A nitrogen-efficient fusion gene SA and its application

Abstract

A nitrogen-efficient fusion gene SA and its application. In the present invention, a fusion gene named SA was constructed by artificially splicing a "source" organ-specific promoter and a key gene of autophagy, and its sequence is shown in SEQ ID NO.1. A plant binary expression vector can be constructed using the SA fusion gene and transformed into a target plant. An example in soybean is provided in the present invention, demonstrating that after the SA fusion gene is transferred into soybean, the efficiency of nutrient remobilization mediated by autophagy in the "source" organs of transgenic soybean can be specifically improved, and the tolerance of transgenic soybean to low nitrogen and low nutrient stresses is significantly enhanced; meanwhile, the yield of transgenic soybean planted under low nitrogen conditions is significantly increased. In addition, this fusion gene can also be transferred into other crops to obtain transgenic new varieties with efficient utilization of nitrogen, which is of great significance for reducing chemical fertilizer application, lowering production costs, and environmental protection in agriculture.

Description

Technical field

[0001] This invention belongs to the field of plant genetic engineering, specifically involving the artificial splicing of a "source" organ-specific promoter and a key gene for autophagy to construct a fusion gene, SA, and its application in transgenic plants. Transgenic plants harboring this fusion gene exhibit improved nitrogen utilization efficiency and yield.

Background technology

[0002] As the world's population continues to grow, the demand for food is also increasing. However, arable land is limited. Therefore, increasing crop yields is a key approach to averting a food crisis. The common approach to increasing crop yields is to increase fertilizer use, which not only increases production costs but also pollutes the environment. Therefore, breeding new high-yielding crop varieties that can efficiently utilize limited nutrients in the soil is crucial to addressing the food crisis.

[0003] Nitrogen is one of the three essential nutrients for plants. Crops require large amounts of nitrogen from the soil during their growth, and nitrogen availability directly controls crop quality and yield. Plants utilize nitrogen through several steps: absorption, assimilation, translocation, and remobilization (Masclaux-Daubresse et al., 2010). Due to factors such as decreased root absorption capacity, nitrogen fertilizer application cannot fully meet the high nitrogen demand of crops during the grain filling period. Increasing grain yield depends not only on nitrogen fertilizer uptake but also on nitrogen reuse during seed maturation (Kichey et al., 2007). Improving nitrogen reuse efficiency ensures that plants reuse nitrogen from their vegetative organs for grain filling, improving plant nitrogen economy and reducing exogenous nitrogen demand after flowering. Nutrient recycling efficiency is a key determinant of crop yield.

[0004] As the most important vegetative organ of a plant, leaves are both the primary photosynthetic organ and the primary "source" for nutrient remobilization after the onset of senescence. During leaf senescence, major organelles and intracellular macromolecules degrade, allowing for a continuous outflow of nutrients to support the growth and development of new organs. Seeds serve as the primary "sink" for this remobilization. Up to 95% of protein synthesis in crop seeds depends on the transport of amino acids formed by protein degradation in senescent leaves to the seeds (Taylor et al., 2010). The efficient mobilization and transport of nitrogen from "source" to "sink" is crucial for the development of crop yield traits and the achievement of "low-fertilizer, high-yield" strategies (Sinclair et al., 2003; Sinclair et al., 2004; Ainsworth et al., 2012).

[0005] Plant cell autophagy is the main form of nutrient remobilization and early transport during leaf senescence, and autophagy is the most efficient subcellular degradation pathway known to date (Mizushima et al., 2008). In this pathway, discarded or damaged proteins and organelles are isolated into autophagosomes and then transported to the vacuole for degradation, thereby achieving the removal of harmful substances in the cell and the recycling of nutrients, and realizing the rational utilization and effective distribution of nitrogen at the cellular level and the whole plant level. The latest research shows that autophagy is the main form of nitrogen remobilization in plants under both nitrogen-sufficient and nitrogen-deficient conditions. Utilization 15 Nitrogen remobilization data indicated by N isotope labeling showed that autophagy contributed approximately 50% to nitrogen remobilization (Guiboileau et al., 2012).

[0006] Isolating, identifying, or creating key autophagy genes that are specifically and highly expressed in "source" organs, and applying strategies to improve the nitrogen utilization efficiency and yield of crops by increasing the efficiency of remobilization of nutrients such as nitrogen in the "source" organs of crops have important application prospects in high-yield and high-efficiency crop molecular breeding. However, no relevant research reports have been reported so far. [Summary of the invention]:

[0007] The purpose of the present invention is to solve the problems of low nitrogen utilization efficiency and excessive application of nitrogen fertilizer during the planting process of crops, and to provide a nitrogen-efficient fusion gene and its application that can improve the nitrogen remobilization efficiency in the "source" organs of crops and be used for the cultivation of nitrogen-efficient and high-yield crops.

