The invention relates to a rape glutamine synthetase gene BnaC04.GS1; 1, glutamine synthetase and application and amplification primer thereof

Abstract

The invention belongs to the technical field of biological genes, and discloses a rape glutamine synthetase gene BnaC04.GS1; the invention discloses a rape glutamine synthetase gene BnaC04.GS1, and the nucleotide sequence of the rape glutamine synthetase gene BnaC04.GS1 is as shown in SEQ ID NO: 1. The invention relates to a glutamine synthetase obtained by coding of 1, and application of the glutamine synthetase in cultivation of high-yield oilseed rape. The invention further discloses application of the rape glutamine synthetase gene BnaC04.GS1; 1 is an amplification primer. The invention provides an oilseed rape glutamine synthetase gene BnaC04.GS1, which has a nucleotide sequence shown in SEQ ID NO.1. The glutamine synthase 1 and the glutamine synthase obtained by coding the glutamine synthase 1 can remarkably increase the nitrogen harvest index and the yield of a single plant of the oilseed rape through overexpression in the oilseed rape, and can quickly and effectively improve the yield of the oilseed rape when being applied to cultivation of high-yield oilseed rape.

Description

Technical Field

[0001] The present invention belongs to the field of biological gene technology, and in particular relates to a rapeseed glutamine synthetase gene BnaC04.GS1;1 , glutamine synthetase and its application in improving nitrogen utilization and yield of rapeseed, and amplification primers. Background Art

[0002] Rapeseed is the world's second largest oil crop, accounting for about 13%-16% of the world's vegetable oil production. It is an important source of edible oil and feed protein. Its planting area and output account for a growing proportion of oil crops year by year, and its planting scale and industrial value are second only to grain crops, which has an important impact on the development of the agricultural economy. In my country, the planting area of ​​rapeseed has remained at about 120 million mu all year round, ranking fifth in the domestic crop planting area, with a total output of 13 million tons, about 120 kg per mu. Among them, rapeseed oil production accounts for about 60% of my country's self-produced vegetable edible oil, making it the most important source of my country's self-produced edible vegetable oil. The main rapeseed production areas in my country are in the middle and lower reaches of the Yangtze River. They are mainly planted in winter fallow fields or rice-oil rotation, which not only avoids the contradiction of "grain and oil competing for land", but also maintains continuous crop production and improves soil. Therefore, developing rapeseed production and increasing rapeseed yields are of great strategic significance to maintaining national grain and oil security.

[0003] In crop production, nitrogen fertilizer plays an extremely important role and is applied in the largest amount. my country's nitrogen fertilizer consumption ranks first in the world, accounting for about one-third of the total global nitrogen fertilizer application. Within a certain range, with the increase in nitrogen application, crop yield tends to increase, but when the nitrogen fertilizer application is too high, the increase in crop yield is not obvious, and even shows a downward trend. Excessive nitrogen application will not only cause resource waste, soil acidification and hardening, and water eutrophication, but also cause crop leggy growth, lodging, and increased pests and diseases, resulting in reduced yield and quality. Due to excessive or unreasonable application of nitrogen fertilizer, the average nitrogen fertilizer utilization efficiency in my country is significantly lower than that in developed countries, at only about 30%. Compared with other grain and oil crops, rapeseed has a higher demand for nitrogen fertilizer, but a lower nitrogen utilization efficiency. The nitrogen requirement for producing a unit weight of rapeseed grain is 263.7% of that of rice, 232.5% of that of wheat, and 111.4% of that of soybeans. This is also one of the important reasons for the annual increase in the application of nitrogen fertilizer for rapeseed. Therefore, improving the nitrogen use efficiency (NUE) of rapeseed under low nitrogen conditions is an important goal of reducing nitrogen fertilizer and increasing its efficiency in field production applications.

[0004] BnaC04.GS1;1 It is a member of the cytoplasmic GS1 gene family of Brassica napus. Its function has not been reported in rapeseed. Developing its function in rapeseed is of great significance for rapeseed cultivation. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a rapeseed glutamine synthetase gene BnaC04.GS1;1 , glutamine synthetase and its application, amplification primers.

[0006] In order to solve the above technical problems, the technical solution proposed by the present invention is: A rapeseed glutamine synthetase gene BnaC04.GS1;1 , whose nucleotide sequence is shown in SEQ ID NO: 1.

[0007] As a general inventive concept, the present invention also provides a method for producing a rapeseed glutamine synthetase gene BnaC04.GS1;1 The amino acid sequence of the glutamine synthetase encoded by the method is shown in SEQ ID NO: 2.

[0008] As a general inventive concept, the present invention also provides a rapeseed glutamine synthetase gene as described above BnaC04.GS1;1 Or the use of the above-mentioned glutamine synthetase in cultivating high-yield rapeseed.

[0009] The above application, preferably, specifically comprises: amplifying the rapeseed glutamine synthetase gene by PCR BnaC04.GS1;1 The CDS sequence was then cloned into the expression vector by homologous recombination to obtain a gene containing BnaC04.GS1;1 The target vector is then transformed into Agrobacterium by heat shock transformation, and finally cloned into rapeseed by genetic transformation for expression to obtain a BnaC04.GS1;1 The overexpression strain is a high-yield rapeseed; the overexpression strain can also be used to further explore the gene function.

