Application of rapeseed BnNRT2.3-like gene and its expression vector in promoting plant nitrogen utilization

Abstract

The present invention belongs to the technical field of plant genetic engineering, and discloses the application of rapeseed BnNRT2.3-like gene and its expression vector in promoting plant nitrogen utilization, wherein the sequence of the BnNRT2.3-like gene is shown in SEQ ID NO.1 and the protein sequence encoded by it is shown in SEQ ID NO.2. Increasing the expression of BnNRT2.3-like in Brassica napus by genetic engineering can promote the absorption of nitrate nitrogen by rapeseed, increase the activity of glutamine synthetase and the chlorophyll content of plants, and make transgenic rapeseed have a larger biomass and be more resistant to sclerotinia; and the gene BnNRT2.3-like plays an important role in the signal transduction pathway of plant response to low nitrogen stress, and overexpression can improve the low nitrogen tolerance of rapeseed. Therefore, BnNRT2.3-like has important application prospects in enhancing the nitrogen utilization rate of Brassica napus, increasing rapeseed oil yield and enhancing the resistance of rapeseed to sclerotinia.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to the isolation and cloning of a nitrate transporter gene BnNRT2.3-like in Brassica napus, the construction of an expression vector and the application of the gene in improving the nitrogen utilization efficiency of plants. Background Art

[0002] Rapeseed has a large demand for nitrogen. If it lacks nitrogen, its growth will be affected, resulting in short plants, yellowing of old leaves, and even reduced yields. At present, the high yield of rapeseed is too dependent on the large-scale application of nitrogen fertilizers, but excessive application of nitrogen fertilizers will not only waste resources, but also reduce the nitrogen utilization rate of rapeseed and its ability to resist diseases and pests. Therefore, improving the nitrogen utilization efficiency of rapeseed is of great practical significance.

[0003] The main nitrogen source for higher plants is nitrate (NO3 - ) and ammonium salts (NH4 + ), usually plants growing in flooded soils prefer NH4 + , terrestrial plants prefer NO3 - Therefore, rapeseed is generally considered to be a nitrate-loving crop. After being absorbed in the roots, nitrate can be transported to the aboveground tissues and organs through the xylem, and then assimilated into amino acids and other nitrogen-containing compounds in plant cells with the participation of multiple enzymes such as nitrate reductase (NR), nitrite reductase (NiR), and glutamine synthetase (GS). Nitrate not only regulates plant growth and development as a nutrient, but also regulates gene transcription as a signal substance, thereby affecting seed germination, plant root growth, stomatal activity, etc. Studies have shown that increasing nitrate can increase the concentration of cytokinins in plant roots, promote plant growth and development, and at the same time affect the transportation of other nutrients in the body and promote the growth of plant roots.

[0004] Nitrate transporters are required for the absorption of nitrate nitrogen by plant roots from the soil, the transport of nitrate nitrogen in plants, and the redistribution of nitrate nitrogen in cells. The main nitrate transporters are the NRT1 family responsible for the low-affinity (LATS) nitrate transport system and the NRT2 family responsible for the high-affinity (HATS) nitrate transport system. NRT2 exists in plants as a gene family. The number of members of the plant NRT2 family is generally small, such as 5 in rice, 7 in Arabidopsis, and 17 in rapeseed. Among them, the NRT2 genes in rice and Arabidopsis have been studied more. OsNRT2.1 and OsNRT2.2 genes are located on rice chromosome 2 and are clustered into a subclass; OsNRT2.3 and OsNRT2.4 genes are located on rice chromosome 1 and are also clustered into a subclass. OsNRT2.1 and OsNRT2.2 genes encode the same amino acids and are both induced by nitrate. The expression of OsNRT2.1 increased with the increase of nitrate absorption, indicating that OsNRT2.1 is a key gene for rice to absorb nitrate. When rice plants were treated with ammonium salt, it was found that the expression of OsNRT2.2 was not inhibited, indicating that OsNRT2.2 can still be transcribed and expressed in flooded soil and is an important gene that can improve the nitrogen utilization efficiency of rice. OsNRT2.3 has two transcripts, OsNRT2.3a and OsNRT2.3b. Tang Zhong et al. found that OsNRT2.3a is located on the plasma membrane and is mainly expressed in the root xylem parenchyma cells. Under low nitrogen conditions, OsNRT2.3a affects the loading of nitrate in the xylem and thus affects the growth of the plant. It is speculated that OsNRT2.3a can promote the long-distance transport of nitrate nitrogen from roots to stems under low nitrogen levels. Fan Xiaorong et al. systematically studied the OsNRT2.3b transporter and found that the OsNRT2.3b gene is also located on the plasma membrane and is mainly expressed in the phloem. It can regulate nitrate transport activity by sensing changes in external pH. Overexpression of the OsNRT2.3b gene can promote rice's absorption of N and significantly improve rice yield and nitrogen use efficiency. Wei et al. found that OsNRT2.4 is a dual-affinity nitrate transporter that can regulate rice root development and affect the distribution of nitrate in rice. In Arabidopsis, Filleur et al. isolated the ANRT2.1 gene from a TDNA mutant. Both AtNRT2.1 and AtNRT2.2 genes are located on chromosome 1 of Arabidopsis and are responsible for the absorption of nitrate in Arabidopsis; AtNRT2.4 and AtNRT2.5 genes are involved in the absorption of nitrate only when the plant is nitrogen starved; AtNRT2.5 is one of the main proteins involved in high-affinity nitrate transport, and AtNRT2.5 is highly induced when Arabidopsis is nitrogen-deficient for a long time.The spatial expression patterns of AtNRT2.1, AtNRT2.4 and AtNRT2.5 genes are different. AtNRT2.1 is mainly expressed in the more mature parts of the taproot, AtNRT2.4 is mainly expressed in the younger parts of the taproot and the ends of the lateral roots, and AtNRT2.5 is mainly expressed in the root hair regions of the taproot and lateral roots. In addition, AtNRT2.4 and AtNRT2.5 are also expressed in the phloem of the stem, affecting the nitrate content of the phloem in the stem. AtNRT2.7 is mainly expressed in the grains and is the only gene in the NRT2 gene family that is located on the tonoplast. It is mainly responsible for the transport of nitrate in the vacuole, regulating the nitrate content in the seeds and controlling seed dormancy. In Brassica napus, Tang et al. identified 17 members of the NRT2 gene family in detail, most of which were upregulated by N starvation, which is very similar to the expression pattern of Arabidopsis NRT2. BnNRT2.1s, BnNRT2.2a and BnNRT2.4a are all specifically expressed in root tissues, while BnNRT2.7a and BnNRT2.7b are mainly expressed in the aerial parts, and BnNRT2.5s is expressed in both stem and root tissues. It can be found that the functions of the cloned NRT2 genes in Arabidopsis and rice have been studied more comprehensively, while the NRT2 genes in rapeseed have been identified, but the functions of most of them have not been systematically studied. Summary of the invention