[0008] The present invention uses molecular biology technology to obtain a fusion gene that drives the expression of a key gene for autophagy driven by a "source" organ-specific promoter, and provides its application in transgenic plants, providing genetic resources and operational strategies for nitrogen-efficient crop breeding. A "source" organ-specific promoter and a key gene for autophagy were cloned separately, and a fusion gene with a length of 2105bp was constructed by artificial splicing, named SA, and its nucleic acid sequence is shown in the sequence SEQ ID NO.1. The present invention transfers the SA fusion gene into the soybean genome, and obtains transgenic soybean lines that specifically overexpress key genes for autophagy in "source" organs such as senescent leaves, thereby improving the remobilization efficiency of macromolecular nutrients in the "source" organs, promoting the nutritional growth of transgenic plants under low nitrogen and low nutrient stress, and ultimately increasing soybean yield, proving that the fusion gene has the potential to improve the tolerance of transgenic crops to low nitrogen and low nutrients and improve yield-related traits.

[0009] The technical solution of the present invention:

[0010] A nitrogen-efficient fusion gene SA, whose nucleic acid sequence is selected from:

[0011] (a) the nucleic acid sequence shown in SEQ ID NO. 1;

[0012] (b) a nucleic acid sequence that is at least 85% homologous to the nucleic acid sequence defined in (a).

[0013] The present invention provides a method for obtaining the fusion gene SA, which is constructed by connecting a "source" organ-specific promoter with a key gene for autophagy through an artificial splicing method.

[0014] The PCR amplification primers for the "source" organ-specific promoter were designed as follows (nucleotide sequences such as SEQ ID NO. 2 and SEQ ID NO. 3, wherein SEQ ID NO. 3 introduces an Nco I cleavage site):

[0015] SEQ ID NO.2: 5'-AAGCTCCTTGGTACCTTTCTCAGGGTAGTG-3'

[0016] SEQ ID NO.3:5'- CCATGG TCATGCTTGCTCTTGCTGTTTTGAT-3'

[0017] The PCR amplification primers were designed for the CDS sequence of the key gene of cell autophagy as follows (SEQ ID NO.4, SEQ ID NO.5, wherein the Nco I restriction site was introduced into SEQ ID NO.4, and the BstP I restriction site was introduced into SEQ ID NO.5):

[0018] SEQ ID NO.4: 5'-ATTTCG CCATGG CCAAAACCTCCTTCAAGCTTC-3'

[0019] SEQ ID NO.5: 5'-CGAGCT GGTCACC TAATGGGATCCGAAGGTGTTCT-3'.

[0020] The steps for constructing the SA fusion gene are as follows:

[0021] First, using primers SEQ ID NO. 2 and SEQ ID NO. 3, soybean genomic DNA was used as a template to amplify the "source" organ-specific promoter portion of the SA fusion gene by PCR, and the amplified product was recovered and TA cloned;

[0022] Second, using primers SEQ ID NO. 4 and SEQ ID NO. 5 and soybean cDNA as template, PCR was used to amplify the key autophagy gene portion of the SA fusion gene, and the amplified product was recovered and TA cloned;

[0023] Third, the TA clone plasmid containing the "source" organ-specific promoter in the fusion gene in step (1) and the TA clone plasmid containing the key gene for autophagy in the fusion gene in step (2) were digested with Hind III / Nco I and Nco I / BstP I, respectively, to recover the nucleic acid fragments;

[0024] Fourth, double-digest the pCAMBIA3301 plasmid with Hind III / BstP I to recover the large vector fragment;

[0025] Fifth, the "source" organ-specific promoter fragment, the cell autophagy key gene fragment and the pCAMBIA3301 vector fragment recovered in step (3) are mixed and ligated under the catalysis of ligase;

[0026] Sixth, the ligation product was transformed into E. coli DH5α competent cells, and the construction of the SA fusion gene on the pCAMBIA3301 vector was obtained through resistance screening.

[0027] The present invention also provides an application of the SA fusion gene, which is used to construct a plant binary expression vector and perform plant transformation, thereby obtaining a transgenic plant whose genome contains the fusion gene nucleic acid sequence described in SEQ ID NO.1 above; that is, the gene is expressed in the transgenic plant to improve the transgenic crop's ability to remobilize nitrogen from the "source" organ.

[0028] One example of the application is the expression of the SA fusion gene in soybeans. This transgenic soybean improves the efficiency of autophagy-mediated nutrient remobilization in the "source" organ and significantly enhances tolerance to low nitrogen and low nutrient stress. Planted in low-nitrogen soil, the soybeans grow more vigorously than non-transformed controls, with larger stem girth, more branches, and higher yield per plant. The specific operation process is as follows:

[0029] Seventh, the pCAMBIA3301 vector containing the SA fusion gene constructed in step 6 was transformed into Agrobacterium LBA4404;

[0030] Eighth, the SA fusion gene was transferred into soybean explants using the Agrobacterium-mediated transformation method at the cotyledonary node (Paz et al., 2006). Resistant buds obtained after selection with 6 mg / L glufosinate were grafted onto wild-type rootstocks and cultured normally (16 h light / 8 h dark, 25°C).