[0010] In the above application, preferably, the amplification primer used in the PCR amplification is OE:: BnaC04.GS1;1 The amplification primers are OE:: BnaC04.GS1;1 The sequences of the forward primer and the reverse primer are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The amplification primer OE:: BnaC04.GS1;1 For amplification of rapeseed glutamine synthetase gene BnaC04.GS1; 1 CDS sequence, suitable for transforming overexpressing material OE:: BnaC04.GS1;1 application.

[0011] In the above application, preferably, the expression vector is P1300.

[0012] The above application, preferably, is to knock out the gene in wild-type rapeseed by CRISPR / Cas9 method. BnaC04.GS1;1, Obtain rapeseed mutants. BnaC04.GS1;1Glutamine synthetase gene BnaC04.GS1;1 Edited target sequence, construct Cas9- BnaC04.GS1;1 The vector was then transformed into Agrobacterium by heat shock transformation, and finally genetically transformed into rapeseed for expression. BnaC04.GS1; Gene-edited transgenic lines.

[0013] In the above application, preferably, the glutamine synthetase gene BnaC04.GS1;1 The edited target sequences are SEQ ID NO:11 and SEQ ID NO:12.

[0014] The above application, preferably, the BnaC04.GS1;1 Gene-edited transgenic lines were identified by PCR amplification, with primers Cas9:: BnaC04.GS1;1 , upstream primer sequence: 5'-CAGATCTCGTTAACCTTAAC -3' (as shown in SEQ ID NO: 13), downstream primer sequence: 5'-GTTGATGCCACTAATATTGATG -3' (as shown in SEQ ID NO: 14).

[0015] As a general inventive concept, the present invention also provides a method for preparing the above-mentioned rapeseed glutamine synthetase gene. BnaC04.GS1;1 The amplification primer is OE:: BnaC04.GS1;1 The sequences of the forward primer and the reverse primer are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

[0016] As a general inventive concept, the present invention also provides a method for preparing the above-mentioned rapeseed glutamine synthetase gene. BnaC04.GS1;1 The amplification primers are G:: BnaC04.GS1;1 The sequences of the forward primer and the reverse primer are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The amplification primer G:: BnaC04.GS1;1 For amplification of rapeseed glutamine synthetase gene BnaC04.GS1;1 CDS sequences are suitable for use in establishing gene expression patterns.

[0017] As a general inventive concept, the present invention also provides a method for preparing the above-mentioned rapeseed glutamine synthetase gene. BnaC04.GS1;1 The amplification primers are GUS:: BnaC04.GS1;1 The sequences of the forward primer and the reverse primer are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The amplification primer GUS:: BnaC04.GS1;1 For amplification of rapeseed glutamine synthetase gene BnaC04.GS1;1The promoter sequence is suitable for use in transforming tissue expression material GUS::BnaC04.GS1;1.

[0018] The present invention uses transgenic technology and plant molecular genetics and physiological and biochemical methods to discover a rapeseed glutamine synthetase gene BnaC04.GS1;1 The encoded enzyme is predicted to be glutamine synthetase, which mainly functions in rapeseed leaves. Different from previous studies, it can be used to improve the nitrogen utilization rate and yield of rapeseed. The rapeseed glutamine synthetase gene was amplified by CRISPRE-CAS9 and other methods. BnaC04.GS1;1 Knockout of the glutamine synthetase gene caused it to lose its function, resulting in a significant decrease in nitrogen harvest index and yield per plant. BnaC04.GS1;1 Overexpression significantly increased the nitrogen utilization rate and single-plant yield of rapeseed, providing an excellent genetic resource for further improving the yield of rapeseed through biotechnology. It has good application prospects in regulating the transfer and reuse of nitrogen, improving the nitrogen harvest index, and increasing the single-plant yield of rapeseed.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) Rapeseed glutamine synthetase gene provided by the present invention BnaC04.GS1;1 Overexpression of the encoded glutamine synthetase in rapeseed can significantly increase the nitrogen harvest index and yield per plant of rapeseed. Its application in the cultivation of high-yield rapeseed can quickly and effectively increase the yield of rapeseed.

[0020] (2) The present invention also provides several rapeseed glutamine synthetase genes BnaC04.GS1;1 PCR amplification primers can effectively and quickly BnaC04.GS1;1 Amplified from cDNA of rapeseed. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is obtained in Example 1 BnaC04.GS1;1 The spatiotemporal expression pattern results included: roots, stems, leaves and leaf veins in the seedling stage; roots, stems, leaves, leaf veins and flower buds in the stalk stage; roots, stems, upper leaves, middle leaves, lower leaves, upper leaf veins, middle leaf veins, lower leaf veins, flower buds, pedicels and flowers in the flowering stage; roots, stems, upper leaves, middle leaves, lower leaves, upper leaf veins, middle leaf veins, lower leaf veins, silique stalks, silique skins and seeds in the silique stage; Figure 2 is obtained in Example 1 BnaC04.GS1;1 The expression pattern results of the silique development cycle include silique skin and seeds at 25 days, 30 days, 35 days, 40 days, 45 days and 50 days after flowering respectively; Figure 3 is obtained in Example 1 BnaC04.GS1;1Tissue localization results: A: seedling, B: root tip at seedling stage; CK is flowering stage, among which C: rosette leaf, D: stem; E: inflorescence; FG: leaf; H: flower; I, J, K: silique); Figure 4 is obtained in Example 2 BnaC04.GS1;1 Results of different mutation types of overexpression strains and mutants: including mutation types of mutant strains and relative expression levels of overexpression strains; Figure 5 The phenotypic identification of each material at the silique stage obtained in Example 2: including low nitrogen treatment (LN: 5mM) and normal nitrogen treatment (MN: 15mM); Figure 6 The results of the analysis of GS enzyme activity, ammonium content, amino acid content, and soluble protein content between the source and the sink of each material at the seedling stage obtained in Example 2; Figure 7 The aboveground total nitrogen and nitrogen harvest index results of the maturity period obtained in Example 2 are analyzed; Figure 8 The results of the field test on grain yield and aboveground biomass at maturity obtained in Example 2 are analyzed; Figure 9 This is the analysis of the harvest index results at maturity of the field test obtained in Example 2. DETAILED DESCRIPTION

