)玉米、γ-PGA外源处理的野生型对照玉米以及野生型对照玉米在正常条件下和盐胁迫处理条件下地下部分的鲜重对比；其中CK：阴性对照自交系KN5585；T1、T2：转基因阳性自交系。
图10为植物表达载体pU130-bar图谱示意图。
具体实施方式
下面结合具体附图和实施例对本发明内容进行详细说明。如下所述例子仅是本发明的较佳实施方式，应该说明的是，下述说明仅仅是为了解释本发明，并非对本发明作任何形式上的限制，凡是依据本发明的技术实质对实施方式所做的任何简单修改、等同变化与修饰，均属于本发明技术方案的范围内。
下述实施例中，所使用的材料、试剂等，如无特殊说明，均从商业途径得到。
实施例1：pgsA、pgsB、pgsC氨基酸序列的获得
从产γ-聚谷氨酸的菌株地衣芽孢杆菌(Bacillus licheniformis)中以公知的方法克隆合成γ-PGA的3个关键酶基因PgsA、PgsB、PgsC；其中，基因PgsA的GenBank ID:AIA08848.1，其氨基酸序列如SEQ ID NO.1所示；基因PgsB的Genebank ID:AIA08846.1，其氨基酸序列如SEQ ID NO.2所示；基因PgsC的Genebank ID:AIA08847.1，其氨基酸序列如SEQ ID NO.3所示。
实施例2：密码子优化
按照已获得的PgsA、PgsB、PgsC基因的氨基酸序列，通过玉米或待转基因的其他植物中的密码子偏好性分析并结合CpG dinucleotides content、GC content、mRNA secondary structure、Cryptic splicing sites、Premature PolyA sites、Internal chi sites and ribosomal binding sites、Negative CpG islands、RNA instability motif(ARE)、Repeat sequences(direct repeat,reverse repeat,and Dyad repeat)、Restriction sites that may interfere with cloning等分析最终确定优化的序列，并根据序列人工从头合成优化后的PgsA、PgsB、PgsC基因；其中，所述密码子优化后的PgsA基因核苷酸序列如SEQ ID NO.4所示；所述密码子优化后的PgsB基因核苷酸序列如SEQ ID NO.5所示；所述密码子优化后的PgsC基因核苷酸序列如SEQ ID NO.6所 示。
实施例3：植物表达载体构建
将密码子优化后的PgsA、PgsB、PgsC基因以公知的方法连入含有bar基因筛选标记的植物表达载体pU130-bar(载体图谱如图10所示)，获得含有目的基因的植物表达载体，命名为植物表达载体PGA001，载体图谱如图1所示；其中所述植物表达载体PGA001的核苷酸序列如SEQ ID NO.7所示；该表达载体各功能元件描述为：
Figure PCTCN2021091993-appb-000005
Figure PCTCN2021091993-appb-000006
上述植物表达载体PGA001构建采用多片段重组酶方法构建，具体构建步骤是：
用限制性内切酶Hindlll和Spel酶切1μg载体pU130-bar，37℃下反应1±0.2小时，使用DC301试剂盒(市售PCR凝胶回收试剂盒)回收酶切载体产物。
实施例2中密码子优化后的pgsA、pgsB、pgsC的PCR产物35S+pgsA+Tnos,35S+pgsB+Tnos,35S+pgsC+Tnos，直接由生物公司合成。