[0031] Ninth, genomic PCR and Southern blot were used to identify the insertion and copy number of exogenous genes, and semi-quantitative RT-PCR was used to detect the expression of SA fusion genes.

[0032] The operation process of the transgenic soybean seedlings obtained after the SA fusion gene transformation and the phenotypic analysis of the seedlings and the determination of physiological indicators is as follows:

[0033] Phenotypic analysis and physiological index determination of SA transgenic soybean seedlings under nitrogen deficiency culture system:

[0034] Tenth, wild-type and transgenic soybean seeds were sterilized and planted in sterilized vermiculite moistened with sterile distilled water, and the seeds were germinated in the dark;

[0035] Eleventh, 4 days after sowing, wild-type and transgenic soybean plants with identical germination were selected and cultured under long-day conditions (16 h light / 8 h dark) at 24°C for 17 days, during which they were continuously watered with distilled water;

[0036] 12. Continuously observe and record the phenotypes of the control and transgenic soybeans. At the same time, sample the transgenic soybeans every two days to measure their plant height, root length, biomass, and total carbon and nitrogen content in cotyledons and primary leaves.

[0037] Phenotypic analysis and physiological index determination of SA transgenic soybean seedlings under low nitrogen culture system:

[0038] Thirteenth, germinate the chlorine-disinfected soybean seeds in sterilized nutrient soil;

[0039] 14. After the primary leaves are fully expanded, select seedlings with the same developmental status, wash them, and transplant them into 10L of sterilized 1 / 20 Hoagland nutrient solution for hydroponics. Replace the 1 / 20 Hoagland nutrient solution every 4 days.

[0040] Fifteenth, the overall and above-ground and underground phenotypes were photographed and physiological indicators such as biomass, root length, and number of lateral branches were measured at 7 days and 24 days after hydroponic treatment.

[0041] Phenotypic observation and physiological index determination of SA transgenic soybean seedlings under low nitrogen culture system:

[0042] 16. Germinate wild-type and transgenic soybean seeds in nitrogen-rich soil;

[0043] 17. After the primary leaves are fully expanded, select materials with consistent growth and transplant them to the low-nitrogen test site. Do not apply fertilizer and manage the site normally.

[0044] 18. Regularly observe the phenotype of field materials and take photos; at harvest time, count the yield trait-related data such as stem girth, number of branches, and grain weight per plant.

[0045] Advantages and positive effects of the present invention:

[0046] The present invention uses a "source" organ-specific promoter and a key gene for cellular autophagy to construct an SA fusion gene through artificial splicing, and then constructs a binary expression vector containing the fusion gene and transforms soybeans. It is demonstrated that the efficiency of autophagy-mediated remobilization of nitrogen and other nutrients in the "source" organ of SA transgenic soybeans is improved, and the tolerance of transgenic soybeans to low nitrogen and low nutrient stress is significantly enhanced. At the same time, multiple yield traits of transgenic soybeans grown under low nitrogen soil conditions are improved, and the final yield of transgenic soybeans is increased. These results show that the introduction of SA fusion genes into major crops can produce new transgenic crop varieties with enhanced tolerance to low nitrogen and low nutrients, improved nitrogen utilization efficiency, and improved yield traits. This will help solve the food crisis, reduce agricultural production costs, and reduce environmental pollution caused by the application of chemical fertilizers, and therefore has important application value.

Description of the drawings

[0047] Figure 1 Molecular identification of SA transgenic soybean lines. A, Genomic PCR. Marker: trans 2k plus DNA molecular weight marker; TL-1, non-transgenic Tianlong No. 1 soybean; SA-1, 3-9: eight SA transgenic soybean lines; +, positive control using the SA construct plasmid as template. B, Southern blot identification. The restriction endonuclease used was EcoRV; hybridization was performed using SA (left) and bar (right) probes, respectively; Marker: digoxigenin-labeled DNA molecular weight marker.

[0048] Figure 2 Phenotypes of SA transgenic soybeans and non-transgenic controls under nitrogen deficiency stress. A, Phenotypes after 4, 7, and 12 days of nitrogen deficiency treatment; B, Belowground biomass at each time point during nitrogen deficiency treatment; C, Total biomass at each time point during nitrogen deficiency treatment. TL-1, non-transgenic Tianlong No. 1 soybean; SA-4 and SA-5, two transgenic SA soybean lines.

[0049] Figure 3 The total nitrogen content changes in cotyledons, primary leaves, first trifoliate leaves, and second trifoliate leaves of SA transgenic soybeans and non-transgenic controls under nitrogen deficiency stress.