[0022] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0023] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0024] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0025] Embodiment 1: A rapeseed glutamine synthetase gene of the present invention BnaC04.GS1;1 , whose nucleotide sequence is shown in SEQ ID NO: 1; the amino acid sequence of the glutamine synthetase encoded by it (hereinafter referred to as "glutamine synthetase BnaC04.GS1; 1") is shown in SEQ ID NO: 2. SEQ ID NO: 1: SEQ ID NO: 2: MSLLTDLVNLNLSETTDKIIAEYIWVGGSGMDMRSKARTLPGPVSDPSELPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRRGNNILVMCDAYTPAGEPIPTNKRHAAAKVFSHPDVVAEVPWYGIEQEYTLLQKDVKWPVGWPIGGFPGPQGPYYCSVGADKSFGRDIVDAH YKACLYAGITISGINGEVMPGQWEFQVGPAVGISAGDEIWRITEVAGVVVSFDPKPIPGDWNGAGAHCNYSTKSMREDGGYEIIKKAIDKLGMRHKEHIAAYGEGNERRLTGHHETADINTFLWGVANRGASIRVGRDTEKEGKGYFEDRRPASNMDPYIVTSMIAETTILWKP* Rapeseed Glutamine Synthetase Gene BnaC04.GS1;1 Glutamine synthetase BnaC04.GS1;1 It was discovered in rapeseed through transgenic technology, plant molecular genetics and physiological and biochemical methods. After research, we also found that its overexpression can significantly increase the yield of rapeseed, and its gene BnaC04.GS1;1 The mutation significantly reduced the nitrogen harvest index and yield of rapeseed, which provides a realistic basis for enhancing the nitrogen transfer and reuse capacity through genetic improvement of varieties and continuously improving the yield of rapeseed.

[0026] 1. Rapeseed Glutamine Synthetase Gene BnaC04.GS1;1 The spatiotemporal expression pattern of To obtain the rapeseed glutamine synthetase gene BnaC04.GS1;1 To investigate the expression pattern of the gene, RNA was extracted from various parts of rapeseed at different growth stages, with three replicates for each sample. Total RNA was extracted using TRIzol, and the total RNA (1 μg) was synthesized into first-strand cDNA using the Hiscript II Q RT SuperMix Kit (Vazyme). Real-time fluorescence quantitative PCR analysis was performed on a StepOnePlus instrument using the ChanQTM SYBR using the qPCR premix (Vazyme) system (Analytik Jena AG). The PCR amplification primers were G:: BnaC04.GS1;1, with forward and reverse primers 5'-AGTCTCGTGATGACCCGTGAG -3' (as shown in SEQ ID NO: 3) and 5'-AGTCTCGTGATGACCCGTGAG-3' (as shown in SEQ ID NO: 4), respectively. Actin was used as an internal standard with primers 5'-CACTATGTTCTCGGGGATTGC -3' (as shown in SEQ ID NO: 5) and 5'-TGGACCTGCCTCATCATACTC -3' (as shown in SEQ ID NO: 6).

[0027] The specific steps are as follows: 1. After tissue sampling, put it into liquid nitrogen for quick freezing; grind the tissue into powder in liquid nitrogen, add 0.1g of sample to a pre-cooled 1.5 mL centrifuge tube, add 1 mL TRIzol, mix quickly, and let it stand at room temperature for 10 minutes; 2. Add 200 μL of chloroform to each tube, shake vigorously for 30 seconds, and let it stand at room temperature (25°C) for 5 minutes; 3. Centrifuge at 4°C 12000rpm for 10 minutes, the sample will separate into three layers, of which the upper aqueous phase is RNA; 4. Carefully aspirate 600μL of supernatant and transfer it to a new centrifuge tube, repeat steps 2 and 3 to obtain purer RNA; 5. Add 500 μL of isopropanol, gently invert and mix, let it stand at room temperature (25°C) for 10 minutes; centrifuge at 4°C 12000 rpm for 10 minutes, and discard the supernatant; 6. Add 1 mL of 75% ethanol to each tube and vortex; 7. 4°C Centrifuge at 12000 rpm for 5 minutes and discard the supernatant; 8. After a brief centrifugation, use a 20μL pipette to carefully aspirate and discard the remaining 75% ethanol; 9. Blow the RNA precipitate on the clean bench until it is translucent and jelly-like. Depending on the amount of precipitate, add 30-40μL sterilized DEPC to dissolve the RNA precipitate; 10. Incubate at 60℃ for 10 minutes; 11. Carefully mix the sample and centrifuge briefly; 12. After the concentration of the RNA sample is measured by the nucleic acid analyzer, store it in a -80℃ refrigerator for later use.