然后进行重组。重组连接体系如下：合成PCR产物pgsA+pgsB+pgsC分别为3μl；酶切载体产物，3μl；重组酶，2μl；重组buffer，4μl；H 2O，2μl。37℃30min后冰上或者4℃保存；
然后转化大肠杆菌DH5α：10μl以上连接产物+100μl大肠杆菌感受态。
感受态DH5α冰箱取出后，迅速放入冰上，5分钟后待菌块溶解加入连接产物，冰上静置25min，42℃热击45s，冰上放置2min(勿晃动)，加入无抗生素的LB 100μl，37℃，200rpm，摇菌1h，涂板，LB+K +，37℃培养一天。
然后进行菌落PCR，检测引物是：
QC-F:GCGGCCGAGCCCCTCATCGAGAA
QC-R:GATCAGGCCCGGCACGATGATG
取阳性菌落进行测序。测序结果正确的质粒用于转化农杆菌。
实施例4：产γ-PGA转基因玉米的获得
(1)玉米胚性愈伤组织的获得
选取玉米自交系KN5585作为受体自交系，取其授粉后10-12d的果穗，去苞叶，在无菌工作台中，先用70％酒精处理5min，再用无菌水冲洗4-5遍，剥取1.5-2mm的幼胚，盾片向上放置在诱导培养基(MS+1mg/L2,4-D+1.38g/L L-脯氨酸+0.5g/L水解酪蛋白+30g/L蔗糖+7g/L琼脂，pH5.8)上，28℃、黑暗培养诱导愈伤组织。2-3周后从诱导出的愈伤组织中，选取生长速度较快、质地松软、松散易碎、颜色鲜艳的胚性愈伤组织转移到继代培养基上(MS+1mg/L2,4-D+0.69g/L L-脯氨酸+0.5g/L水解酪蛋白+30g/L蔗糖+7g/L琼脂，pH5.8)继代培养，每2周继代一次，用于农杆菌侵染。
(2)含植物表达载体的农杆菌的制备
选择EH105农杆菌为转化菌株，在50μl EHA105商业化农杆菌感受态细胞(博迈德)中加入含PGA001表达载体的质粒3μl，冰浴30分钟，液氮速冻5分钟，37℃水浴5分钟；再加入800μlYEP液体培养基，28℃、250rpm振荡培养3小时；取出菌液涂布于含利福平及卡那霉素的固体YEP培养基上，置28℃黑暗倒置培养3-4天，取菌落进行菌落PCR验证，并 进行测序，对测序正确的农杆菌保存，用于转化；
(3)农杆菌侵染与共培养
挑取含有PGA001表达载体的农杆菌单菌落，加入5mL的YEP(Kan)培养基中，培养过夜；室温下，6000rpm，离心5min，收集菌体；将菌体悬浮于含有100mM乙酰丁香酮(AS)的侵染液(MS+1mg/L2,4-D+68.5g/L蔗糖+36g/L葡萄糖+100mM AS,pH5.2)中，并使OD 600＝0.6-0.8，混匀，待用；将制得的侵染液在25℃摇床，180rpm活化1小时，用于侵染；将玉米的胚性愈伤组织收集在一起放入一个无菌的三角瓶中，将侵染液倒入，将大的愈伤组织块打散，摇匀使得愈伤组织充分接触农杆菌，侵染15min；将愈伤组织取出，在滤纸上吸干多余的菌液，然后接种到共培养培养基(MS+1mg/L 2,4-D+100mM AS+0.5g/L MES+20g/L蔗糖+7.5g/L琼脂，pH5.2)上，22℃黑暗培养3天。
(4)恢复培养与抗性愈伤组织的筛选