[0050] Figure 4 Figure 2 shows the vegetative growth phenotypes of SA transgenic soybeans and non-transgenic controls grown under low-nutrient hydroponic conditions. AC, basic aboveground and belowground phenotypes after 7 days of low-nutrient hydroponic treatment; DH, basic aboveground and belowground phenotypes after 24 days of low-nutrient hydroponic treatment. TL-1, non-transgenic Tianlong No. 1 soybean; SA-4 and SA-5, two transgenic SA soybean lines. Arrows in G indicate lateral branches.

[0051] Figure 5Basic phenotypic and yield-related traits of SA transgenic and non-transgenic soybeans grown in low-nitrogen soils. A, basic phenotype at 122 days of age; B, main stem girth; C, number of primary branches; D, grain weight per plant. TL-1, non-transgenic Tianlong No. 1 soybean; SA, SA transgenic soybean. [Specific implementation]:

[0052] Example 1: Cloning of soybean "source" organ-specific promoters

[0053] The 1800 bp promoter was amplified using soybean genomic DNA as a template by PCR. The amplified product was recovered and cloned by TA.

[0054] (1) PCR amplification of target fragment

[0055] Specific primers were designed based on the soybean "source" organ-specific promoter sequence, the sequences of which are shown in SEQ ID No. 2 and SEQ ID No. 3, wherein the downstream primer introduces the Nco I cutting site.

[0056] Soybean genomic DNA was extracted by the CTAB method, and the genomic DNA was used as a template to perform PCR amplification using the above primers to prepare a gene promoter fragment.

[0057] PCR reaction system:

[0058]

[0059] PCR reaction procedure:

[0060] 94℃ for 5 minutes;

[0061] 94°C for 30 seconds, 58°C for 110 seconds, 25 cycles;

[0062] 72°C for 10 minutes;

[0063] (2) Cloning of target fragments and identification of positive clones

[0064] ① Recovery of target fragments

[0065] The target DNA fragment was recovered by agarose gel electrophoresis using an agarose gel recovery kit purchased from Axygen. Specific steps are described in the product manual.

[0066] ②Connection

[0067] The following reagents were added to the reaction system and reacted at 16°C overnight to achieve the ligation of the target fragment with the pMD-18T vector (purchased from Takara).

[0068]

[0069] ③ Transformation and identification of positive clones

[0070] Prepare competent E. coli DH5α cells using standard CaCl2 induction and transformation methods. Transform the competent cells with 10 μL of the ligation product, spread evenly onto LB plates containing Amp, and incubate inverted at 37°C for 12-14 hours. Single colonies on the transformation plates were selected and the plasmids were extracted using standard methods. Double digestion with Hind III and Nco I yielded a 2.7 kb pMD-18 vector fragment and an 1800 bp promoter fragment. PCR amplification was performed using the plasmid extract as template using the aforementioned PCR primers and amplification conditions. Agarose gel electrophoresis confirmed the presence of an 1800 bp gene promoter fragment, indicating a positive clone containing this promoter sequence.

[0071] ④ Sequencing verification

[0072] The positive clones were identified and sent to Beijing Liuhe BGI Genomics Co., Ltd. for DNA sequencing. The nucleic acid sequence is shown in SEQ ID NO.1.

[0073] Example 2: Cloning of key soybean autophagy genes

[0074] First, using soybean cDNA as a template, the 360bp functional gene was amplified by PCR, and the amplified product was recovered and TA cloned.

[0075] (1) PCR amplification of target fragment

[0076] Specific primers were designed based on the sequences of known soybean autophagy key genes, the sequences of which are shown in SEQ ID No. 4 and SEQ ID No. 5. An Nco I restriction site was introduced into the upstream primer, and a BstP I restriction site was introduced into the downstream primer.

[0077] Glycine max RNA was extracted by Trizol method and reverse transcribed into cDNA. The cDNA was used as template and PCR amplified with the above primers to prepare gene fragments.

[0078] PCR reaction system:

[0079]

[0080] PCR reaction procedure:

[0081] 94℃ for 5 minutes;

[0082] 94°C for 30 seconds, 58°C for 30 seconds, 25 cycles;

[0083] 72°C for 10 minutes;

[0084] (2) Cloning of target fragments and identification of positive clones

[0085] ① Recovery of target fragments

[0086] The target DNA fragment was recovered by agarose gel electrophoresis using an agarose gel recovery kit purchased from Axygen. Specific steps are described in the product manual.

[0087] ②Connection

[0088] The following reagents were added to the reaction system and reacted at 16°C overnight to achieve the ligation of the target fragment with the pMD-18T vector (purchased from Takara).

