CN114480485B - Genetic engineering application of paddy rice glutamine aminotransferase gene OsGAT2 - Google Patents
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Abstract
The invention discloses a genetic engineering application of a rice glutamine aminotransferase gene OsGAT 2. Application of a rice glutamine aminotransferase gene OsGAT2 in improving rice yield and nitrogen utilization efficiency. The rice glutamine aminotransferase gene OsGAT2 is expressed at the joint of rice rhizomes. By taking Japanese sunny rice as a background, the plant height is obviously increased and the nitrogen utilization efficiency is improved after the gene is knocked out by a genetic engineering CRISPR-Cas9 technology. In low nitrogen fertilizer applied lands, mutant tillering numbers and individual yield are significantly increased. Has important significance for increasing the yield of rice and improving the physiological utilization efficiency of nitrogen.
Description
Technical Field
The invention belongs to the fields of genetic engineering technology and botanics, and relates to a rice glutamine amino transferase gene OsGAT2 and application thereof in improving yield and nitrogen utilization efficiency of rice during low nitrogen fertilizer application.
Background
Nitrogen is one of the most important mineral elements in plants, and is an essential element constituting important organic compounds such as chlorophyll, nucleic acid, and enzymes in plants. When nitrogen is deficient in gramineae plants, the plants are reduced in tillering, long and thin in stems, few in spike grains, insufficient in seeds, and prone to premature senility, so that yield is reduced (SK De Datta, 1986). In actual agricultural production, applying nitrogen fertilizer is a guarantee for ensuring high yield and excellent yield of crops. However, the excessive application of nitrogen fertilizer also creates a number of environmental and production problems. The excessive application of the nitrogen fertilizer can accelerate the acidification of soil and destroy the ecological stability of the environment. Meanwhile, the utilization efficiency of the plant to nitrogen is reduced, the growth period is influenced, the late maturity of the greedy green is caused, the nutrition growth of the plant is overlong, the reproductive growth is delayed, the normal growth period is disturbed, and the yield and the physiological utilization efficiency of the nitrogen are reduced. Therefore, the method analyzes the nitrogen absorption path and mechanism of plants, researches the functions of related genes, and plays an important role in improving the yield and applying the nitrogen fertilizer in actual production.
Aminotransferases (EC 2.6.1. X) are an important and diverse class of proteins commonly found in animals, plants and microorganisms, and the physiological function of these enzymes is to catalyze the transfer of amino donors to amino acceptor compounds: generally, amino acids are the amino donor and 2-oxo acids are the amino acceptor, and the reaction products are the corresponding 2-oxo acids and amino acids, respectively (Givan, 1980). These enzymes play a central role in metabolism and they transfer amino groups into amino acids, amino sugars, purine and pyrimidine nucleotides, coenzymes and antibiotics. At least 16 members of the family of aminotransferases are currently known, and the catalyzed reaction plays an indispensable role in a variety of metabolic pathways in plants, such as amino acid synthesis and metabolism, secondary metabolism, carbon nitrogen assimilation, light respiration, glyoxylate detoxification and stress resistance (Liepman a H et al 2004).
The nitrogen in rice is transported in rice body mainly in the form of amino acid and small amount of nitrate Nitrogen (NO) 3 - ). Among these, asparagine (asparamgine, asn) and glutamine (gin) are potent nitrogen storage and transport compounds, and are also the most important forms of nitrogen transport from the root to the ground. Glutamine is the most abundant amino acid that transports nitrogen in xylem and phloem juice of rice (p.j.lea et al, 2007). The members of the glutamine aminotransferase family (GATs) are numerous and function differently in rice. Among these, more asparagine synthetase (Asparagine synthetase) and glutamate synthetase (Glutamate synthetase, GOGAT) are reported. Rice coding genes of asparagine synthetase (Asparagine synthetase) are OsASN1 and OsASN2 (Ohashi M et al 2015), and overexpression of OsASN1 can promoteThe uptake and assimilation of nitrogen by rice seedlings enhances the tolerance of the seedlings to nitrogen deficiency, and the yield increases under nitrogen deficiency conditions (Lee S et al 2020). Mutation of glutamate synthase (Glutamate synthetase, goat) in the coding gene in rice will result in a significant reduction in spikelet numbers, yield and biomass (Tamura W et al 2011).