使用含有抗生素的无菌水冲洗愈伤组织表面3-5次，待加入的水不再浑浊时倒掉液体，将愈伤组织转入铺有滤纸的平皿中，在超净台中吹干愈伤表面水分。转入恢复培养基(MS+1mg/L2,4-D+0.69g/L L-脯氨酸+0.5g/L水解酪蛋白+20g/L蔗糖+250mg/L头孢霉素+7.5g/L琼脂，pH5.8)，28℃暗培养7-10天。恢复培养后将转化愈伤组织转入添加有草铵膦(PPT)的筛选培养基(MS+1mg/L2,4-D+0.69g/L L-脯氨酸+0.5g/L水解酪蛋白+20g/L蔗糖+250mg/L头孢霉素+10mg/L PPT+7.5g/L琼脂，pH5.8)上，开始筛选培养两周，然后更换新的筛选培养基，25-28℃暗培养15天，将愈伤组织打散转入新的筛选培养基继续筛选，总共筛选2轮。
(5)抗性愈伤组织的分化和生根
将抗性愈伤组织转入分化培养基(MS+0.5mg/L6-BA+0.5g/LMES+10mg/LPPT+250-300mg/L的头孢霉素+20g/L蔗糖+7g/L琼脂，pH5.8)，25-28℃暗培养7-10天，然后转入光照培养箱25-28℃培养，直至再生芽长至3-5cm时转入生根培养基(MS+20g/L蔗糖+7g/L琼脂，pH5.8)。待长出大量健壮根后，炼苗2-3天，洗净根部培养基后移栽在灭菌的营养土中，室内炼苗7天后移栽到大田(炼苗期间用薄膜覆盖花盆以提高湿度)。
(6)取移栽成活的转基因幼苗的幼叶进行PAT/bar蛋白快速检测试纸条(Artron)检测，转基因阳性玉米自交收获种子。获得T1代转基因玉米植株后，经过两代严格自交，最终获得转基因玉米纯合株系。
实施例5：转基因玉米的检测
转基因检测方法包括PAT/bar蛋白快速检测试纸条检测、PCR检测、RT-PCR检测、产物γ-PGA含量的检测。
PAT/bar蛋白快速检测试纸条(Artron)检测参照试剂盒说明书进行；PCR、RT-PCR检测参照(解光宁.玉米泛素受体ZmDA1,ZmDAR1对籽粒发育的影响[D].山东大学,2017。)所述方法。产物γ-PGA含量的检测参考NY/T 3039-2016方法进行。
将玉米植株在60℃真空烘干后，研碎，称取10g混合均匀的试样，分别测定试样中经盐酸水解后的和未经水解的游离谷氨酸的含量，两者之差即为聚谷氨酸的含量。
试验结果见图1和表1
表1、转基因玉米材料中γ-PGA的含量(mg/100g干重)
Figure PCTCN2021091993-appb-000007
由上述结果可以看出PgsA、PgsB、PgsC三个基因已经成功转入到玉米中(图1)，并在转基因玉米中成功检测到了γ-PGA。
实施例6：转基因玉米表型鉴定
选择转基因阳性玉米株系对其整个发育期(萌发期、苗期、拔节期等)以及种子的表型进行鉴定。
试验结果见图2、表3，对正常生长条件下的转基因阳性玉米和阴性对照玉米的表型进行观察，确定在玉米中异源合成γ-PGA对玉米生长发育的影响，结果如图2所示。
结果显示，转基因阳性玉米株系具有更好的萌发率，更高的株高，更发达的根系，说明在玉米中合成γ-PGA可以促进玉米的生长；发明人还对其在正常灌溉条件下的产量进行评估，发现在玉米中合成γ-PGA可以提高正常生长条件下玉米的产量，主要是提高了穗粒数、穗粒重、百粒重(表3)。
实施例7转基因玉米抗逆性鉴定
转基因玉米的抗旱性检测
对转基因阳性玉米和阴性对照玉米分别进行干旱胁迫处理，并对其表型进行研究。干旱处理分PEG模拟干旱处理和断水干旱胁迫处理。
PEG模拟干旱处理主要选萌发期和三叶期这两个时期，玉米培养液选择Hogland营养液(海博生物)，萌发期干旱胁迫处理选择的PEG6000的浓度分别为14％、18％。选取转基因材料及对照材料的种子表面消毒，分别在含0mmol/L、12％PEG、14％PEG溶液的滤纸上萌发，统计萌发率，观察生长势。苗期模拟干旱处理选择的PEG6000的浓度为18％；苗期干旱处理采用盆栽断水处理，并测定游离脯氨酸、可溶性糖、叶绿素含量和ABA含量等生理生化指标。田间干旱处理实施地点为三亚南繁基地，选择开花前断水处理的方法，具体实施办法为：断水15天，复水一次，再断水15天，一直到收获，对抗逆表型和产量进行测定。