[0005] In view of this, the present invention has isolated and cloned a novel compound from Brassica napus that can promote plants, especially rapeseed, to absorb and utilize NO3 - The high-affinity nitrate transporter gene BnNRT2.3-like plays an important role in the signal transduction pathway of plant response, especially rapeseed to low nitrogen stress. Overexpression of this gene can improve rapeseed's tolerance to low nitrogen, so it has great economic value in the efficient molecular breeding of plant nitrogen nutrition.

[0006] The technical solution of the present invention is as follows:

[0007] The inventors analyzed the reported functions of Arabidopsis high-affinity nitrate transporters in improving plant nitrogen efficiency and found that AtNRT2.3 plays an important role in plant nitrogen utilization, but the functions of homologous genes in rapeseed have not been reported.

[0008] The present invention discovered a rapeseed BnNRT2.3-like gene that can promote plant nitrogen absorption and utilization from the Brassica napus Westar variety through a large number of sequence comparison analyses. The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0009] The present invention further constructs a recombinant expression vector based on the BnNRT2.3-like gene sequence.

[0010] In a specific embodiment, the above-mentioned recombinant expression vector contains promoter Double CaMV 35S. The construction method of the expression vector is: using the primer pair shown in SEQ ID NO.5-6 to amplify the Double CaMV 35S fragment from the pCAMBIA1300s vector, and then connect it to the EcoRI / HidIII restriction site of the pCAMBIA2301 vector to obtain a recombinant plasmid; using the primer pair shown in SEQ ID NO.7-8 to amplify the BnNRT2.3-like gene, the obtained product and the recombinant plasmid are respectively digested, recovered and connected to obtain a BnNRT2.3-like overexpression vector, which is recorded as pCAMBIA2301-1300-D35s-BnNRT2.3-like-NOS.

[0011] The present invention also provides a method for improving nitrogen utilization of Brassica napus, specifically comprising: connecting the sequence of the BnNRT2.3-like gene into an expression vector to obtain a BnNRT2.3-like overexpression vector; transferring the BnNRT2.3-like overexpression vector into a host bacterium and transforming Brassica napus, screening transgenic plants and identifying their expression levels by qPCR.

[0012] Preferably, the host bacterium is Agrobacterium GV3101.

[0013] Preferably, the method for transforming Brassica napus in the above technical solution is: infecting the hypocotyl of Brassica napus with a bacterial solution of Agrobacterium GV3101 containing a BnNRT2.3-like overexpression vector.

[0014] Preferably, the primer sequences used in the qPCR identification are shown in SEQ ID NOs. 13-14.

[0015] By verifying the transcription level of BnNRT2.3-like gene in positive transgenic seedlings, it was found that the expression of BnNRT2.3-like gene was significantly increased. Further experiments proved that the BnNRT2.3-like gene overexpression strain was significantly more resistant to low nitrogen stress than the wild-type plants, and its taproot length, aboveground dry weight, glutamine synthetase activity, chlorophyll content and leaf nitrate content were significantly higher than those of the wild-type plants; and in potted plants and fields with normal water and fertilizer management, the leaf area of ​​BnNRT2.3-like overexpression plants was significantly higher than that of wild-type plants. Therefore, the recombinant expression vector can be used to promote nitrogen utilization in rapeseed.