Disclosure of Invention
The invention aims to provide engineering application of a rice glutamine aminotransferase gene OsGAT2, which mainly has the functions of increasing tillering under the condition of low nitrogen fertilizer, improving yield and improving nitrogen nutrient utilization efficiency.
Application of rice glutamine aminotransferase gene OsGAT2 in improving rice yield and nitrogen utilization efficiency; the rice glutamine aminotransferase gene OsGAT2 genome sequence has the accession number of Os06g48620 in an MSU database, and the coded protein sequence has the accession number of Os06g48620 in the MSU database.
As a preferable mode of the invention, the application of the rice glutamine aminotransferase gene OsGAT2 in improving the plant height, tiller number and/or nitrogen utilization efficiency of rice is provided.
As a further preferred aspect of the present invention, the rice glutamine aminotransferase gene OsGAT2 in knocked out or mutated rice can improve the plant height, tiller number and/or nitrogen utilization efficiency of the rice.
The beneficial effects are that:
the rice glutamine aminotransferase gene OsGAT2 is expressed at the joint of rice rhizomes. By taking Japanese sunny rice as a background, the plant height is obviously increased and the nitrogen utilization efficiency is improved after the gene is knocked out by a genetic engineering CRISPR-Cas9 technology. In low nitrogen fertilizer applied lands, mutant tillering numbers and individual yield are significantly increased. Has important significance for increasing the yield of rice and improving the physiological utilization efficiency of nitrogen.
Drawings
Fig. 1: expression level of OsGAT2 gene in rice.
A: expression conditions of different parts of OsGAT2 in different periods; b: osGAT2 expression under nitrogen deficiency treatment. Values represent mean ± SD (n=3),. Times.p < 0.05
Fig. 2: and (5) identifying mutation sites of the OsGAT2 gene knockout mutant.
Light boxes in the gene structure represent exons, dark boxes represent 5'-UTR and 3' -UTR, and black lines represent introns. Underlined in the base sequence indicates the Spacer sequence and the deletion line represents the base deletion.
Fig. 3: osgat2 mutant phenotype at maturity under nitrogen deficient conditions.
A: photographing the osgat2 mutant; b: the osgat2 mutant strain height; c: effective tillering number; values represent mean ± SD (n=5), p < 0.05, p < 0.01
Fig. 4: osgat2 mutant yield under nitrogen-deficient conditions.
A: osgat2 mutant biomass (DW); b: yield of individual plants; values represent mean ± SD (n=5), p < 0.05, p < 0.01
Fig. 5: efficiency of nitrogen utilization of osgat2 mutant.
Osgat2 mutant cereal grass ratio; b: concentration of cereal/diazonium; c: agronomic physiological utilization efficiency; the values represent mean ± SD (n=5), p < 0.01, p < 0.001
Gat2-1, gat2-2, gat2-3 in the above figures represent 3 constructs of OsGAT2 mutant materials using CRISPR/Cas9 technology, respectively.
Detailed Description
Example 1: expression study of OsGAT2
1) Extraction of Total RNA and Synthesis of cDNA
The rice variety Nipponbare is selected. Selecting full and healthy mature seeds, removing shells, soaking the mature seeds in 70% alcohol solution for 30s for preliminary disinfection, and disinfecting the mature seeds by using sodium hypochlorite solution containing 1% active chlorine for 1h. Cleaning seeds in a super clean bench with sterilized water for 5-6 times, transferring the seeds into 1/2MS culture medium for culturing, selecting seedlings with consistent growth vigor after the seedlings grow to two leaves and one heart, transferring the seedlings into international standard nutrient solution for culturing, simultaneously treating with different nitrogen concentrations, respectively using NH 4 + The concentration of the nutrient solution is 0.25mM and 2.5 mM. At different time periods according to the requirementThe different parts were sampled and ground to powder with liquid nitrogen. Leaf total RNA was extracted by Trizol (Life technologies, carisbad, calif., USA) and cDNA was synthesized by reverse transcription using PrimeScript TMRT kit (Takara, shiga, japan).