游离脯氨酸、可溶性糖、叶绿素、ABA含量的测定方法参考(Wang B，Li Z，Ran Q，et al.ZmNF-YB16 Overexpression Improves Drought Resistance and Yield by Enhancing Photosynthesis and the Antioxidant Capacity of Maize Plants[J].Frontiers in Plant Science,2018,9:709；Li Z，Liu C，Zhang Y，et al. The bHLH family member ZmPTF1 regulates drought tolerance in maize by promoting root development and ABA synthesis[J].Journal of Experimental Botany,2019(19):19.)进行。
抗旱性分析结果见图3-图5。
结果表明，转基因阳性玉米在PEG处理下仍可以较好的萌发，而阴性对照玉米的萌发却受到了明显的抑制(图3)。在苗期的PEG模拟干旱处理试验中，发明人将转基因阴性对照玉米一部分外源施加50mg/L的γ-PGA，另一部分作为对照，与转基因阳性玉米同时在18％PEG溶液处理下进行培养，结果显示，在18％PEG溶液处理条件下，阴性对照玉米的生长明显受到了抑制，而转基因阳性玉米和外源施加γ-PGA的处理组的生长状况明显优于对照材料(图4A)。发明人对18％PEG处理5d后的转基因阳性玉米株系、外源施加γ-PGA的阴性玉米植株以及阴性对照株系进行了地上部分和地下部分的鲜重统计，发现转基因阳性玉米株系在PEG处理条件下的生物量明显高于阴性对照玉米，甚至略高于外源施加γ-PGA的玉米组(图4B-4C)。说明γ-PGA在玉米中的异源合成可以达到外源施加γ-PGA提高玉米抗旱性的效果。这表明，转基因玉米的抗逆性提高是因为其产生了γ-PGA。
发明人还对三叶期的转基因阳性玉米和阴性对照玉米进行了盆栽断水处理试验。发现在干旱胁迫早期条件下，转基因阳性玉米相对转基因阴性对照玉米表现出较好的生长状态。经历8天的断水处理后，阴性对照玉米几乎萎蔫致死，恢复浇水，转基因阳性玉米仍可以迅速恢复生长，而对照玉米几乎全部死亡(图5)。对断水处理6天的叶片的ABA、可溶性糖、脯氨酸和叶绿素含量进行测定，结果显示，在干旱胁迫条件下，在玉米中异源合成γ-PGA可以显著提高玉米叶片中ABA、可溶性糖、脯氨酸和叶绿素的含量，从而提高玉米的抗旱性(表2)。
表2、干旱处理对转基因阳性玉米和阴性对照玉米中ABA、可溶性糖(Soluble sugar)、脯氨酸(Proline)、叶绿素(Chlorophyll)含量的影响
Figure PCTCN2021091993-appb-000008
大田干旱断水试验结果见图6。结果表明，在大田干旱条件下，转基因阳性玉米也表现出良好的抗旱性。
转基因玉米抗盐性研究包括萌发期和苗期：对萌发期玉米选择不同浓度的NaCl溶液进行盐胁迫处理。选取转基因阳性材料及阴性对照材料的种子表面消毒后，分别在含150mM NaCl、200mM NaCl溶液的滤纸上萌发，并观察萌发情况；苗期抗盐试验采取溶液胁迫处理：将玉米种子表面消毒使其萌发后转移至营养液(Hogland玉米营养液)中培养，将培养至三叶期的玉米分别移至含200mM NaCl的营养液中培养，并统计表型变化。