[0028] RNA quality detection and concentration determination: 1. Electrophoresis detection of RNA integrity: 1% agarose gel fast electrophoresis to detect RNA molecular integrity and observe whether the 18S and 28S bands are clear; 2. UV spectrophotometer to measure A260 and A280 to quantitatively analyze RNA purity and concentration; use a solvent that dissolves RNA to read BLANK, and NanoDrop to determine RNA concentration; the ideal effect of RNA separation is A260 / A280=1.8~2.0.

[0029] Total RNA was used to synthesize the first strand of cDNA using the reverse transcription kit TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix. The final concentration of cDNA for each sample was 1µg. cDNA was stored at -20°C for future use. The reverse transcription system was as follows: Total RNA -, Oligo (dT) primer 1µL, 2×Ts reaction mix 10µL, Enzyme mix 1µL, gDNA remover 1µL, RNA-free water -. Reaction conditions: incubate at 42°C for 15min, inactivate at 85°C for 5s, and store in a -20°C refrigerator for future use.

[0030] According to the full length of gene cDNA, qPCR primers were designed using the primer5 software. Primers with good specificity and no complementary bases were selected and synthesized at the Qingke Primer Synthesis Company to obtain amplification primers G:: BnaC04.GS1;1 Using cDNA of rapeseed at different stages as template, primer specificity was detected by agarose gel electrophoresis. The reaction system was as follows: cDNA 1µL, G:: BnaC04.GS1;1 0.5 µL of forward primer (10 µM), G:: BnaC04.GS1;1 Reverse primer (10 µM) 0.5 µL, 2× Easy Taq® supermix 12.5 µL, ddH2O 10.5 µL. Reaction conditions: 94°C, 2 min; 94°C, 30 sec; 55°C, 30 sec; 72°C, 1 min; 34 cycles in total, 72°C, 5 min; 4°C.

[0031] After the primers of fluorescent quantitative PCR were detected correctly, the first strand of cDNA synthesized by reverse transcription was used as a template and quantified by two-step method on LightCycler®96 real-time fluorescent quantitative PCR instrument. Three technical replicates were set for each gene and each sample. The reaction system was as follows: cDNA 1, forward primer (10 μM) (as shown in SEQ ID NO: 3) 0.3, reverse primer (10 μM) (as shown in SEQ ID NO: 4) 0.3, TransStart®Top Green qPCR Supermix 7.5, ddH2O 5.9. Reaction conditions: (1) pre-denaturation stage: 94℃ 30 s, 94℃ 15 s, 60℃ 30 s, 40 cycles; (2) melting curve stage: 60℃ 60 s, 97℃ 1 s; (3) cooling stage: 40℃ 1 min.

[0032] The results are as follows Figure 1-2As shown, the rapeseed glutamine synthetase gene BnaC04.GS1;1 It is highly expressed in leaf tissues throughout the growth period, especially in old leaves during the flowering and silique stages. In addition, it is highly expressed in roots during the flowering period and in silique skins and seeds during the silique stage. ,BnaC04.GS1;1 The expression level is maintained at a high level in the silique peel.

[0033] 2. Rapeseed Glutamine Synthetase Gene BnaC04.GS1;1 Positioning For research BnaC04.GS1;1 To enhance tissue specificity of expression, we constructed a gene carrying Pro BnaC04.GS1;1 The transformation vector was obtained by PCR amplification. BnaC04.GS1;1 The promoter (2.0 kb) was amplified using primers GUS:: BnaC04.GS1;1 The forward and reverse primers were 5'-TGCC AAGCTT GCATGCCATAATAATAGATAAA -3' (HindIII site italics) (as shown in SEQ ID NO: 7) and 5'-ACCCGG GGATCC CCGGGGGCTACGAACACCGA -3' (BamH1 recognition site is italicized) (as shown in SEQ ID NO: 8). The fragment was cut using HindIII and BamH1 endonucleases, and the amplified fragment was cloned into the pCAMBIA1300 vector to generate Pro BnaC04.GS1;1 The generated constructs were transformed into Agrobacterium tumefaciens GV3101 strain and then introduced into wild-type Arabidopsis thaliana (cv. Col-0) by Agrobacterium tumefaciens-mediated transformation. The specific steps are as follows: Use the sequenced rapeseed database (https: / / yanglab.hzau.edu.cn / BnIR / gene_search_darmor) BnaC04.GS1;1 The design was based on the 2.0 kp sequence before the start codon ATG. BnaC04.GS1;1 Promoter-specific primers GUS:: BnaC04.GS1;1 , and added two restriction sites, Hind Ⅲ and BamH1, and used the genomic DNA of young leaves of rapeseed cultured in nutrient solution as template, and used PrimeSTAR high-fidelity polymerase for PCR amplification. The PCR reaction system was as follows: template DNA 3 µL (200ng / µL), Buffer (5×) 10 µL, dNTP Mixture (2.5 mM each) 4 µL, GUS:: BnaC04.GS1;1 2.5 µL of forward primer, GUS:: BnaC04.GS1;1Reverse primer 2.5 µL, PrimeSTAR 0.5 µL, ddH2O, 26.5 µL.