[0089]

[0090] ③ Transformation and identification of positive clones

[0091] Prepare competent E. coli DH5α cells using standard CaCl2 induction and transformation methods. Transform the competent cells with 10 μL of the ligation product, spread evenly onto LB plates containing Amp, and incubate inverted at 37°C for 12-14 hours. Single colonies on the transformation plates were selected and plasmids were extracted using standard methods. Double digestion with Nco I and BstP I yielded a 2.7 kb pMD-18 vector fragment and a 360 bp fragment of the functional gene. PCR amplification was performed using the plasmid extract as a template using the aforementioned PCR primers and amplification conditions. A positive clone containing the functional gene sequence was identified by agarose gel electrophoresis, yielding a 360 bp fragment.

[0092] ④ Sequencing verification

[0093] The positive clones were identified and sent to Beijing Liuhe BGI Genomics Co., Ltd. for DNA sequencing. The nucleic acid sequence is shown in SEQ ID NO.1.

[0094] Example 3: Construction of SA fusion gene using pCAMBIA3301 vector

[0095] (1) The vector plasmid pCAMBIA3301 (purchased from a reagent company) was extracted from the corresponding Escherichia coli engineering bacteria, and double-digested with Hind III / BstP I to recover the large vector fragment.

[0096] (2) The plasmid was extracted from the TA clone prepared in Example 1, double-digested with Hind III / Nco I, and the promoter fragment was recovered by agarose gel electrophoresis.

[0097] (3) The plasmid was extracted from the TA clone prepared in Example 2, double-digested with Nco I / BstP I, and the functional gene fragment was recovered by agarose gel electrophoresis.

[0098] (4) The three fragments were ligated at 16°C overnight under the catalysis of ligase to complete the construction of the expression vector SA-pCAMBIA3301.

[0099]

[0100] (5) The ligation mixture was used to transform Escherichia coli DH5α competent cells using the same method as in Example 1.

[0101] (6) Single clones on the transformation plate (Kan resistance) were selected, and plasmids were extracted according to conventional methods. The plasmid DNA was double-digested with Hind III and BstP I to produce two fragments, one of which was a 12 kb pCAMBIA3301 vector fragment and the other was a 2.1 kb SA fusion gene fragment.

[0102] (7) PCR reaction was performed using the plasmid as a template, using the same method as in Example 1.

[0103] (8) The positive clones identified by enzyme digestion and PCR were sent to a sequencing company for sequencing.

[0104] (9) Plasmids were extracted from positive clones, and the expression vector SA-pCAMBIA 3301 was transformed into Agrobacterium LBA4404 using conventional methods and used to transform soybean.

[0105] Example 4: Preparation of transgenic soybeans

[0106] (1) The binary expression vector SA-pCAMBIA3301 containing the SA fusion gene constructed in Example 3 was used for the Agrobacterium-mediated transformation of soybean cotyledonary nodes (Paz et al., 2006). Resistant buds obtained by selection with 6 mg / L glufosinate were grafted onto wild-type rootstocks and cultured under normal conditions (16 h light / 8 h dark, 25°C).

[0107] (2) PCR detection of transgenic soybeans: Leaves of transgenic soybeans and non-transgenic soybeans of Tianlong No. 1 were cut and genomic DNA was extracted from the leaves according to the method in the Molecular Cloning Laboratory Manual (3rd Edition) (Huang Peitang et al., 2002). PCR reactions were performed using specific primers. The primer sequences are shown in SEQ ID No. 2 and SEQ ID No. 5.

[0108] The reaction system was the same as in Example 1. The PCR products were subjected to agarose gel electrophoresis. A 2.1 kb SA fusion gene band appeared in the transgenic plants, but not in the non-transgenic plants, indicating that the target fragment had been integrated into the plant genome ( Figure 1 A).

[0109] (3) Identification of the copy number of exogenous gene inserted into transgenic soybeans: Leaves of homozygous lines of SA transgenic soybeans and non-transgenic soybeans of Tianlong No. 1 were taken, and genomic DNA of the leaves was extracted according to the method of Molecular Cloning Experiment Guide (3rd Edition) (Huang Peitang et al., 2002). Southern blot experiments were performed, and the restriction endonuclease used was EcoR V. Specific probes for SA and bar genes were used for hybridization. The test results showed that all 8 SA transgenic soybean lines had exogenous gene insertions, and the copy number of the exogenous gene ranged from 1 to 3 ( Figure 1 B).

[0110] Example 5: Phenotypic analysis and physiological index determination of SA transgenic soybean seedlings under nitrogen deficiency conditions

[0111] (1) Seeds were harvested from individual plants of the transgenic soybean lines obtained in Example 4. After sowing, 100 mg / L glufosinate was applied to the leaves for resistance screening. The genome was extracted and tested by PCR. Positive plants (i.e., T1 generation) were individually transferred to soil for culture. Seeds were harvested from individual plants. The obtained seeds were sown again and screened for resistance with 100 mg / L glufosinate. The lines in which all plants tested positive were considered homozygous (T2 generation).