[0016] In addition, overexpression of the BnNRT2.3-like gene enhanced rapeseed's resistance to Sclerotinia sclerotiorum, so this gene and its recombinant expression vector are expected to be used to improve rapeseed's resistance to Sclerotinia sclerotiorum or for breeding of varieties resistant to Sclerotinia sclerotiorum.

[0017] The beneficial effects of the present invention are:

[0018] (1) The BnNRT2.3-like gene cloned in the present invention has not been reported before, which enriches the demand for this type of nitrate transporter NRT2 gene in rapeseed, and also provides new genetic resources for nitrogen efficient breeding of other crops, which has a guiding and reference role in improving the nitrogen utilization efficiency of other crops.

[0019] (2) The BnNRT2.3-like overexpression strain obtained by transforming rapeseed with the overexpression vector pCAMBIA2301-1300-D35s-BnNRT2.3-like-NOS constructed by the present invention was studied from the perspective of the functional acquisition of BnNRT2.3-like, which provides raw materials for the study of the nitrate transport function of NRT2.3-like, and is of great significance.

[0020] (3) Through a series of studies on the nitrogen efficiency of BnNRT2.3-like overexpression strains, the present invention fully proves that BnNRT2.3-like is involved in the transport and utilization of nitrogen by plants and plays an important role. By regulating the expression of the BnNRT2.3-like gene, it is possible to regulate the accumulation of nitrogen in plants, increase the activity of nitrogen assimilation-related enzymes and improve rapeseed's resistance to sclerotinia.

[0021] (4) The development and utilization of BnNRT2.3-like will help solve the problems of low nitrogen utilization efficiency and low resistance to sclerotinia in rapeseed production, and help reduce a series of problems caused by excessive use of nitrogen fertilizers. The cloning of the BnNRT2.3-like gene will help breed rapeseed varieties with high nitrogen utilization efficiency and certain disease resistance, and also provide a theoretical and practical basis for the application of BnNRT2.3-like in nitrogen efficiency of major oil crops (rapeseed). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The result of comparing the amino acid sequence encoded by the sequenced rapeseed BnNRT2.3-like gene with the known Arabidopsis thaliana AtNRT2.3-like gene;

[0023] Figure 2 Schematic diagram of the construction process of the plant transformation vector pCAMBIA2301-1300-D35s-BnNRT2.3-like-NOS in Example 2;

[0024] Figure 3Schematic diagram of the Agrobacterium-mediated genetic transformation system of Brassica napus hypocotyls in Example 3;

[0025] Figure 4 This is a diagram showing the identification and expression level analysis of the positive seedlings of the rapeseed BnNRT2.3-like overexpressing transgenic strain in Example 3;

[0026] Figure 5 This is a comparison of the phenotype and nitrogen accumulation of WT and BnNRT2.3-like overexpressing plants after harvest in the field in Example 3;

[0027] Figure 6 Identification of the resistance of the BnNRT2.3-like overexpressing transgenic strain to Sclerotinia sclerotiorum in Example 3;

[0028] Figure 7 This is a comparison of leaves of wild-type and BnNRT2.3-like (OE) plants in Example 3 under normal management in potted seedling stage;

[0029] Figure 8 This is a comparison diagram of the phenotypes of rapeseed plants in Example 4 between the wild type and BnNRT2.3-like (OE) plants after 7 days of hydroponic low nitrogen stress treatment;

[0030] Fig. 9 This is a comparison of plant biomass, taproot length and nitrogen utilization efficiency of the wild type and BnNRT2.3-like (OE) after 7 days of low nitrogen stress treatment in Example 4;

[0031] Fig.10 NR activity, GS activity, nitrate nitrogen content and chlorophyll content in the leaves of wild-type and BnNRT2.3-like (OE) plants 7 days after low nitrogen stress treatment in Example 4. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0033] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0034] Example 1 Isolation and cloning of BnNRT2.3-like gene

[0035] The Brassica napus Westar variety (originally from Canada, a publicly available variety, now preserved and provided by the Rapeseed Research Laboratory of Huazhong Agricultural University) was used as the experimental material. Total RNA from the leaves of Brassica napus Westar was extracted using the Super Total RNA Extraction Kit (purchased from Promega, USA). After RNA extraction, it was treated with DNaseI (purchased from Promega), and RNA integrity was detected by 1.2% (w / v) agarose gel (EtBr) electrophoresis (5V / cm). The concentration of nucleic acids was determined on an IMPLEN NanoPhotometer-N50 series ultra-micro UV spectrophotometer (made in Germany). The RNA 260 / 280 ratio was between 1.9 and 2.1, and RNA with a 260 / 230 ratio greater than 2.0 and a concentration greater than 500 ng / μL was selected for the next step of analysis.