抗盐性分析结果见图7-图8。结果表明，无论在较低浓度还是高浓度NaCl条件下，转基因玉米种子的萌发情况都比对照阴性玉米好，说明转基因玉米在萌发期具有更好的抗盐性，苗期水培抗盐试验结果也显示转基因阳性玉米具有明显的抗盐性。
转基因阳性(内源合成γ-PGA)玉米、γ-PGA外源处理的野生型对照玉米抗盐和抗旱性对比：转基因阳性(内源合成γ-PGA)玉米、γ-PGA外源处理的野生型自交系抗旱和抗盐性对比试验采取溶液胁迫处理的方法。首先将玉米种子表面消毒后萌发，再将萌芽小苗插入到营养液(Hogland玉米营养液)中培养，待植株长至三叶期时，开始进行处理，转基因玉米株系分别转移到含有18％PEG或200mmol/LNaCl的营养液中进行处理，对照玉米自交系分别转移至含有20mg/Lγ-PGA、18％PEG、18％PEG+20mg/Lγ-PGA、200mmol/L NaCl、200mmol/L NaCl+20mg/Lγ-PGA的营养液中进行处理，未经任何处理的野生型玉米和转基因阳性玉米株系作为对照，期间观察表型变化，最后进行生物量测定。
结果见图9。结果表明，转基因玉米株系与外源γ-PGA处理的野生型玉米的抗旱性和抗盐性相当，甚至比外源添加γ-PGA的野生型玉米的抗性更好一些，说明转PgsA、PgsB、PgsC基因使其在玉米中内源合成γ-PGA在提高玉米抗旱性和抗盐性上具有更好的效果。
实施例8：转基因玉米在正常条件下和干旱条件下产量分析
对大田中种植的转基因阳性玉米植株和转基因阴性对照玉米植株在开花前进行干旱处理，具体实施办法为断水15天后复水，然后再断水15天，然后复水。最后对产量进行统计。
结果见图2F、图6C、表3。
结果显示，在干旱条件下，转基因阳性玉米的产量显著提高。与正常灌溉条件下产量相比，转基因玉米受干旱的影响较小，而阴性对照玉米在干旱条件下产量显著下降。
表3、转基因阳性玉米与阴性对照玉米在正常灌溉条件下和干旱条件下的产量统计
Figure PCTCN2021091993-appb-000009
实施例9：转基因阳性玉米与阴性对照玉米种子中淀粉和直链淀粉的含量检测
对转基因阳性玉米与阴性对照玉米种子中的淀粉和直链淀粉的含量进行测定，方法参考(解光宁.玉米泛素受体ZmDA1,ZmDAR1对籽粒发育的影响[D].山东大学,2017。)。
转基因阳性玉米与阴性对照玉米种子中淀粉和直链淀粉的含量检测对比发现转基因玉米可以显著提高玉米种子中淀粉和直链淀粉的含量。
结果见表4。
表4、转基因阳性玉米与阴性对照玉米种子中淀粉和直链淀粉含量
Figure PCTCN2021091993-appb-000010
综上所述，本发明首次利用合成生物学原来和基因工程技术提供了一种在植物中合成γ-PGA的方法，且成功的在转基因玉米中检测到γ-PGA的合成，证明了γ-PGA可以在植物中异源合成；且本发明选择主要的粮食作物玉米作为供试材料，获得的转基因玉米证实可以明显提高其抗旱、抗盐性，在干旱条件下可以明显提高其产量；在非胁迫的正常灌溉条件下的产量也没有下降，甚至还略微提高；本发明还对转基因玉米籽粒的品质进行了测定，发现在玉米中合成γ-PGA还可以提高玉米籽粒中的淀粉含量和直链淀粉含量。上述研究结果提示，本发明方法可以广泛应用于作物抗逆分子育种、农作物生物量提高等方面，具有重要的理论意义和巨大的经济价值。