2) Quantitative determination of OsGAT2 expression
The real-time PCR method is adopted. Using qPCR />Green master mix kit (Vazyme, south tokyo, china).
The specific primer sequences of the OsGAT2 genes are as follows:
upstream primer F1:5'-ACACCAGAGGAAGATGAATG-3' (SEQ ID NO. 1)
Downstream primer R1:5'-GGTTCCACGAATGGTACTTA-3' (SEQ ID NO. 2)
The control reference OsActin gene specific primer sequences used were:
OsActin-F:5’-CAACACCCCTGCTATGTACG-3’(SEQ ID NO.3)
OsActin-R:5’-CATCACCAGAGTCCAACACAA-3’(SEQ ID NO.4)
the amplification reaction was performed in a Thermo Lifetech ABI QuantStudioTM Flex system (Life Technologies, carisbad, calif., USA). The method comprises the following steps: the temperature is 95 ℃ for 30s; the temperature is 95 ℃ for 5s; the temperature is 60 ℃ for 30s,40 periods; the temperature is 95 ℃ for 15s; the temperature is 60 ℃ for 60s; the temperature was 95℃for 15s. After completion of the reaction, a dissolution profile was performed to detect primer specificity. The PCR quantitative result shows that OsGAT2 is expressed at the joints of germs and radicle of rice sprouting for 3 days and leaves, roots and rhizomes of rice water culture for 9 days, and the relative expression amount in the leaves is high (figure 1A). Under nitrogen deficiency conditions, expression of OsGAT2 was significantly up-regulated in each site (fig. 1B).
Example 2: creation and identification of OsGAT2 mutant plants
1) Creation of OsGAT2 mutant plants
The CRISPR/Cas9 technology is utilized to construct the OsGAT2 mutant material. First, according to the gene sequence, a CRISPR-Cas9 system is utilized to design a NGG or CCN structural target sequence. Designing a primer:
Spacer 1-F:5’-GCCGATCGATAATCTTGGGGCCA-3’(SEQ ID NO.5)
Spacer 1-R:5’-AAACTGGCCCCAAGATTATCGAT-3’(SEQ ID NO.6)
Spacer 2-F:5’-GGCAGATTTTGTTTTCGCAATTTC-3’(SEQ ID NO.7)
Spacer 2-R:5’-AAACGAAATTGCGAAAACAAAATC-3’(SEQ ID NO.8)
these 2 pairs of spacers were annealed by PCR to form oligonucleotide double strands, which were ligated into the portal vectors PYLgRNA-OsU3/LacZ and PYLgRNA-OsU a, which were capable of expressing snRNA by driving the U3 and U6 promoters. The recombinant plasmid is subjected to Gateway LR reaction under the action of LR Clonase II enzyme and is transferred into a final expression plasmid pYLCRISPR/Cas9-MH containing Cas 9. The expression vector with correct sequencing is transferred into agrobacterium EHA105 by an electrotransfer method, and the OsGAT2 CRISPR/Cas9 mutant rice material is obtained by a rice transgenic technology.