[0036] cDNA is synthesized using II Q RT SuperMix for qRNA (+gDNA wiper) kit (purchased from Vazyme, China). 1 μg of total RNA was used as a template and mixed with 4 μL of 4×gDNA wiper Mix, DEPC-water to a total volume of 16 μL; 2°C, 2 min, placed on ice for 2-3 min; then 5×Hiscript II qRTSuper Mix II 4 μL was added and mixed to a total volume of 20 μL; then 50°C, 15 min, 85°C, 5 sec. Each cDNA was diluted to 200 μL and stored at -20°C for later use.

[0037] The target band was amplified using the TA cloning forward primer BnNRT2.3-like-F (5'-ATGGCTTCTAATGAAGAA-3', SEQ ID NO: 3) and the reverse primer BnNRT2.3-like-R (5'-TTACGGAAACGTGAAACA-3', SEQ ID NO: 4), and amplified using TransTaq HiFi DNA Polymerase (Quanshijin (Beijing) Biotechnology Co., Ltd.), where the PCR reaction conditions were: 94°C pre-denaturation for 3 min; 94°C, 30 sec, 60°C, 30 sec, 72°C, 1 min 10 sec, 32 cycles; 72°C extension for 5 min. The PCR product was then cloned into the pMD18-T vector (purchased from Takara Biotechnology (Dalian) Co., Ltd.). The recovery, connection and transformation of the target fragment were performed with reference to the UNIQ-10 column DNA gel recovery kit (purchased from Sangon Biotechnology (Shanghai) Co., Ltd.). The target fragment and T vector connection system is: 4.5μL target fragment, 0.5μL pMD-18T vector, 5μL SolutionⅠ (purchased from Takara Biotechnology (Dalian) Co., Ltd.) at 16°C overnight connection. The connection product was transformed into DH5α competent state by heat stimulation, and the bacterial solution was applied to LB solid plate containing 100mg / L Amp antibiotic. After about 10-12h growth, single colonies were selected for PCR, and the primers were universal primers M13F / R. The positive colonies were sent to Sangon Biotechnology (Shanghai) Co., Ltd. for sequencing.

[0038] After sequencing, it was found that the nucleotide sequence amplified from the BnNRT2.3-like gene was 1704 bp long (as shown in SEQ ID NO.1), containing an open reading frame with a full length of 1987 bp. The protein sequence of 567 amino acids corresponding to the ORF was determined by BlastX (http: / / www.ncbi.nlm.nih.gov) to be consistent with the BnNRT2.3-like protein. The protein sequence encoded by the gene is shown in SEQ ID NO.2.

[0039] The amino acid sequence was aligned using ClustalX (Thompson JD, Gibson TJ, Plewniak F, et al. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 1997, 25: 4876-82) and it was found that the target band had a 93% homology with Arabidopsis thaliana NRT2.3 at the amino acid level, which was consistent with the results of alignment with the NCBI database ( Figure 1 In the figure, Bn is Brassica napus L., and At is Arabidopsis thaliana).

[0040] Example 2 Construction of plant overexpression vector

[0041] According to the CaMV 35S sequence (Gene ID: AJ007626) published by NCBI, the following primers were designed using Primer Premier 5.0 software:

[0042] 5'-GAATTCTTAATTAAGAGCTCGCATGCC-3' (SEQ ID NO: 5);

[0043] and 5'-GGTACCGTCCCCGTGTTCTCCAA-3' (SEQ ID NO: 6).

[0044] The Double CaMV 35S fragment was amplified from the pCAMBIA1300s vector (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) by PCR using the above primers, and then ligated to the EcoRI / HidIII restriction site of the pCAMBIA2301 vector (from Professor Wang Jing of Huazhong Agricultural University) to finally obtain the final vector pCAMBIA2301-1300s (11634bp).

[0045] SalⅠ / BamHI restriction sites and corresponding protective bases were added to both ends of the BnNRT2.3-like TA cloning amplification primers, wherein the primers are specifically as follows:

[0046] OE-BnNRT2.3-like-F: 5'-TCTCGAGCTTTCGCGAGCTCATGGCTTCTAATGAAGAA-3' (SEQID NO: 7);

[0047] OE-BnNRT2.3-like-R: 5'-AGGTCGACTCTAGAGGATCCTTACGGAAACGTGAAACA-3' (SEQ ID NO: 8).

[0048] The TA-positive clone colony plasmid of BnNRT2.3-like was used as a template for PCR amplification. The length of the obtained PCR product was 1744bp, which was double-digested with SalⅠ / BamHI. The pCAMBIA2301-1300s vector was also double-digested. The digestion reaction system was: 10μg of gene fragment or vector, 2.5μL of SalⅠ, 2.5μL of BamHI, 15μL of 10*M buffer, and ddH20 was added to a total volume of 150μL. After mixing, it was placed at 37℃ for 8-12h. The double-enzyme-cut gene fragment and pCAMBIA2301-1300s plasmid fragment were recovered respectively, and the BnNRT2.3-like double-enzyme-cut gene fragment was ligated with T4 ligase and constructed into the SalⅠ / BamHⅠ restriction site of the pCAMBIA2301-1300s vector, thereby obtaining the Brassica napus BnNRT2.3-like transgenic overexpression vector pCAMBIA2301-1300s-D35s-BnNRT2.3-like-NOS, which contains a kanamycin-resistant gene sequence in its T-DNA region and the promoter of BnNRT2.3-like overexpression is Double CaMV35S.