Claims (6)

  1. 一种γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法，步骤是：
    (1)克隆合成γ-聚谷氨酸的基因：从产γ-聚谷氨酸的菌株中克隆合成γ-PGA的3个关键酶基因PgsA、PgsB、PgsC；其中，基因PgsA的GenBank ID:AIA08848.1，其氨基酸序列如SEQ ID NO.1所示；基因PgsB的Genebank ID:AIA08846.1，其氨基酸序列如SEQ ID NO.2所示；基因PgsC的Genebank ID:AIA08847.1，其氨基酸序列如SEQ ID NO.3所示；
    (2)密码子优化：按照PgsA、PgsB、PgsC基因的氨基酸序列，通过玉米或待转基因的其他植物中的密码子偏好性分析，人工从头合成优化后的PgsA、PgsB、PgsC基因；
    (3)构建植物表达载体：将密码子优化后的PgsA、PgsB、PgsC基因连入含有bar基因筛选标记的植物表达载体pU130-bar，获得含有目的基因的植物表达载体，命名为植物表达载体PGA001；
    (4)植物抗逆性和产量提高的转基因植物的获得；
    其特征在于：
    步骤(1)所述产γ-聚谷氨酸的菌株是地衣芽孢杆菌(Bacillus licheniformis)或解淀粉芽孢杆菌(Bacillus amyloliquefaciens)；
    步骤(2)所述密码子优化后的PgsA基因核苷酸序列如SEQ ID NO.4所示；所述密码子优化后的PgsB基因核苷酸序列如SEQ ID NO.5所示；所述密码子优化后的PgsC基因核苷酸序列如SEQ ID NO.6所示；
    步骤(3)所述植物表达载体PGA001的核苷酸序列如SEQ ID NO.7所示；
    步骤(4)所述植物抗逆性和产量提高的转基因植物是转基因玉米，获得该转基因玉米的方法是：
    1)含植物表达载体PGA001的农杆菌菌株制备
    选择EH105农杆菌为转化菌株，在50μl该农杆菌感受态细胞中加入含PGA001表达载体的质粒3μl，冰浴30分钟，液氮速冻5分钟，37℃水浴5分钟；再加入800-1000μl YEP液体培养基，25-28℃、180-250rpm振荡培养3小时；取出菌液涂布于含利福平及卡那霉素的固体YEP培养基上，置25-28℃黑暗倒置培养3-4天，取菌落进行菌落PCR验证，并进行测序，对测序正确的农杆菌保存，用于转化；
    2)愈伤组织的准备
    选取玉米自交系KN5585作为受体自交系，取其授粉后10-12d的果穗，去苞叶，在无菌工作台中，先用70％酒精处理5-6min，再用无菌水冲洗4-5遍，剥取1.5-2mm的幼胚，盾片向上放置在幼胚诱导培养基上，28℃、黑暗培养诱导愈伤组织；2-3周后将诱导出的愈伤组 织，选取生长速度较快、质地松软、松散易碎、颜色鲜艳的胚性愈伤组织转移到继代培养基上继代培养，每2周继代一次，用于农杆菌侵染；
    3)农杆菌侵染
    挑取含有PGA001表达载体的农杆菌单菌落，加入5-6mL的YEP(Kan)培养基中，培养过夜；室温下，5000-6000rpm，离心5-10min，收集菌体；将菌体悬浮于含有乙酰丁香酮(AS)的侵染液中，并使OD 600＝0.6-0.8，混匀，待用；将制得的侵染液在25-28℃，摇床180rpm活化1-2小时，用于侵染；将玉米的胚性愈伤组织收集在一起放入一个无菌的三角瓶中，然后倒入侵染液，将大的愈伤组织块打散摇匀，使愈伤组织充分接触农杆菌，侵染15-20min；将愈伤组织取出，在滤纸上吸干多余的菌液，然后接种到共培养培养基上19-22℃黑暗培养3天；
    4)恢复培养
    使用含有抗生素的无菌水冲洗愈伤组织表面3-5次，待加入的水不再浑浊时倒掉液体，将愈伤组织转入铺有滤纸的平皿中，在超净台中吹干愈伤表面水分，转入恢复培养基，28℃暗培养7-10天；
    5)筛选培养
    恢复培养后将转化愈伤组织转入添加有草铵膦的筛选培养基上，筛选培养两周，然后更换新的筛选培养基25-28℃暗培养15-20天，将愈伤组织打散再转入新的筛选培养基继续筛选，总共筛选2轮，培养30-40天；
    6)分化生根
    将抗性愈伤组织转入分化培养基，25-28℃暗培养7-10天，然后转入光照培养箱25-28℃培养，直至再生芽长至3-5cm时转入生根培养基；
    7)炼苗移栽后自交收种
    待长出大量健壮根后，炼苗2-3天，洗净根部培养基后移栽在灭菌的营养土中，室内炼苗7天后移栽到大田；期间取其幼叶进行PAT/bar蛋白快速检测试纸条检测，转基因阳性玉米自交收获种子；获得T1代转基因玉米植株后，经过两代严格自交，最终获得转基因玉米纯合株系。
  2. 根据权利要求1所述γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法，其特征在于：所述筛选培养中的筛选培养基配方为：MS+1mg/L2,4-D+0.69g/L L-脯氨酸+0.5g/L水解酪蛋白+20g/L蔗糖+250mg/L头孢霉素+10mg/L草铵膦+7.5g/L琼脂，pH5.8。
  3. 根据权利要求1所述γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法，其特征在于：所述分化生根中的分化培养基配方为：MS+0.5mg/L 6-BA+0.5g/LMES+10mg/L草铵膦+250-300mg/L的头孢霉素+20g/L蔗糖+7g/L琼脂，pH5.8。
  4. 一种能使γ-聚谷氨酸在植物中异源合成并提高植物抗逆性和产量的植物表达载体，其特征在于：所述植物表达载体命名为植物表达载体PGA001，其核苷酸序列如SEQ ID NO.7所示。
  5. 一种利用权利要求1所述γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法获得的植物转基因品系。
  6. 根据权利要求5所述利用γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法获得的植物转基因品系，其特征在于：所述植物转基因品系是转基因玉米纯合株系；所述抗逆性是指抗旱、抗盐性。
PCT/CN2021/091993 2021-04-28 2021-05-07 γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法 WO2022227102A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110465460.1 2021-04-28
CN202110465460.1A CN113512562B (zh) 2021-04-28 2021-04-28 γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法

