Application of NRT1.1 gene in improvement of soybean nitrogen absorption efficiency

Abstract

The invention belongs to the technical field of soybean genetic breeding. More specifically, the invention relates to an application of an NRT1.1 gene in improvement of soybean nitrogen absorption efficiency. The invention discloses an application of an NRT1.1 gene in improving the nitrogen absorption efficiency of soybeans. The NRT1.1 gene comprises one or more of an NRT1.1 a gene, an NRT1.1 b gene and an NRT1.1 d gene; the sequence of the NRT1.1 a gene is as shown in SEQ ID NO.1, the sequence of the NRT1.1 b gene is as shown in SEQ ID NO.2, and the sequence of the NRT1.1 d gene is as shown in SEQ ID NO.4. The invention further discloses a preparation method of the gene. The research finds that the nitrogen absorption efficiency of the soybean can be remarkably improved by overexpressing the NRT1.1 a, NRT1.1 b or NRT1.1 d gene through the root system, and the improvement effect is remarkably superior to that of the NRT1.1 c gene. The NRT1.1 gene disclosed by the invention is used for improving soybeans, a soybean germplasm resource capable of efficiently absorbing nitrogen is created, and the NRT1.1 gene has good application prospect and value in soybean breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of soybean genetic breeding, and more specifically, relates to the application of NRT1.1 gene in improving soybean nitrogen absorption efficiency. Background Art

[0002] Soybean (Glycine max) is an important grain and oil crop. Therefore, it is urgent to solve the contradiction between supply and demand of soybean production by increasing soybean yield.

[0003] Nitrogen is one of the macronutrients necessary for plant growth and an important component of amino acids and proteins in plants. Its average content accounts for about 1.5% of the dry weight of plants. Applying nitrogen fertilizer is an important means to increase crop yields. However, excessive application of nitrogen fertilizer not only leads to a decrease in plant nitrogen use efficiency (NUE), but also causes environmental pollution, posing a serious challenge to the sustainable development of agriculture. In order to obtain sufficient nitrogen sources, plant roots absorb nitrogen in different forms from the soil, including nitrate nitrogen (NO3 - ), ammonium nitrogen (NH4 + ), soluble polypeptides and complex insoluble nitrogen compounds, etc., NO3 - It is the main inorganic nitrogen source used by soybeans. - The absorption and translocation of nutrients are of great significance to improving crop NUE.

[0004] Plants absorb nitrate nitrogen from the soil mainly through nitrate transporters (NRTs). There are four nitrate transporter families in plants: nitrate transporter 1 family (NRT1), nitrate transporter 2 family (NRT2), chloride channel family (CLC) and slow anion channel-associated homologous protein family (SLAC / SLAH). These four gene families and some transcription factors and regulatory proteins together constitute a complex nitrate regulatory network. There are a large number of genes in the nitrate transporter 1 family (NRT1). Plants generally have multiple NRT1 genes. For example, millet contains 8 NRT1 genes. The ability of different NRT1 genes to absorb and transport nitrate varies greatly. At present, there is no report on the function of NRT1.1 in soybean. Summary of the invention

[0005] The first object of the present invention is to provide an application of NRT1.1 gene in improving nitrogen absorption efficiency of soybean.

[0006] The second object of the present invention is to provide the application of NRT1.1 gene in improving soybean yield.

[0007] The third object of the present invention is to provide an application of the NRT1.1 gene as a target in constructing a soybean line with improved nitrogen absorption efficiency.

[0008] The fourth object of the present invention is to provide the use of an agent for promoting the expression of NRT1.1 gene.

[0009] The fifth object of the present invention is to provide an agent for constructing a soybean plant that absorbs nitrogen efficiently and its application.

[0010] The sixth object of the present invention is to provide a method for cultivating soybean varieties that efficiently absorb nitrogen.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention has found that overexpression of NRT1.1a, NRT1.1b, and NRT1.1d genes in the root system can significantly improve the nitrogen absorption efficiency of soybeans, and the effect of improving the nitrogen absorption efficiency is better than overexpression of the NRT1.1c gene more than 10 times. Therefore, the present invention provides the following application schemes:

[0013] The application of NRT1.1 gene in improving the nitrogen absorption efficiency of soybean, wherein the NRT1.1 gene includes one or more of NRT1.1a gene, NRT1.1b gene and NRT1.1d gene; the NRT1.1a gene sequence is shown in SEQ ID NO.1, the NRT1.1b gene sequence is shown in SEQ ID NO.2, and the NRT1.1d gene sequence is shown in SEQ ID NO.4.