[0049] The construction process of the above-mentioned overexpression vector and the specific structure of each vector are as follows Figure 2 As shown in the figure, LB is the left border of T-DNA, 35S is the cauliflower virus 35S promoter, MCS is the multiple cloning site, NOS is the terminator, and RB is the right border of T-DNA.

[0050] Example 3 Obtaining transgenic rapeseed plants overexpressing BnNRT2.3-like

[0051] In this example, the recombinant plasmid (i.e., overexpression vector) pCAMBIA2301-1300s-D35s-BnNRT2.3-like-NOS was transferred into Agrobacterium GV3101 by conventional hypocotyl infection method, and then screened and differentiated into seedlings. The specific steps are as follows:

[0052] (1) Sowing: Wash the Brassica napus Westar seeds with 75% alcohol for 1 minute, then wash the seeds with 0.1% mercuric chloride solution for 5 minutes, and finally wash the seeds with sterile water for 5 times. Use sterilized tweezers to place the seeds in M0 solid culture medium, and culture the inoculated seeds in the dark at 24°C for 5 days. The M0 solid culture medium specifically includes: MS inorganic salts and trace elements 4.404 g / L formulated in 1962, adjusted to pH 5.8-5.9, added with 7 g / L agar, and sterilized by conventional high-pressure steam sterilization.

[0053] (2) Activation of Agrobacterium: 3 days after sowing, take the Agrobacterium strain GV3101 stored at -80°C and streak it on LB solid medium, and culture it at 28°C for 6 hours; pick a single colony in 5mL LB liquid medium, and culture it at 28°C, 200rpm, shaking for 20-24 hours to allow the Agrobacterium to grow to the logarithmic phase; take 500 μL of the cultured bacterial solution and expand it in 50mL LB liquid medium for about 12 hours. The LB solid medium contains the antibiotics rifampicin (50 mg / mL), gentamicin (50 mg / mL) and kanamycin (50 mg / mL).

[0054] (3) Preparation of infection solution: Pour the activated Agrobacterium solution into two 50 mL sterile centrifuge tubes, centrifuge at 3500 rpm for 15 min, remove the supernatant, place on ice, and use 1 mL of DM liquid medium (wash the solution, centrifuge and remove the supernatant, add 2-3 mL of DM liquid medium to resuspend the bacteria, and adjust the OD of the infection solution to 0. 600 The value is about 0.6. The preparation method of DM liquid culture medium is as follows: MS inorganic salt (Murashige and Skoog, 1962) and trace elements (Murashige and Skoog, 1962) 4.404g / L, sucrose 30g / L, adjust the pH of the culture medium to 5.8-5.9, sterilize at 121℃ for 20min; after sterilization, add 2,4-D 1mg / L, AS 100mmol / L, KT 0.3mg / L in the sterile operating table.

[0055] (4) Infecting explants: Place the infection solution prepared in step (3) on ice. Use a scalpel to cut the hypocotyls of the dark-cultured Brassica napus seedlings prepared in step (1) (the length of each hypocotyl should preferably be 0.8-1.0 cm). Use sterile tweezers to transfer the cut hypocotyls into a plate containing the infection solution. Infect for 15-30 min (shake every 3 min) depending on the concentration of the solution.

[0056] (5) Co-culture: Transfer the infected explants to a plate containing filter paper (sterilized in advance), dry the infection fluid visible on the surface of the explants, and then transfer them to M1 solid culture medium and culture them in the dark at 24°C for 40 to 48 hours. The M1 solid culture medium specifically contains: MS inorganic salts formulated by Murashige and Skoog in 1962, 4.404 g / L of trace elements, 18 g / L of mannitol, 30 g / L of sucrose, 2 mg / L of 2,4-D, 0.3 mg / L of KT, adjust the pH of the culture medium to 5.8 to 5.9, add 7 g / L of agar, and add 100 mmoL / L of AS after sterilization.

[0057] (6) Screening: The explants after co-cultivation were transferred to M2 solid medium for 20 days of screening culture at 24°C, 16 h light / 8 h dark culture. The M2 solid medium specifically contained MS inorganic salts formulated by Murashige and Skoog in 1962, 4.404 g / L of trace elements, 18 g / L of mannitol, 30 g / L of sucrose, 2 mg / L of 2,4-D, 0.3 mg / L of KT, the pH of the medium was adjusted to 5.8-5.9, and 7 g / L of agar was added.

[0058] (7) Differentiation culture: The screened explants were transferred to M3 solid culture medium and differentiation culture was started, with subculturing every 15-20 days until differentiation and budding (culture conditions were consistent with the screening conditions in step (6)). The M3 solid culture medium specifically comprises: MS inorganic salts formulated by Murashige and Skoog in 1962, 4.404 g / L trace elements, 10 g / L glucose, 0.25 g / L xylose, 0.6 g / L yeast extract (MES), pH of the culture medium was adjusted to 5.8-5.9, 7 g / L agar was added, and after sterilization, 2 mg / L zeatin (ZT), 0.1 mg / L IAA (indoleacetic acid), 250 mg / L TMT reagent, and 25 mg / L Kan were added.