[0112] (2) After sterilization, homozygous transgenic soybean and non-transgenic control seeds were planted in sterilized vermiculite, watered with distilled water, and allowed to germinate in the dark;

[0113] (3) Select homozygous transgenic soybean and non-transgenic control plants with the same germination and continue to cultivate them in distilled water under long-day conditions (16 h light / 8 h dark) at 25°C. Continuously observe the phenotypes of homozygous transgenic soybean and non-transgenic control plants. Take samples every other day, carefully wash off the vermiculite remaining on the roots, take photos to record the phenotypes, and measure the fresh weight of the aboveground and underground parts. The sum of the two is the total biomass ( Figure 2 ).

[0114] The results showed that under nitrogen deficiency culture conditions, SA transgenic soybean seedlings had obvious advantages in nutritional growth and root development compared with non-transgenic controls, and had higher biomass, indicating that transgenic soybeans have stronger tolerance to nitrogen deficiency stress.

[0115] (4) Measure the total nitrogen content of the cotyledons, primary leaves and trifoliate leaves of each plant sampled every other day in (3) and draw a content change curve ( Figure 3 ).

[0116] The total nitrogen content was determined using a Vario EL / micro cube elemental analyzer produced by the German company elemantar. The determination method was as follows: the plant material with recorded fresh weight was placed in a paper bag and withered at 105°C for 2 hours, then dried at 85°C to constant weight (about 48 hours), the dry weight was recorded, and then ground into powder for determination on the analyzer.

[0117] The results showed that as the nitrogen deficiency treatment time prolonged, the total nitrogen content of cotyledons and leaves of each layer gradually decreased. The total nitrogen content in SA transgenic soybeans decreased faster than that in the non-transgenic controls, while the initial total nitrogen content of new leaves was higher than that in the non-transgenic controls, indicating that each layer of leaves eventually acted as a "source" organ for nitrogen mobilization. The SA transgenic strain had a faster nitrogen mobilization and outward output rate, and its support for new leaves was more significant.

[0118] Example 6: Phenotypic analysis and physiological index determination of SA transgenic soybean seedlings under low nutrient conditions

[0119] (1) Germinating chlorine-disinfected soybean seeds in sterilized nutrient soil;

[0120] (2) After the primary leaves have fully expanded, select seedlings of uniform developmental status, carefully rinse the roots with sterile water, and transplant them into hydroponic tanks containing 10 L of sterilized 1 / 20 Hoagland nutrient solution. Each tank should contain 12 treated plants, including 6 transgenic plants and 6 controls. The hydroponic tanks should be surface-disinfected with 2% sodium hypochlorite solution in advance. The 1 / 20 Hoagland nutrient solution should be replaced every 4 days.

[0121] (3) The overall and aboveground and underground phenotypes were photographed and physiological indicators such as biomass, root length, and number of lateral branches were measured at 7 and 24 days after hydroponic treatment.

[0122] The results showed that under this treatment system, transgenic soybeans showed higher above-ground and underground fresh weight, more developed root system, and more vigorous lateral branch growth ( Figure 4 This suggests that overexpression of the SA fusion gene strengthens support for "sink" organs such as roots and lateral branches, thereby improving the plant's tolerance to low nutrition.

[0123] Example 7: Phenotypic analysis and physiological index determination of SA transgenic soybean seedlings under low nitrogen cultivation conditions

[0124] (1) The homozygous transgenic soybean seeds obtained in Example 5 and the non-transgenic control seeds were germinated in small cups in nitrogen-rich soil.

[0125] (2) After the primary leaves are fully expanded, select materials with consistent growth and transplant them to the low nitrogen test site with a row spacing of 40 cm and a plant spacing of 15 cm. No fertilizer is applied and other management is normal. The soil nitrogen content in the test site is 0.84 ± 0.05 mg g -1 According to the standards of the second national soil survey, it belongs to the fourth level of nitrogen nutrient, which is a relatively low nitrogen level.

[0126] (3) Observe the phenotype and take photos at the end of growth (seedling age 122 days) Figure 5 A).

[0127] The results showed that SA transgenic soybeans grew more vigorously than non-transgenic controls in low-nitrogen soils.

[0128] (4) At harvest, the seeds were analyzed and physiological indicators such as the main stem girth and the number of primary branches were counted; yield traits such as single plant grain weight were counted and data analysis was performed ( Figure 5 BD).

[0129] The results showed that SA transgenic soybeans had a larger stem girth and more primary branches when harvested in low-nitrogen soil, indicating more vigorous vegetative growth; in addition, the single-plant yield of SA transgenic soybeans was significantly higher than that of the non-transgenic control, indicating that SA overexpression has the potential to increase yield.