[0034] Add the above ingredients in sequence, flick to mix, and centrifuge briefly. The PCR reaction conditions are 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 sec, 58℃ annealing for 30 sec, 72℃ extension for 2.5 min, 40 cycles; 72℃ extension for 10 min. BnaC04.GS1;1 The PCR amplified product of the promoter was detected by electrophoresis and recovered by gel recovery kit, and then the expression vector p1300 plasmid and BnaC04.GS1;1 The fragments recovered from the PCR amplification products were then detected by gel electrophoresis to recover the target fragments. The two target fragments of the correct size were ligated at 16°C overnight and then transferred into DH5ɑ E. coli competent cells (ligation system: p1300 plasmid was 4 µL; proOs BnaC04.GS1;1 4 µL of the recovered fragment of the PCR amplification product; 1 µL of T4 ligase; 1 µL of 10×T4 Buffer). The antibiotic used at this time was kanamycin. The positive clones were picked for bacterial liquid PCR detection and sent for sequencing. After sequencing identification, the bacterial liquid was expanded and the plasmid was extracted. The plasmid was named proBnaC04.GS1;1–p1300 and transformed into Agrobacterium GV3101. The antibiotics were kanamycin and rifampicin. After the bacterial liquid was positively identified, glycerol was added to store the bacterial liquid at -80°C for later use. The strains obtained by genetic transformation were cultured throughout the growth period, and the positive plants were added to the prepared GUS dye solution to ensure that all tissues of the sample were completely immersed in the GUS dye solution. They were placed in a 37°C incubator and stained with tin foil for about 2 hours in the dark. When the sample showed obvious blue color, the dye solution was poured out and recovered, and it was soaked in 70% ethanol for 1 hour, and then decolorized with 80% ethanol for 1 hour. Finally, 70% ethanol was added to soak until the sample was completely decolorized, and the decolorized sample was stored in anhydrous ethanol. The GUS staining site was photographed with a camera or stereo microscope.

[0035] Depend on Figure 3 It can be seen that rapeseed glutamine synthetase gene BnaC04.GS1;1 The expression level is high in cotyledons and roots, especially root tips, during the seedling stage, and is higher in leaves, calyx and siliques during the flowering stage, especially in the mesophyll tissue of old leaves, large vascular tissue and both ends of siliques.

[0036] Embodiment 2: Rapeseed glutamine synthetase gene of the present invention BnaC04.GS1;1 or its enzyme body (rapeseed glutamine synthetase BnaC04.GS1;1) in the cultivation of high-yield rapeseed (to improve nitrogen harvest index and single plant yield), including BnaC04.GS1;1 Construction of overexpression and mutant plants, seedling phenotypes, and yield analysis at maturity.

[0037] 1. BnaC04.GS1;1 Acquisition of overexpression and gene knockout materials In order to verify the rapeseed glutamine synthetase gene of the present invention BnaC04.GS1;1 In order to investigate the role of Westar in rapeseed, we first used Agrobacterium tumefaciens-mediated transgenic technology to obtain overexpression strains, including amplification of Westar cDNA as a template. BnaC04.GS1;1 The PCR amplification primers were OE:: BnaC04.GS1;1, and the forward primer and reverse primer were 5'-ATTTTGGAGAGAACACGGGGGACTTTGCAACATGAGTCTTCTTACAGATCTCGTTA -3' (as shown in SEQ ID NO: 9) and 5'-GGAACCACTCCCTGAAGCGGCCGCTGTACAAGGTTTCCAGAGGATTGTGGTCTCT -3' (as shown in SEQ ID NO: 10), respectively. BnaC04.GS1;1 The overexpression vector was used to transform Agrobacterium tumefaciens GV3101, and the positive strain was used to genetically transform wild-type rapeseed Westar (WT) to obtain BnaC04.GS1;1 Overexpression transgenic lines were obtained, including OE1, OE2, OE5, OE6, OE7, and OE8. BnaC04.GS1;1 Specifically, the target sequence 1: 5'-ACCCTTCGGAGCTACCAAAGTGG -3' (as shown in SEQ ID NO: 11) and the target sequence 2: 5'-GTGCGATGCTTACACTCCAGCGG -3' (as shown in SEQ ID NO: 12) were used to construct a CRISPR Cas9 vector, transform Agrobacterium tumefaciens GV3101, and the positive strain was used to genetically transform wild-type rapeseed Westar (WT) to obtain BnaC04.GS1;1 Gene-edited transgenic strains. Transgenic strains were identified by PCR amplification, and the identification primers were Cas9:: BnaC04.GS1;1 , upstream primer sequence: 5'- CAGATCTCGTTAACCTTAAC -3' (as shown in SEQ ID NO: 13), downstream primer sequence: 5'- GTTGATGCCACTAATATTGATG -3' (as shown in SEQ ID NO: 14). After identification, two knockout strains KO1 and KO2 with different mutation types were obtained. Figure 4As shown, overexpression lines BnaC04.GS1;1 The relative expression levels of the knockout strains KO1 and KO2 were significantly higher than those of the wild type WT, and three strains, OE1, OE2, and OE7, were subsequently selected for testing. The mutation type of the knockout strain KO1 was 1 base inserted in the first target and 6 bases missing in the second target, and the mutation type of the knockout strain KO2 was 1 base missing in the first target and 1 base missing in the second target, all of which resulted in the premature termination of their coding protein sequences to varying degrees.