[0059] (8) Rooting culture: When a clear growth point can be found on the seedlings that have differentiated and sprouted, carefully cut off the seedlings with a scalpel at the junction of the callus tissue and the bud (be careful not to damage the growth point during the operation), and then transfer the seedlings to M4 solid culture medium for rooting. Among them, M4 solid culture medium is specifically: MS inorganic salts and trace elements 2.202g, sucrose 10g / L, IBA (indolebutyric acid) 0.5mg / L, adjust the pH of the culture medium to 5.8-5.9, add 7g / L agar, and add 250mg / L TMT and 25mg / L Kan after sterilization.

[0060] The above Agrobacterium-mediated genetic transformation process of Brassica napus hypocotyls is as follows Figure 3 As shown, Figure A shows that the Brassica napus Westar seeds disinfected with 75% alcohol and 0.1% mercuric chloride are sown on the culture medium, Figure B shows that the hypocotyls of the rapeseed infected with Agrobacterium GV3101 containing the transformation vector are placed in the M1 culture medium for co-culture, Figure C shows that the hypocotyls after co-culture are transferred to the M2 culture medium and cultured for 15 to 20 days with a 16-h light / 8-h dark culture cycle, Figure D shows that the explants after selective culture are placed in the M3 culture medium and cultured for more than 15 days with a 16-h light / 8-h dark culture cycle until buds emerge, and Figure E shows that the explants after differentiation culture buds are placed in the fresh M4 culture medium and cultured for more than 15 days with a 16-h light / 8-h dark culture cycle until they differentiate into seedlings.

[0061] The transgenic plants were further identified through the following process:

[0062] ①. The conventional CTAB method was used to extract genomic DNA from Brassica napus leaves. The specific steps were as follows: Take young and tender Brassica napus leaves with a length of 1 to 2 cm, place them in a pre-cooled mortar, add liquid nitrogen 2 to 3 times during the process to grind them into a fine slurry, transfer them to a 1.5 mL centrifuge tube, and add 700 μL of 2x CTAB solution. Incubate at 70°C for 30 minutes, shake gently every 6 minutes, and incubate at 70°C for 30 minutes, shake gently every 10 minutes. Cool to room temperature, add 700 μL of Tris-saturated phenol: chloroform: isoamyl alcohol with a volume ratio of 25:24:1, repeatedly invert and mix, and then shake gently for about 40 times. Centrifuge at 3100 rpm at room temperature for 15 minutes. Take about 500 μL of the supernatant and add an equal volume of chloroform: isoamyl alcohol with a ratio of 24:1. After shaking, centrifuge at 100 rpm at room temperature for 15 minutes. Discard the supernatant, add 1mL of frozen -20℃ anhydrous ethanol, place in an ice bath at -20℃ for 30min, and centrifuge at 12000rpm for 10min at room temperature. Soak and wash with 75% alcohol and repeatedly blow and beat the precipitate for 3min to remove salt. Pour away the alcohol, air dry, and add 30-50μL ddH2O to each sample to dissolve. The extracted rapeseed genomic DNA was tested for concentration using Nanodrop micro-nucleic acid detector.

[0063] ②. Positive transgenic plant detection: Using the extracted DNA as a template, PCR was used to amplify the Kan gene on the vector for positive seedling identification. The primers used were:

[0064] KAN-F: 5'-ACTGGGCACAACAGACAATCG-3' (SEQ ID NO: 9);

[0065] KAN-R: 5'-GCATCAGCCATGATGGATACTTT-3' (SEQ ID NO: 10).

[0066] Test results such as Figure 4 As shown in Figure A: The five transformed plants (OE is the abbreviation of overexpression) OE-1, OE-18, OE-25, OE-36 and OE-50) were able to amplify electrophoresis bands of the expected size (289bp), while the non-transgenic wild-type control had no corresponding electrophoresis band, indicating that the transgenic rapeseed genome already contained exogenous gene DNA fragments.

[0067] ③. qPCR identification of transgenic rapeseed overexpressing BnNRT2.3-like

[0068] a. Extraction of genomic RNA from Brassica napus leaves: using Super total RNA extraction kit (purchased from Promega, USA) was used to extract total RNA from leaves of WT and BnNRT2.3-like overexpressing positive strains identified in ②. RNA was reverse transcribed into cDNA using II Q RT SuperMix for qRNA (+gDNA wiper) kit (purchased from Vazyme, China).