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[0148] Yan Xiong, Anthony L. Contento and Diane C. Bassham. AtATG18a is required for the formation of autophagosomes during nutrient stress and enescence in Arabidopsis thaliana. The Plant Journal, 2005, 42: 535~546. Sequence Listing <110> Nankai University <120> A nitrogen-efficient fusion gene SA and its application <141> 2018-06-01 <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 2165 <212> DNA <213> Artificial Sequence() <220> <221> promoter <222> (1)..(1805) <223> Soybean "source" organ-specific promoter sequence <220> <221> CDS <222> (1806)..(2165) <223> Key genes for soybean cell autophagy <400> 1 ccttggtacc tttctcaggg tagtgcttct tgatatggcc aaccttttga catttgaaac 60 atcggacttg cttcttggtt tcaggccttg acttcttcca tttaccttct cctttcttac 120 cttttccctt cattgctgct agaccaaatg acacctcctc tgtaatcttt gaatcttgga 180 gtttttggaa ttctttggtt caaattgagt tgaccacctc ctccaaggta attagctgat 240 ctttcccata cagaagagca tctctaaagt gctcgaaggt cttaggtaaa gcatttagaa 300 gaatgacaac tttatcttca tcttctagtt tcactccgat attctaaaga tcatcaatgt 360 tcttgttgaa ttctgcgagt tgttcctcga tcgtccttga ttccgccatc ttgaatgaat 420 agagttgttg tttgagacac aacctatggg tcaaagacct ggtcatgtac agtgattcca 480 actttgccca tatcccagcc gcagttttct cctttgcaat ctcccttagt gccttaccac 540 caagacatag tatgattgca cttcttgcct tgtcaatcat gctcttcttc tctgcatgcg 600 tcaaggttgg cggcatgttc gaatcccctt tcaacgcttc atcacatcct tgttgaatca 660 agattgcttc catcttgatc ttccatagtc cgaagtcatt ggagccagaa aacttctcaa 720 tgttaaactt cgttgccatt tcttgaatga tcttgttaac ctttgcgtta cttcccacat 780 acagcaccac ttgttggttc aagaactgtg atactgcaaa gcaagatcaa aattggaaca 840 aaggatataa tcatccacac acaaggcaca aaactaaatg ttgttattat tcttttggaa 900 tcaatattgg ctatgcacat atactctcac tcaatggcaa acatgcagggg gatatata 960 tatatatata ttcacagtgt aaaagatgt atcatataat aaatttatta actttaaaaa 1020 taattattg aaaaatcaca gtaagttaat gatattact gattagtta aaaaatcaaa 1080 ttttaatgat atacataaaattaactc taattaaaa agtataaaa ccctgttttc 1140 ttccaaaca cttaatatat caatctgat atattaata gaaaccgtgt tatccgcaca 1200 agttgacaat ttatattc tctataacgt ttaaagtttt tataaaaagt tgactaagcc 1260 aactgttaga ggctgccaat caccacttg cagagagtgt gtgtataaaa attaaataag 1320 tagttttgga ttcttgtata ttttcaata atgcttatct cctctaacaa tatgtcttaa 1380 aaattaaaat ccaattgtca tgcattattg tttctttatt cattatata ttcataaaa 1440 ttatacaaat gggcagccac tccggcgttg ccttactt aaacagaaca tacaatagtg 1500 tttatttaat cattatcttt daddy daddy daddy daddy daddy 1560 tcagcacccc ttttgt aacttcgtgt gaacttcac ttattcatt tctcaccata 1620 caattaaag tcatctctg cgttatatct tggctgcca tcatgttttc cccaccaact 1680 ctttcatgcc atatagaatg atagctactt aattatatct ttctccgcag ctactttatt 1740 tcttctcata aattctgagg tttatagtt tagtcatcat CAAACAGCA agAGCAAGCA 1800 tgacc atg gcc aaa acc tcc tc aag ctt cag cat cct tg gag aga Met Ala Lys Thr Ser Phe Lys Leu Gln His Pro Leu Glu Arg 1 5 10 agg cag gct gaa gct tct cgc att aga gag aaa tat cct gat aga ata 1895 Arg Gln with Glu Alone Ser with Arg Glu Lys Tyr Pro Asp with Arg 15 20 25 30 Cct Gtg Att Gtg Gag Aaa GCT Gaa Aga Agt Gac Att Ccca Gac Att Gat Gat 1943 Pro Val Ile Val Glu Lys Ala Glu Arg Ser Asp Ile Pro Asp Ile Asp 35 40 45 aag aaa aaa tac ctt gtc cct gct gat tg act gtt ggc cag ttt gtt 1991 Lys Lys Lys Tyr Leu Val Pro Ala Asp Leu Thr Val Gly Gln Phe Val 50 55 60 tat gtt gtt cgc aaa agg att aag ctc agt gca gag aag gct att ttt 2039 Tyr Val Val Arg Lys Arg Ile Lys Leu Ser Ala Glu Lys Ala Ile Phe 65 70 75 gtt ttc atc aac aac act cta cct cca act gct gca ttg atg tct gct 2087 Val Phe Ile Asn Asn Thr Leu Pro Pro Thr Ala Ala Leu Met Ser Ala 80 85 90 att tat gag gaa aat aag gat caa gat ggc ttt ctt tac atg act tac 2135 Ile Tyr Glu Glu Asn Lys Asp Gln Asp Gly Phe Leu Tyr Met Thr Tyr 95 100​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ccatggtcat gcttgctctt gctgttttga t 31 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence() <220> <400> 4 atttcgccat ggccaaaacc tccttcaagc ttc 33 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence() <220> <400> 5 cgagctggtc acctaatggg atccgaaggt gttct 35