Publications (1)

Publication Number Publication Date
WO2022227102A1 true WO2022227102A1 (zh) 2022-11-03

Family

ID=78063749

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/091993 WO2022227102A1 (zh) 2021-04-28 2021-05-07 γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法

Country Status (2)

Country Link
CN (2) CN115725519A (zh)
WO (1) WO2022227102A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115725519A (zh) * 2021-04-28 2023-03-03 齐鲁工业大学 γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101033472A (zh) * 2005-03-04 2007-09-12 三得利株式会社 产生聚谷氨酸的转基因植物
CN102181475A (zh) * 2011-03-04 2011-09-14 天津大学 一种对转入玉米的Bar基因进行筛选的方法
CN107384840A (zh) * 2017-09-07 2017-11-24 北京农业生物技术研究中心 一株抗旱促生复合菌剂及其应用
CN113512562A (zh) * 2021-04-28 2021-10-19 齐鲁工业大学 γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101033472A (zh) * 2005-03-04 2007-09-12 三得利株式会社 产生聚谷氨酸的转基因植物
CN102181475A (zh) * 2011-03-04 2011-09-14 天津大学 一种对转入玉米的Bar基因进行筛选的方法
CN107384840A (zh) * 2017-09-07 2017-11-24 北京农业生物技术研究中心 一株抗旱促生复合菌剂及其应用
CN113512562A (zh) * 2021-04-28 2021-10-19 齐鲁工业大学 γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 26 May 2014 (2014-05-26), ANONYMOUS : "gamma polyglutamic acid biosynthesis synthase C [Bacillus licheniformis]", XP055981915, retrieved from Genbank Database accession no. AIA08847 *
DATABASE Protein 26 May 2014 (2014-05-26), ANONYMOUS: "gamma polyglutamic acid biosynthesis synthase A [Bacillus licheniformis]", XP055981907, retrieved from Genbank Database accession no. AIA08848 *
DATABASE Protein 26 May 2014 (2014-05-26), ANONYMOUS: "gamma polyglutamic acid biosynthesis synthase B [Bacillus licheniformis]", XP055981911, retrieved from Genbank Database accession no. AIA08846 *
ZHANG JING-JING;BAI YOU-LU;YANG LI-PING;LU YAN-LI;WANG LEI;LI GE;ZHANG YIN-JIE: "Mechanism of Spraying γ-Poly Glutamic Acid Increasing Yield and Nutrient Uptake of Summer Maize", JOURNAL OF PLANT NUTRITION AND FERTILIZERS, vol. 25, no. 11, 25 November 2019 (2019-11-25), pages 1856 - 1867, XP055981901, DOI: 10.11674/zwyf.19073 *