[0014] The application of the above NRT1.1 gene in improving soybean yield.

[0015] The above-mentioned NRT1.1 gene is used as a target in constructing soybean lines with improved nitrogen absorption efficiency.

[0016] The application of the reagent for promoting the expression of the NRT1.1 gene in improving the nitrogen absorption efficiency of soybean.

[0017] Application of the reagent for promoting the expression of the NRT1.1 gene in improving soybean yield.

[0018] The present invention provides a reagent for constructing a soybean plant capable of efficiently absorbing nitrogen, which contains a reagent for promoting the expression of the NRT1.1 gene.

[0019] As an alternative embodiment, the reagent includes an overexpression vector of the NRT1.1 gene.

[0020] The use of the above reagents in constructing soybeans with high nitrogen absorption efficiency should also be within the protection scope of the present invention.

[0021] The present invention provides a method for cultivating soybean varieties with high nitrogen absorption efficiency, and overexpresses NRT1.1 gene in soybean; the NRT1.1 gene includes one or more of NRT1.1a gene, NRT1.1b gene and NRT1.1d gene; the NRT1.1a gene sequence is shown in SEQ ID NO.1, the NRT1.1b gene sequence is shown in SEQ ID NO.2, and the NRT1.1d gene sequence is shown in SEQ ID NO.4.

[0022] As an optional implementation scheme, the overexpression vector of the NRT1.1 gene is transformed into Agrobacterium, and then the Agrobacterium is used to infect soybeans to obtain soybean varieties that absorb nitrogen efficiently.

[0023] The present invention has the following beneficial effects:

[0024] The present invention obtains composite plants of soybeans with overexpression of NRT1.1a in the root system, soybeans with overexpression of NRT1.1b in the root system, soybeans with overexpression of NRT1.1c in the root system, and soybeans with overexpression of NRT1.1d in the root system through a hairy root rooting method, while the aboveground parts are non-transgenic. The above-mentioned plants are used to study the functions of soybean NRT1.1a gene, NRT1.1b gene, NRT1.1c gene, and NRT1.1d gene in improving the nitrogen absorption efficiency of soybean. The present invention has found that the nitrogen absorption efficiency of soybean can be significantly improved by overexpression of NRT1.1a, NRT1.1b or NRT1.1d gene in the root system, and the improvement effect is significantly better than that of NRT1.1c gene. Soybean is improved by using NRT1.1a, NRT1.1b, and NRT1.1d genes to create soybean germplasm resources that absorb nitrogen efficiently, which has good application prospects and value in soybean breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 These are the phenotypic verification results of soybean plants NRT1.1a OE, NRT1.1b OE, NRT1.1c OE, and NRT1.1d OE.

[0026] Figure 2 These are the results of measuring nitrogen absorption efficiency of soybean plants of NRT1.1a OE, NRT1.1b OE, NRT1.1c OE, and NRT1.1d OE. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0028] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0029] The experimental methods of the hairy root composite plant technology in the following examples refer to the literature "Chen Jiaxin, Mei Hao, Huang Caixiang, et al. Method for efficiently cultivating soybean chimeric plants using transgenic hairy roots [J]. Acta Botanica Sinica, 2024, 59(1): 89-98.".

[0030] The RUBY reporter gene contained in the vector used in the hairy roots in the following examples has been disclosed in the document "Wang D, Zhong Y, Feng B, Qi X, Yan T, Liu J, Guo S, Wang Y, Liu Z, Cheng D, Zhang Y, Shi Y, Zhang S, PanR, Liu C, Chen S. The RUBY reporter enables efficient haploid identification inmaize and tomato. Plant Biotechnol J. 2023Aug; 21(8): 1707-1715.", and the pCAMBIA1300 vector is commercially available.

[0031] The method for determining the nitrogen absorption efficiency of soybean in the following examples is based on the literature "Liu Y, Hu B, Chu C. 15N-nitrate Uptake Activity and Root-to-shoot Transport Assay in Rice[J]. Bio-Protocol, 2016, 6: 1897."

[0032] Example 1 Creation of composite soybean plants with overexpression of NRT1.1a, NRT1.1b, NRT1.1c and NRT1.1d genes in their roots

[0033] The nucleic acid sequence of soybean NRT1.1a gene is shown in SEQ ID NO.1 (5'-3').