[0069] b. In order to determine whether BnNRT2.3-like is overexpressed in rapeseed, the identified transgenic plants were analyzed using the real-time fluorescence quantitative PCR method. Green Realtime PCR Master Mix-Plus-Kit (Takara Biotech (Dalian) Co., Ltd.), using the rape housekeeping gene BnActin7 (Brassica napusactin-7) as the internal standard gene, and its primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0070] The qPCR primers for BnActin7 (Gene ID: 106418315) are:

[0071] BNACTIN7-F1: 5'-TCTTCCTCACGCTATCCCTCG-3' (SEQ ID NO: 11),

[0072] BNACTIN7-R1: 5'-AGCCGTTCCAGCTCTTGC-3' (SEQ ID NO: 12);

[0073] The QPCR primers for the BNNRT2.3LIKE gene are (the pair of primers are QPCR specific primers designed based on the nucleotide sequence sequenced in Example 1):

[0074] Q-BNNRT2.3-LIKE-F:5'-ATGTTTTTGAGACCGTCTAGCG-3'(SEQ ID NO:13),

[0075] Q-BNNRT2.3-LIKE-R: 5'-GGTTCGCATCAGAGTTCCAG-3' (SEQ ID NO: 14).

[0076] PCR program: pre-denaturation at 95°C for 30 sec, followed by 40 cycles (95°C for 10 sec, 60°C for 10 sec, 72°C for 26 sec).

[0077] Test results such as Figure 4 As shown in Figure B, the BnNRT2.3-like expression levels of OE-18, OE-25, OE-36 and OE-50 strains were significantly higher than the non-transgenic wild type (WT) control, indicating that these strains are independent BnNRT2.3-like overexpressing transgenic strains.

[0078] ④. Field cultivation of transgenic plants

[0079] The identified transgenic plants and wild types were cultivated in the field under the same field water and fertilizer management. When the plants were harvested, the field yield and agronomic traits of BnNRT2.3-like overexpressing rapeseed were recorded, and the effects of BnNRT2.3-like overexpression on plant phenotype and nitrogen accumulation were analyzed.

[0080] The comparison of plant height, number of effective siliques per plant, silique length, grain weight per silique, and grain yield per plant between BnNRT2.3-like overexpressing rapeseed and wild type at harvest is shown in the following table:

[0081]

[0082] As can be seen from the above table, compared with the wild type, the BnNRT2.3-like overexpressing plants had significant increases in plant height, number of effective siliques per plant, silique length, grain weight per silique, and grain yield per plant.

[0083] Figure 5The following are comparisons of plant phenotypes and nitrogen accumulation after harvest, where A shows the phenotypic comparison of WT plants (left) and BnNRT2.3-like overexpressing plants (right) after harvest at maturity, B shows the total biomass of WT and BnNRT2.3-like overexpressing plants after harvest, and C shows the nitrogen accumulation of the aboveground parts of WT and BnNRT2.3-like overexpressing plants after harvest. This shows that under the same field water and fertilizer management, the growth of BnNRT2.3-like overexpressing plants at maturity is significantly better than that of the wild type and the total biomass is significantly increased, but the nitrogen accumulation of the aboveground parts increases but not significantly.

[0084] ⑤. Identification of resistance of BnNRT2.3-like overexpressing transgenic rapeseed to Sclerotinia sclerotiorum

[0085] The seedling leaves of WT plants and BnNRT2.3-like overexpressing plants were inoculated with Sclerotinia sclerotiorum in vitro, and the disease phenotype was observed 36 hours after inoculation. Figure 6 Middle A) and lesion size ( Figure 6 (Fig. B).

[0086] In the figure, WT+S. sclerotiorum and BnNRT2.3-like (OE)+S. sclerotiorum respectively represent the inoculation of S. sclerotiorum with wild type and BnNRT2.3-like overexpression strain. Figure 6 It can be seen that 36 hours after inoculation, the mycelial expansion area of ​​the leaves of the BnNRT2.3-like (OE) strain was significantly smaller than that of the control, indicating that after overexpression of BnNRT2.3-like, the plant's resistance to Sclerotinia sclerotiorum was enhanced.

[0087] In addition, it was found that in potted plants and fields under normal water and fertilizer management, the leaf area of ​​BnNRT2.3-like (OE) plants was significantly larger than that of wild-type plants. Figure 7 shown.

[0088] Example 4 Tolerance of BnNRT2.3-like overexpressing transgenic rapeseed to low nitrogen environment

[0089] In this example, the physiological response of the BnNRT2.3-like overexpressing strain in a low nitrogen environment was detected by the following process:

[0090] The seeds of the test materials were dried in the sun for 2 hours, then soaked in pure water for 1 day, and then sown on gauze soaked in pure water. Water was added every day to ensure that the gauze was moistened. About 7-9 days after sowing, seedlings with root lengths that met the requirements were selected and moved into the nutrient solution. Two treatments, low nitrogen stress (-N, 0.3mM N) and normal nitrogen supply (+N, 6mM N), were set for nutrient solution culture, with 5 biological replicates for each treatment. - (Ca(NO3 - )2) was the only nitrogen source, and the macro-elements and trace elements were respectively Afdonine and Anon nutrient solution formula (conventional), and the iron salt was 0.05 mM EDTA-Fe; the specific culture method was referred to Yang Ningmei (Yang Ningmei, Gene expression profile and co-expression network analysis of Brassica napus in response to nitrogen deficiency stress [D], 2019).