[0038] The wild type (WT), overexpression lines (OE1, OE2, OE7) and mutant lines (KO1, KO2) were cultured under nitrogen deficiency. BnaC04.GS1;1 The expression level of GS was measured, and then the GS enzyme activity, ammonium ion content, free amino acid content and soluble protein in each part of the wild type (WT), overexpression (OE1, OE2, OE7) and mutant (KO1, KO2) were measured, as follows: 1. GS enzyme activity determination method: First, prepare the reagents required for the reaction, including 1. Extraction buffer (0.05 mol / L pH 8.0 Tris-HCl buffer containing 2 mmol / L Mg 2+ , 2 mmol / L DTT and 0.4 mol / L sucrose): Accurately weigh 1.5295 g Tris (trishydroxymethylaminomethane), 0.1245 g MgSO4•7H2O, 0.1543 g DTT (dithiothreitol) and 34.25 g sucrose, dissolve them in deionized water, adjust the pH to 8.0 with 0.05 mol / L HCl, and finally make up to 250 mL. 2. Preparation of reaction mixture A (0.1 mol / L Tris-HCl buffer, pH=7.2) (containing 80 mmol / L Mg 2+ , 20 mmol / L L-glutamic acid, 20 mmol / L cysteine ​​and 2 mmol / L EGTA): Weigh 3.0590 g Tris, 4.9795 g MgSO4, 0.8628 g L-glutamic acid, 0.6057 g cysteine ​​and 0.1920 g EGTA, dissolve them in 200 mL distilled water, adjust the pH to 7.4 with 0.1 mol / L HCl, and finally adjust the volume to 250 mL. 3. Preparation of reaction mixture B (containing 80 mmol / L hydroxylamine hydrochloride, pH = 7.2): containing 80 mmol / L Mg 2+, 20 mmol / L L-glutamic acid, 20 mmol / L cysteine ​​and 2 mmol / L EGTA): Weigh 3.0590 g Tris, 4.9795 g MgSO4, 0.8628 g L-glutamic acid, 0.6057 g cysteine, 0.1920 g EGTA and 1.39 g hydroxylamine, dissolve them in 200 mL distilled water, adjust the pH to 7.4 with 0.1 mol / L HCl, and make up to 250 mL. 3. Color developer (mixture of 0.2 mol / L TCA, 0.37 mol / L FeCl3 and 0.6 mol / L HCl): Accurately weigh 3.3176 g TCA (trichloroacetic acid) and 10.1021 g FeCl3•6H2O, dissolve them in deionized water, add 5 mL concentrated hydrochloric acid, and make up to 100 mL. 4.40 mmol / L ATP solution: Weigh 0.1210g ATP and dissolve it in 5mL distilled water (prepare before use). Extraction of crude enzyme solution: Weigh 1g of plant material in a mortar, add 3mL extraction buffer, grind it into a homogenate on an ice bath, transfer it to a centrifuge tube, centrifuge it at 15,000g for 20 min at 4°C, and the supernatant is the crude enzyme solution. Reaction: Take 1.6mL of reaction mixture B in a centrifuge tube, add 0.7mL crude enzyme solution and 0.7mL ATP solution, mix well and incubate at 37℃ for 30 min. After the reaction is completed, add 1mL color developer immediately to terminate the reaction. Shake well, leave it at room temperature for 5 min, centrifuge it at 5000g for 10min at room temperature, take the supernatant and measure the absorbance at 540 nm, and add 1.6mL reaction mixture A as a control. The rest is the same as the experimental group. Result calculation: GS enzyme activity = A / (P*V*T), where A: absorbance at 540nm; P: soluble protein content in enzyme solution (mg / mL); V: volume of crude enzyme solution added to the reaction system (mL); T: reaction time (h).

[0039] 2. Determination of ammonium ion content: Weigh 0.1g old leaves and new leaves in 10ml centrifuge tubes, add steel balls, freeze in liquid nitrogen for 20min, break in tissue grinder, extract with 5ml 0.1% dilute hydrochloric acid for half an hour, filter, take 200ul of supernatant, dilute to 3ml with 0.1% dilute hydrochloric acid, add 0.5ml phenol solution and 0.5ml sodium hypochlorite alkaline solution, shake well, water bath at 37℃ for 30min, and perform colorimetry at 625nm. Prepare 0, 0.125, 0.25, 0.5, 0.75, 1, 1.25 ug / mL ammonium nitrogen standard solutions, take 200ul of the above ammonium nitrogen standard solutions, dilute to 3ml with 0.1% dilute hydrochloric acid, add 0.5ml phenol solution and 0.5ml sodium hypochlorite alkaline solution, shake well, water bath at 37℃ for 30min, and perform colorimetry at 625nm. According to the content of ammonium nitrogen standard solution (ug / mL) and the corresponding absorbance value, a linear equation Y=aX+b is established, that is, Y=content of ammonium nitrogen standard solution (ug / mL), X=absorbance value, and the standard curve formula is obtained. According to the absorbance value of the sample, the colorimetric solution content of the sample on the standard curve is obtained, that is, the ammonium nitrogen content (ug / mL), and the ammonium ion content of each sample is calculated. Calculation formula: Sample nitrogen content (ug / g) = C*(V / a) / W*14 / 18; C: colorimetric solution content (ug / mL) checked from the mark; V: total volume of sample digestion solution (mL); a: the amount of sample nitration dilution used in colorimetry; W: sample weight (g).