2) Identification of OsGAT2 mutant plants
Acquisition of transgenic T 0 And (5) extracting rice genome DNA from leaves of the seedlings. Designing and identifying primers according to the OsGAT2 mutation site:
OsGAT2-F:5’-TTGATCACAACAATGGGGAT-3’(SEQ ID NO.9)
OsGAT2-R:5’-GCCTGTATTTCAACTGCAAA-3’(SEQ ID NO.10)
the genome DNA is used as a template, the OsGAT2 mutation site is amplified by PCR, the product is compared with a wild type sequence after sequencing, and 3 effective mutant strains which are named gat2-1, gat2-2 and gat2-3 are selected. gat2-2 deleted 9 bases on exon 1, gat2-3 deleted 12 bases on exon 1, gat2-6 deleted 9 bases on exon 1 (FIG. 2). Amplifying the Cas9 carrier sequence by taking genomic DNA of the OsGAT2 mutant strain as a template, wherein the Cas9 carrier sequence does not exist in the genome of all mutant strains. Cas9 vector sequence primer:
Cas9-F:5’-ACAAGGGCAGGGATTTCG-3’(SEQ ID NO.11)
Cas9-R:5’-ACTGGTGGATGAGGGTGGC-3’(SEQ ID NO.12)
example 3: evaluation of biomass, single plant yield, cereal straw ratio, plant Nitrogen concentration and Nitrogen utilization efficiency
The OsGAT2 mutant line and the original background Japanese sunny wild type material are planted in a white horse scientific research teaching base of Nanjing university one plant per hole, and are provided with normal nitrogen supply treatment (250 kg/ha) and field nitrogen deficiency treatment (75 kg/ha). And observing the growth conditions of each mutant strain and the whole growth period of the wild type, and counting agronomic characters such as plant height, effective tiller number and the like in the mature period. And collecting the whole overground part samples of each material in the mature period, rapidly placing the overground part samples in a105 ℃ oven, deactivating enzymes for 30min, placing the overground part samples in a 70 ℃ oven, drying the overground part samples to balance weight, and respectively weighing the individual plant yields of each mutant strain and the wild type strain. The nitrogen content and nitrogen utilization efficiency of plants are measured by adopting a Kjeldahl method, and after drying and digestion, the total nitrogen is measured by a flow analyzer (model AA3, brann+Luebbe company, germany). The calculated nitrogen utilization efficiency is the physiological utilization efficiency of nitrogen (plant yield/total nitrogen uptake by plants). The analysis results showed that under the nitrogen deficiency condition, the strain height of the OsGAT2 mutant increased by about 7cm (figure 3B), the effective tiller number increased by about 2-3 (figure 3C), the biomass increased by about 34.5% compared with the wild type (figure 4A), and the single plant yield increased by about 36.1% (figure 4B). Under normal nitrogen conditions, the OsGAT2 mutant cereal grass ratio is increased by about 43.4% compared with the wild type (figure 5A), and meanwhile, the nitrogen content in plant cereal and grass is obviously reduced (figure 5B), the average nitrogen content in grass is reduced by about 18%, and the average nitrogen content in cereal is reduced by about 13.6%. Compared with the wild type, the physiological utilization efficiency of the OsGAT2 mutant rice on nitrogen is obviously improved by about 29.2 percent (figure 5C).
Reference to the literature
[1]De Datta S K.Improving nitrogen fertilizer efficiency in lowland rice in tropical Asia[J]. Fertilizer research,1986,9(1-2):171-186.
[2]Givan C V.Aminotransferases in Higher Plants-ScienceDirect[J].Amino Acids and Derivatives,1980:329-357.
[3]Lea P J,Miflin B J.Alternative route for nitrogen assimilation in higher plants[J].Nature,1974, 251:614-616,420(6913):316-320.
[4]Lee S,Park J,Lee J,et al.OsASN1 Overexpression in Rice Increases Grain Protein Content and Yield under Nitrogen-Limiting Conditions[J].Plant Cell Physiol,2020,61(7):1309-1320.
[5]Liepman A H,Olsen L J.Taylor&Francis Online::Genomic Analysis of Aminotransferases in Arabidopsis thaliana-Critical Reviews in Plant Sciences-Volume 23,Issue 1[J].Critical Reviews in Plant Sciences,2004.
[6]Ohashi M,Ishiyama K,Kojima S,et al.Asparagine synthetase1,but not asparagine synthetase2, is responsible for the biosynthesis of asparagine following the supply of ammonium to rice roots[J]. Plant Cell Physiol,2015,56(4):769-778.
[7]Tamura W,Kojima S,Toyokawa A,et al.Disruption of a Novel NADH-Glutamate Synthase2 Gene Caused Marked Reduction in Spikelet Number of Rice[J].Frontiers in plant science,2011,2: 57.
Sequence listing
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Claims (2)
1. Knock-out of the Rice glutamine aminotransferase GeneOsGAT2Application of improving rice yield and/or nitrogen utilization efficiency under low nitrogen condition; the rice glutamine aminotransferase geneOsGAT2The genomic sequence has accession number Os06g48620 in the MSU database.
2. The use according to claim 1, characterized in that the rice glutamine aminotransferase gene in rice is knocked out or mutatedOsGAT2，Can improve the plant height, tiller number and/or nitrogen utilization efficiency of the rice.
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