Also Published As

Publication number Publication date
CN113512562A (zh) 2021-10-19
CN113512562B (zh) 2022-05-17
CN115725519A (zh) 2023-03-03

Similar Documents

Publication Publication Date Title
CN109456982B (zh) 水稻OsMYB6基因及其编码蛋白在抗旱和抗盐中的应用
CN109837296B (zh) 玉米基因ZmNAC77的一个耐盐耐旱新功能及其应用
CN113005126B (zh) DgSPL3基因及其克隆方法和应用
CN104611359A (zh) ZmSPL1蛋白及其编码基因在调控玉米籽粒发育中的应用
CN112626084B (zh) 草莓MYB转录因子FvMYB24基因、表达蛋白及应用
WO2022227102A1 (zh) γ-聚谷氨酸在植物中异源合成提高植物抗逆性和产量的方法
CN106591324B (zh) 谷子SiASR4基因及应用
CN113604480A (zh) 一种玉米转录因子ZmHsf28及应用
AU2020100800A4 (en) Use of aegilops tauschii hmt1 gene
CN103045639B (zh) AtTGA4基因在提高植物抗逆性中的应用
CN104878042A (zh) 一种提高植物抗逆性的方法及其应用
CN115807006B (zh) 基因片段b在培育植物新材料中的应用
JP2012507263A (ja) 変化した植物構造を示すグルタミン酸デカルボキシラーゼ（ｇａｄ）トランスジェニック植物
CN114686494B (zh) SlERF.H2基因及其所编码的蛋白质在调控番茄耐盐性中的应用
KR20100053456A (ko) 선발마커가 제거된 형질전환 벼의 제조방법
CN112458101B (zh) 一种鸭茅锌指蛋白基因DgZFP125及其表达载体和应用
CN115896045A (zh) 杜梨E3泛素连接酶基因PbrATL18在植物抗干旱和炭疽病遗传改良中的应用
WO2003097827A1 (fr) Procede de culture de plans de tomates halophiles
Liu et al. Saussurea involucrata SIDhn2 gene confers tolerance to drought stress in upland cotton
Mandal et al. Osmotin: A PR gene impart tolerance to excess salt in Indica Rice
CN104498508A (zh) 小麦渐渗系应答非生物胁迫调控基因TaGBF及其应用
CN112608371B (zh) 多效性基因SbSnf4及其在提高高粱茎杆含糖量和生物产量中的应用
CN114107333B (zh) 一种大麦受体类激酶HvSERK1在根毛生长中的应用
Rihan Plant tissue culture and artificial seed production techniques for cauliflower and their use to study molecular analysis of abiotic stress tolerance
CN113604475B (zh) 棉花gh_d03g1517基因在促进抗旱和耐盐中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21938575

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21938575

Country of ref document: EP

Kind code of ref document: A1