[0040] 3. Determination of free amino acid content: Prepare acetic acid-sodium acetate buffer (pH=5.4): weigh 54.4g of sodium acetate and put it into a 100ml beaker, add ammonia-free distilled water to 50ml mark, heat and stir in a water bath to dissolve. After cooling, transfer to a 100ml volumetric flask, add 30ml of glacial acetic acid, and then dilute to 100ml with ammonia-free distilled water; hydrated ninhydrin solution: weigh 3.0g of recrystallized ninhydrin and put it into a beaker, add 75ml of n-propanol, and stir to dissolve. Then add 150ml of n-butanol and 300ml of ethylene glycol, and finally add 45ml of acetic acid-sodium acetate buffer with pH=5.4, mix well, store in a brown bottle, and store at 4℃ for use. It is valid within 10 days; standard amino acids: weigh 0.0025g of sodium glutamate, dissolve it in distilled water and make up to 50ml, and store it in a refrigerator at -20℃, that is, the mother solution. Take 5mL of the mother solution and make up to 50ml with distilled water, which is the standard solution of 5μg / ml amino acids; 0.1% ascorbic acid: weigh 50mg of ascorbic acid, dissolve it in 50ml of ammonia-free distilled water, and use it as soon as it is prepared; 10% acetic acid (V / V), take 10mL of anhydrous acetic acid to 60mL of distilled water, dilute and make up to 100mL; 60% ethanol (V / V), take 60mL of anhydrous ethanol and make up to 100mL with distilled water. Sample preparation: Weigh 0.1 g of old leaves and new leaves respectively, put them into a 2 mL EP tube, add steel beads, freeze them quickly with liquid nitrogen, and then grind them thoroughly with a tissue grinder. Then add 1 ml of 10% acetic acid, grind them at low temperature (no need to freeze them quickly with liquid nitrogen, just put the sample grinder and samples on ice), and continue to grind them into a homogenate with a tissue grinder. Centrifuge them at 12,000 g, 4°C for 10 min, and take the supernatant for determination.

[0041] Prepare standard curve: Take 6 1.5ml EP tubes and operate according to Table 1.

[0042] Table 1 Reagents and dosages for preparing standard curve

[0043] Test steps: Take 10μL of standard solution, add 190μL of deionized water, 300μL of hydrated ninhydrin, and 10μL of 0.1% ascorbic acid in sequence, mix well after adding the reagents, cover tightly, sew the plastic wrap tightly, heat in boiling water for 15 minutes, take out and quickly cool with cold water, so that the red color formed during heating is gradually oxidized by the air and fades away, and then turns blue-purple, turn the EP tube upside down several times after cooling, and add 490μL of 60% ethanol. After turning the EP tube upside down several times to mix thoroughly, take the supernatant and measure the absorbance at a wavelength of 570nm to draw a standard curve. According to the amino acid standard solution content (ug) and the corresponding absorbance value, establish the linear equation Y=aX+b, that is, Y=amino acid standard solution content (ug), X=absorbance value, obtain the standard curve formula, and check the colorimetric solution content of the sample on the standard curve according to the absorbance value of the sample, that is, the amino acid content (ug), and calculate the amino acid content of each sample.

[0044] The sample measurement operation was carried out according to Table 2.

[0045] Table 2 Sample measurement operation

[0046] After adding the reagent, mix well, cover tightly, seal with plastic wrap, heat in boiling water for 15 minutes, take out and quickly cool with cold water, so that the red color formed during heating is gradually oxidized by the air and fades away, and then turns blue-purple, invert the EP tube several times after cooling, and add 490μL 60% ethanol. Invert the EP tube several times to mix thoroughly, take the supernatant and measure the absorbance at a wavelength of 570nm. Record A test tube and A blank tube respectively, and calculate ΔA=A test tube-A blank tube Calculation: Amino acid content (μg / g) = C*(V1 / V2) / W Where: C is the amino acid content found on the standard curve (μg); V1 is the volume of the sample extract, 1 mL; V2 is the sample volume during measurement, 0.01 mL, ml; W is the fresh weight of the sample, g.

[0047] 4. Soluble protein determination method: weigh 0.1g of fresh sample, add steel beads, freeze in liquid nitrogen for 20min, break on a tissue grinder, add 1mL extraction buffer to grind into homogenate, centrifuge at 3000g for 10min at room temperature, and keep the supernatant for later use. Take 100μL of sample extract in a 2mL centrifuge tube, add 1mL Coomassie Brilliant Blue reagent, shake well, place for 2min, and then colorimetric at 595nm, measure absorbance, and determine the protein content through the standard curve.

[0048] like Figure 5 As shown, under low nitrogen and normal nitrogen culture conditions, overexpression BnaC04.GS1;1It promotes the growth of rapeseed, and its above-ground parts grow more vigorously and have more siliques, while the mutant strains grow relatively weakly and have fewer siliques.