[0034] SEQ ID NO.1:

[0035]

[0036] The nucleic acid sequence of soybean NRT1.1b gene is shown in SEQ ID NO.2 (5'-3').

[0037] SEQ ID NO.2:

[0038]

[0039] The nucleic acid sequence of soybean NRT1.1c gene is shown in SEQ ID NO.3 (5'-3').

[0040] SEQ ID NO.3:

[0041]

[0042] The nucleic acid sequence of soybean NRT1.1d gene is shown in SEQ ID NO.4 (5'-3').

[0043] SEQ ID NO.4:

[0044]

[0045] Using soybean W82 variety as the background, the NRT1.1a, NRT1.1b, NRT1.1c, and NRT1.1d genes were overexpressed respectively through the hairy root composite plant technology, and soybean composite plants with root overexpression of NRT1.1a (denoted as NRT1.1aOE), soybean composite plants with root overexpression of NRT1.1b (denoted as NRT1.1b OE), soybean composite plants with root overexpression of NRT1.1c (denoted as NRT1.1c OE), and soybean composite plants with root overexpression of NRT1.1d (denoted as NRT1.1d OE) were obtained.

[0046] The specific preparation method of soybean with overexpression gene in root system is as follows:

[0047] (1) Acquisition of NRT1.1a, NRT1.1b, NRT1.1c, and NRT1.1d gene fragments

[0048] According to the CDS sequence of NRT1.1a, NRT1.1b, NRT1.1c, and NRT1.1d genes, the stop codon of the sequence was removed, and upstream and downstream primers with recombination adapters were designed respectively. The primer information is shown in Table 1. PCR amplification was performed using soybean root cDNA as a template to obtain the target genes (NRT1.1a gene, NRT1.1b gene, NRT1.1c gene, and NRT1.1d gene).

[0049] Table 1 Primer sequences for amplifying NRT1.1a, NRT1.1b, NRT1.1c, and NRT1.1d genes

[0050]

[0051]

[0052] (2) Construction of overexpression vector

[0053] The vector pCAMBIA1300-RUBY was linearized by restriction endonucleases HindⅢ and KpnⅠ, and the target gene was connected to the vector after restriction digestion by homologous recombination. The ligation product was transformed into Escherichia coli DH5α, and the positive single clone was detected and the plasmid was extracted.

[0054] (3) Construction of soybeans with overexpression of genes in roots

[0055] The plasmid in step (2) is transformed into K599 Agrobacterium, and a positive single clone is obtained by detection. The K599 Agrobacterium is used to infect soybean hypocotyls to obtain transgenic hairy roots. The method is based on the reference "A method for efficiently cultivating soybean chimeric plants using transgenic hairy roots" to obtain soybeans with overexpressed genes in the root system.

[0056] In addition, the empty vector (pCAMBIA1300-RUBY) was transformed into K599 Agrobacterium, and K599 Agrobacterium was used to infect soybean hypocotyls to obtain transgenic hairy roots and negative control soybean plants, which were recorded as RUBY.

[0057] Example 2 Phenotypic verification of soybean root overexpression of NRT1.1 gene composite plants

[0058] 1. Experimental methods

[0059] RNA was extracted from the lateral roots of soybean hairy roots with overexpressed genes in the root system (NRT1.1a OE, NRT1.1b OE, NRT1.1c OE, and NRT1.1d OE), and reverse transcribed into cDNA. qRT-PCR was used to detect the expression levels of NRT1.1a, NRT1.1b, NRT1.1c, and NRT1.1d genes. The qRT-PCR primers are shown in Table 2.

[0060] Table 2 qRT-PCR primer sequences of NRT1.1a, NRT1.1b, NRT1.1c, NRT1.1d genes

[0061]

[0062] (II) Experimental results

[0063] The phenotype verification results of NRT1.1a OE, NRT1.1b OE, NRT1.1c OE, and NRT1.1d OE plants are as follows Figure 1 As shown, the results showed that the expression levels of NRT1.1a, NRT1.1b, NRT1.1c, and NRT1.1d in transgenic soybean hairy roots were higher than those in the negative control plants (RUBY). Compared with the negative control plants, the expression level of NRT1.1a gene in soybeans with overexpression of NRT1.1a gene in roots increased by about 3 times, the expression level of NRT1.1b gene in soybeans with overexpression of NRT1.1b gene in roots increased by about 4 times, the expression level of NRT1.1c gene in soybeans with overexpression of NRT1.1c gene in roots increased by about 12 times, and the expression level of NRT1.1d gene in soybeans with overexpression of NRT1.1d gene in roots increased by about 12.5 times.