Claims

1. A nitrogen-efficient fusion gene SA, whose nucleic acid sequence is: The nucleic acid sequence shown in SEQ ID NO.1; The fusion gene SA is a fusion gene constructed by ligating a source organ-specific promoter and a key autophagy gene through an artificial splicing method. The specific operation steps are as follows: (1) Using primers SEQ ID NO. 2 and SEQ ID NO.3, with soybean genomic DNA as a template, the source organ-specific promoter part of the SA fusion gene was amplified by PCR method, and the amplified product was recovered and subjected to TA cloning; (2) Using primers SEQ ID NO.4 and SEQ ID NO.5, with soybean cDNA as a template, the key autophagy gene part in the SA fusion gene was amplified by PCR method, and the amplified product was recovered and subjected to TA cloning; (3) The TA cloning plasmid containing the source organ-specific promoter in the fusion gene in step (1) and the TA cloning plasmid containing the key autophagy gene in the fusion gene in step (2) were double-digested with Hind 111 / Nco I and Nco I / BstP I respectively, and the above nucleic acid fragments were recovered respectively; (4) The pCAMBIA3301 plasmid was double-digested with Hind 111 / BstP I, and the large vector fragment was recovered; (5) The recovered source organ-specific promoter fragment, key autophagy gene fragment in step (3) and the recovered pCAMBIA3301 vector fragment in step (4) were mixed and subjected to a ligation reaction under the catalysis of a ligase; (6) The ligation product was transformed into Escherichia coli DH5a competent cells, and the construction of the SA fusion gene on the pCAMBIA3301 vector was obtained through resistance screening.

2. The primers of the fusion gene SA described in claim 1, characterized in that 4 primers were designed for the creation of the SA fusion gene, and the specific primer sequences are the nucleic acid sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.

5.

3. The method for constructing the fusion gene SA described in claim 1, characterized in that The fusion gene is a fusion gene constructed by ligating a source organ-specific promoter and a key autophagy gene through an artificial splicing method. The specific operation steps are as follows: (1) Using the primers SEQ ID NO. 2 and SEQ ID NO.3 described in claim 2, with soybean genomic DNA as a template, the source organ-specific promoter part of the SA fusion gene was amplified by PCR method, and the amplified product was recovered and subjected to TA cloning; (2) Using the primers SEQ ID NO.4 and SEQ ID NO.5 described in claim 2, with soybean cDNA as a template, the key autophagy gene part in the SA fusion gene was amplified by PCR method, and the amplified product was recovered and subjected to TA cloning; (3) Double digest the TA cloning plasmid containing the promoter of the source organ specific gene in the fusion gene in step (1) and the TA cloning plasmid containing the key gene of autophagy in the fusion gene in step (2) with Hind III / Nco I and Nco I / BstP I respectively, and recover the above nucleic acid fragments; (4) Double digest the pCAMBIA3301 plasmid with Hind III / BstP I and recover the large vector fragment; (5) Mix the promoter fragment of the source organ specific gene, the key gene fragment of autophagy recovered in step (3) and the pCAMBIA3301 vector fragment recovered in step (4), and carry out a ligation reaction under the catalysis of ligase; (6) Transform the ligation product into competent cells of Escherichia coli DH5α, and obtain the construction of the SA fusion gene on the pCAMB1A3301 vector through resistance screening.

4. Application of the nitrogen-efficient fusion gene SA described in claim 1, characterized in that, use this fusion gene to construct a plant binary expression vector and carry out plant transformation, so as to obtain a transgenic plant whose genome contains the fusion gene with the nucleic acid sequence shown in SEQ ID NO.

1.

5. The application according to claim 4, characterized in that, transform soybeans with the plant binary expression vector in claim 4 to obtain transgenic soybeans with the SA fusion gene inserted into the genome. The efficiency of autophagy-mediated nutrient remobilization in the source organs of these transgenic soybeans is improved, and the tolerance to low nitrogen and low nutrient stress is significantly enhanced; when planted in low nitrogen soil, they grow more vigorously than the non-transformed control, with an increased stem girth, more branches, and an increased yield per plant.

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