[0049] Depend on Figure 6 It can be seen that in the nutrient solution culture test, as the low nitrogen treatment time increases, the free ammonium content and soluble protein content in the old leaves of the overexpression strain are always lower than those of the wild type WT, while the amino acid content and soluble protein in the new leaves gradually accumulate, and the free ammonium content in the new leaves is significantly lower than that of the wild type WT. The free ammonium content, amino acid content and soluble protein in the old and new leaves of the mutant are opposite to those of the overexpression strain. On the 6th and 10th days of low nitrogen, the GS enzyme activity in the old and new leaves of the overexpression strain was significantly higher than that of the wild type WT, while the GS activity in the new leaves of the mutant was significantly lower than that of the wild type WT, and after the 10th day of low nitrogen, the GS enzyme activity in the old leaves of the mutant decreased. The above results show that the rapeseed glutamine synthetase gene of the present invention BnaC04.GS1;1 Through its glutamine synthetase activity, it regulates the transfer of organic nitrogen from old leaves to new leaves under low nitrogen conditions, thereby improving the plant's low nitrogen tolerance.

[0050] At maturity, wild-type WT, BnaC04.GS1;1 The mutants (KO1, KO2) and overexpression lines (OE1, OE2, OE7) were analyzed for total nitrogen and yield-related traits.

[0051] The results are as follows Figure 7-Figure 9 As shown in the figure, under low nitrogen and normal nitrogen conditions, the total nitrogen content of the overexpressed strains was significantly higher than that of the wild type WT, about 60% higher than that of the wild type WT, while the mutants were the opposite, significantly lower than the wild type WT, about 30%-40% lower; under low nitrogen and normal nitrogen conditions, the nitrogen harvest index of the overexpressed strains was significantly higher than that of the wild type WT, while mutation of the gene only showed a decreasing trend of the nitrogen harvest index under low nitrogen conditions ( Figure 7 ). Under two nitrogen levels, the grain yield per plant of the overexpression strain was significantly higher than that of the wild type WT, increasing by about 30% and 60% respectively, while that of the mutant was significantly lower than that of the wild type WT, decreasing by about 35% and 25% respectively. In addition, the aboveground biomass of the overexpression strain was significantly higher than that of the wild type WT, while the opposite was true for the mutant ( Figure 8 ). In addition, the harvest index of the mutant strain was significantly lower than that of the wild type WT at both nitrogen levels, while that of the overexpression strain was significantly higher than that of the wild type WT under normal nitrogen conditions ( Fig. 9 These results indicate that overexpression BnaC04.GS1;1The gene can promote the accumulation of total nitrogen in the aboveground part and increase the nitrogen harvest index, thereby effectively increasing the grain yield and harvest index. The mutation of this gene will reduce the plant's nitrogen absorption, especially the nitrogen harvest index under low nitrogen conditions, which will have an adverse effect on rapeseed, resulting in a decrease in yield and a decrease in the harvest index. The values ​​are the mean ± standard deviation of three repeated experiments, and the P value is a statistical test for significant differences. Student's t-test analysis *P<0.05, **P<0.01.

[0052] The above describes the rapeseed glutamine synthetase of this embodiment. BnaC04.GS1;1 It plays a positive role in increasing rapeseed yield and provides genes and germplasm resources for further improving rapeseed yield through genetic breeding.

Claims

1. A rapeseed glutamine synthetase gene BnaC04.GS1;1 , characterized in that, The nucleotide sequence thereof is shown in SEQ ID NO:

1.

2. A rapeseed glutamine synthetase gene according to claim 1 BnaC04.GS1;1 The glutamine synthetase encoded by Its amino acid sequence is shown in SEQ ID NO:

2.

3. A rapeseed glutamine synthetase gene as claimed in claim 1 BnaC04.GS1;1 Or use of the glutamine synthetase as claimed in claim 2 in cultivating high-yield rapeseed.

4. The use according to claim 3, characterized in that The application specifically includes: amplifying the rapeseed glutamine synthetase gene by PCR BnaC04.GS1;1 The CDS sequence was then cloned into the expression vector by homologous recombination to obtain a gene containing BnaC04.GS1;1 The target vector is then transformed into Agrobacterium by heat shock transformation, and finally cloned into rapeseed by genetic transformation for expression to obtain a BnaC04.GS1;1 The overexpression strain is a high-yield rapeseed; the amplification primer used in the PCR amplification is OE:: BnaC04.GS1;1 The sequences of the forward primer and the reverse primer are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

5. The use according to claim 4, characterized in that The expression vector is P1300.

6. A rapeseed glutamine synthetase gene for use in claim 1 BnaC04.GS1;1 The amplification primer is characterized in that The amplification primers are OE:: BnaC04.GS1;1 The sequences of the forward primer and the reverse primer are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

7. A rapeseed glutamine synthetase gene for use in claim 1 BnaC04.GS1;1 The amplification primer is characterized in that The amplification primer is G:: BnaC04.GS1;1 The sequences of the forward primer and the reverse primer are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

8. A rapeseed glutamine synthetase gene for use in claim 1 BnaC04.GS1;1 The amplification primer is characterized in that The amplification primers are GUS:: BnaC04.GS1;1 The sequences of the forward primer and the reverse primer are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

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