[0064] Example 3 Nitrogen absorption efficiency of hairy root composite plants

[0065] 1. Experimental methods

[0066] The method for determining nitrogen absorption efficiency is based on the reference "Liu Y, Hu B, Chu C. 15N-nitrate Uptake Activity and Root-to-shoot Transport Assay in Rice[J]. Bio-Protocol, 2016, 6: 1897." The specific operation is as follows:

[0067] The hairy roots of soybeans with overexpressed genes were taken after 20 days of growth and 5 mM 15 Treat with N-KNO3 (abundance 98%) solution for 1.5 hours, wash with 0.1mM CaSO4 solution 3 times, divide the plant into aboveground part and root part (the root system is rinsed with deionized water), set up 5 replicates, put into a kraft envelope, and dry in an oven at 105℃ for 30 minutes, bake at a constant temperature of 70℃ until the dry weight is constant, and weigh the dry weight of the aboveground part and the root part with a 1 / 10,000 balance. Grind the aboveground plant dry samples and the root plant dry samples separately, then pass through a 100-mesh sieve, weigh about 10 mg for each replicate, and use an element analyzer-isotope mass spectrometer to determine the content of the sample. 15 N content, calculate nitrogen absorption efficiency.

[0068] (II) Experimental results

[0069] The soybean plants of NRT1.1a OE, NRT1.1b OE, NRT1.1c OE and NRT1.1d OE were 15 The N absorption efficiency was determined. The results were as follows: Figure 2 As shown in the figure, the results showed that compared with the negative control plant (RUBY), the nitrogen absorption efficiency of soybeans could be significantly improved by overexpressing NRT1.1a and NRT1.1b genes by about 4 times in the root system. However, the nitrogen absorption efficiency of soybeans was still not as good as that of soybean plants with about 12 times overexpression of NRT1.1c genes in the root system. The nitrogen absorption efficiency of NRT1.1d OE soybean plants was comparable to that of NRT1.1a OE soybean plants and slightly lower than that of NRT1.1b OE soybean plants.

[0070] It was demonstrated that NRT1.1a, NRT1.1b, and NRT1.1d genes have stronger nitrate transport capabilities than NRT1.1c gene and are more valuable in improving soybean nitrogen absorption efficiency.

[0071] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of NRT1.1 gene in improving nitrogen absorption efficiency of soybean, It is characterized in that The NRT1.1 gene includes one or more of the NRT1.1a gene, the NRT1.1b gene, and the NRT1.1d gene; the NRT1.1a gene sequence is shown in SEQ ID NO.1, the NRT1.1b gene sequence is shown in SEQ ID NO.2, and the NRT1.1d gene sequence is shown in SEQ ID NO.

4.

2. Use of the NRT1.1 gene described in claim 1 in increasing soybean yield.

3. Use of the NRT1.1 gene described in claim 1 as a target in constructing a soybean line with improved nitrogen absorption efficiency.

4. Use of an agent for promoting the expression of the NRT1.1 gene of claim 1 in improving the nitrogen absorption efficiency of soybeans.

5. Use of an agent for promoting the expression of the NRT1.1 gene according to claim 1 in increasing soybean yield.

6. A reagent for constructing a soybean plant that efficiently absorbs nitrogen, It is characterized in that Contains an agent for promoting the expression of the NRT1.1 gene as claimed in claim 1.

7. The reagent according to claim 6, It is characterized in that The reagent includes an overexpression vector of the NRT1.1 gene.

8. Use of the reagent according to claim 6 or 7 in constructing soybeans with high nitrogen absorption efficiency.

9. A method for cultivating soybean varieties that efficiently absorb nitrogen, It is characterized in that The NRT1.1 gene is overexpressed in soybean; the NRT1.1 gene includes one or more of the NRT1.1a gene, the NRT1.1b gene, and the NRT1.1d gene; the NRT1.1a gene sequence is shown in SEQ ID NO.1, the NRT1.1b gene sequence is shown in SEQ ID NO.2, and the NRT1.1d gene sequence is shown in SEQ ID NO.

4.

10. The method according to claim 9, It is characterized in that The overexpression vector of the NRT1.1 gene in claim 7 is transformed into Agrobacterium, and then the Agrobacterium is used to infect soybeans to obtain soybean varieties that efficiently absorb nitrogen.

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