[0091] Figure 8 The phenotypes of rapeseed plants of wild-type (WT) and BnNRT2.3-like (OE) plants under hydroponic low-nitrogen stress for 7 days were shown. It was found that the two lower leaves of the wild-type rapeseed had begun to turn yellow and the cotyledons had turned yellow or fallen off at 7 days, while only one leaf of the BnNRT2.3-like overexpression plant showed a yellowing trend, indicating that under low-nitrogen stress, BnNRT2.3-like overexpression can promote taproot elongation and delay leaf yellowing.

[0092] Fig. 9 Figure 1 shows the plant biomass, taproot length and nitrogen use efficiency of the wild type (WT) and BnNRT2.3-like (OE) after 7 days of low nitrogen stress treatment. Figure A shows the taproot length of WT and BnNRT2.3-like (OE) after 7 days of low nitrogen stress treatment, Figure B shows the biomass of WT and BnNRT2.3-like (OE) plants after 7 days of low nitrogen stress treatment, and Figure C shows the nitrogen use efficiency of WT and BnNRT2.3-like (OE) plants after 7 days of low nitrogen stress treatment. Fig. 9 It can be seen that under low nitrogen stress treatment, the biomass and nitrogen utilization efficiency of BnNRT2.3-like (OE) plants were significantly higher than those of WT, and the main root of BnNRT2.3-like (OE) plants was significantly longer than that of WT, indicating that BnNRT2.3-like can promote plant root growth and improve plant nitrogen utilization efficiency in a low nitrogen environment, thereby promoting the absorption and utilization of nitrate nitrogen by plants.

[0093] After 7 days of low nitrogen treatment, fresh functional leaves with consistent parts were taken to extract nitrate reductase and glutamine synthetase and measure the enzyme activities. At the same time, the nitrate content and chlorophyll concentration of the leaves were measured for analysis. Fig.10Figure 2 shows the nitrite reductase (NR) activity, glutamine synthetase (GS) activity, nitrate nitrogen content and chlorophyll content of leaves of wild type (WT) and BnNRT2.3-like (OE) plants after 7 days of low nitrogen stress treatment. Figure A shows the NR activity of leaves of WT and BnNRT2.3-like (OE) plants after 7 days of low nitrogen stress treatment, Figure B shows the GS activity of leaves of WT and BnNRT2.3-like (OE) plants after 7 days of low nitrogen stress treatment, Figure C shows the chlorophyll content of leaves of WT and BnNRT2.3-like (OE) plants after 7 days of low nitrogen stress treatment, and Figure D shows the nitrate nitrogen content of leaves of WT and BnNRT2.3-like (OE) plants after 7 days of low nitrogen stress treatment.

[0094] from Fig.10 It can be seen that the leaves of BnNRT2.3-like (OE) plants after 7 days of low nitrogen treatment had significantly higher nitrate nitrogen content, NR activity, GS activity and chlorophyll content than WT, indicating that overexpression of BnNRT2.3-like can promote the assimilation of nitrate nitrogen by plants under low nitrogen environment, and plays an important role in plant nitrogen assimilation and improving nitrogen utilization efficiency under low nitrogen stress.

[0095] In summary, compared with wild-type plants, BnNRT2.3-like overexpressing plants not only have better growth and significantly increased total biomass, but also overexpression of BnNRT2.3-like genes enhances rapeseed's resistance to Sclerotinia sclerotiorum. Under low nitrogen stress, the taproot length, aboveground dry weight, glutamine synthetase activity, chlorophyll content and leaf nitrate content of BnNRT2.3-like overexpressing plants were significantly higher than those of wild-type plants. It can be seen that overexpression of BnNRT2.3-like genes can not only play an important role in the process of plant absorption and utilization of nitrate nitrogen, but also provide a new idea for improving rapeseed's resistance to Sclerotinia sclerotiorum, which is of great significance for breeding rapeseed nitrogen-efficient lines.

[0096] The above description is a preferred embodiment of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a recombinant expression vector comprising a rapeseed BnNRT2.3-like gene in improving rapeseed resistance to bacterial sclerotinia, characterized in that: The sequence of the BnNRT2.3-like gene is shown in SEQ ID NO.1, and the protein sequence encoded by it is shown in SEQ ID NO.2, and the rapeseed is Brassica napus.

2. The use according to claim 1, characterized in that: The recombinant expression vector contains the promoter DoubleCaMV 35S.

3. The use according to claim 2, characterized in that: The construction method of the recombinant expression vector is as follows: using the primer pair shown in SEQ ID NO.5-6 to amplify the Double CaMV 35S fragment from the pCAMBIA1300s vector, and then connecting it to the EcoRI / HidIII restriction site of the pCAMBIA2301 vector to obtain a recombinant plasmid; using the primer pair shown in SEQ ID NO.7-8 to amplify the BnNRT2.3-like gene, and the obtained product and the recombinant plasmid are respectively digested, recovered, and then connected to obtain the obtained product.

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