Application of overexpressed GmNAC181 gene in regulation and control of K < + > absorption and / or transport of crops

Abstract

The invention relates to the technical field of molecular biology, in particular to application of an overexpressed GmNAC181 gene in regulation and control of K < + > absorption and / or transport of crops. The invention provides application of an overexpressed GmNAC181 gene as shown in SEQ ID NO.1 in regulation and control of K < + > absorption and / or transport of crops. The overexpressed GmNAC181 gene can improve the K < + > absorption of soybeans.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to the effect of overexpression of GmNAC181 gene on regulating crop K + Application in absorption and / or transport. Background Art

[0002] Potassium is one of the three essential mineral elements for soybeans. It participates in multiple physiological processes and plays a vital role in the formation of soybean yield. However, with the increase in planting intensity, the content of available potassium in my country's soil has gradually decreased, and potassium deficiency in cultivated land has become one of the important factors limiting the sustainable production of crops. At the same time, in order to promote the increase in soybean yield, the existing technology generally adopts the method of applying a large amount of chemical fertilizers, which increases the input of agricultural production and ultimately leads to a decrease in the income of soybean planting. In addition, in the actual agricultural production process, the effective utilization rate of potassium fertilizer is only about 50%, and nearly half of the potassium fertilizer cannot be effectively used by plants. Therefore, exploring potassium absorption efficient genes and creating potassium efficient soybean germplasm resources are one of the important ways to develop high-yield and high-efficiency soybeans in the future. Using molecular technology to create potassium-efficient soybean germplasm resources is particularly important for coping with soil potassium deficiency and reducing potassium fertilizer application in the future.

[0003] Based on this, the present invention is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide a method for regulating crop K by overexpressing GmNAC181 gene. + Application in absorption and / or transport.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for regulating crop K by overexpressing the GmNAC181 gene shown in SEQ ID NO.1. + Application in absorption and / or transport.

[0007] Preferably, the amino acid sequence encoded by SEQ ID NO.1 is as shown in SEQ ID NO.2.

[0008] Preferably, the nucleotide sequence of the primer pair for amplifying the GmNAC181 gene is shown in SEQ ID NOs. 3-4.

[0009] The present invention also provides a recombinant expression vector, which comprises the GmNAC181 gene and an empty vector;

[0010] The empty vector is pTF101 vector.

[0011] The present invention also provides a method for overexpressing the recombinant expression vector in regulating crop K+ Application in absorption and / or transport.

[0012] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the GmNAC181 gene and an empty vector bacterium;

[0013] The empty-load bacteria are Agrobacterium rhizogenes.

[0014] The present invention also provides the recombinant bacteria in regulating crop K + Application in absorption and / or transport.

[0015] The present invention also provides a kit, which comprises the GmNAC181 gene.

[0016] The present invention also provides the kit for regulating crop K + Application in absorption and / or transport.

[0017] Beneficial effects of the present invention:

[0018] The present invention provides a method for regulating crop K by overexpressing the GmNAC181 gene shown in SEQ ID NO.1. + Absorption and / or transport, overexpression of GmNAC181 gene can improve soybean K + absorb. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the relative expression level detection diagram of GmNAC181 gene;

[0020] Figure 2 Schematic diagram of potassium deficiency phenotypes of different plants cultured at different potassium concentrations for 14 days and 18 days;

[0021] Figure 3 The potassium accumulation in the leaves of different plants after being cultured at different potassium concentrations for 14 and 18 days. DETAILED DESCRIPTION

[0022] The present invention provides a method for regulating crop K by overexpressing the GmNAC181 gene shown in SEQ ID NO.1. + Applications in absorption and / or transport;

[0023] The SEQ ID NO.1 is:

[0024] ATGGGAAACCCAGAATCCAATTTGCCACCCGGTTTTAGGTTCCACCCAACCGATGAGGAGCTCATTCTTCACTACCTTAGTAAAAAGGTAGCATCCATACCCTTGCCCGTTTCCATCATTGCAGAGGTTGATATCTACAAGTTAGATCCATGGGACTTACCAGCTAAGGCAACCTTTGGGGAGAAAGAATGGTATTTTTTTAGTCCTAGAGACCGAAAGTACCCTAATGGTGCAAGGCCAAACAGGGCAGCTGCTTCAGGTTATTGGAAGGCCACTGGCACTGATAAGACCATAGTGACATCATTGCAAGGAGGAGCACAAGAGAGCGTTGGTGTGAAGAAGGCTTTGGTGTTCTACAAAGGAAGACCCCCAAAGGGTGTGAAGACCAATTGGATCATGCATGAATATCGCCTTGTAGACAACAACAAACCCATTAAGCTCAAAGACTCCTCCATGAGATTGGATGACTGGGTTCTGTGCCGGATTTACAAGAAATCCAAACATCCTCTAACTTCAACAGAGGCATCAATAGGGGTAGGTGATGTAGATCAAGCAGAGGAGCACGTATTCAAAGAGACCCTTTTGCCAATCTCAAAATCTCCAACACCTCCACCTCAAAACACATTGATATCTCAGAAATCTGTGTCCTTCTCCAACTTGTTGGATGCCATGGACTACTCTATCCTAAGCAGTTTCTTATCTGAAAATCATACCACCCACCCACCAGGGGTTGGATCAAGTACAGGCTTCAACACTGGGAATTTGGATCAGCAACCATCTCCAATCGACAACAGCAATGGTTACATGTTTCAGAAGAACAACCCCCAATTGAACCCTTCAGTCTCCAACATGGAAAACATGATGAGGCCAAAGCGCCAAGTTTCAAACATGGATGAGGAAACCTTGTACCCATCAAAGAAATACCTTAGTTCCTCTTGCAACTTCCCTACTAACATCAACAACCAATACGAGAACCCACAATGGAACTACCTCATGAAGCAGTCTTTGCTGAATCAACGGTTAATGCTTGGTCCTCATCTTCAATTTCAAGGATAA;

[0025] In the present invention, the amino acid sequence encoded by SEQ ID NO.1 is as shown in SEQ ID NO.2; SEQ ID NO.2 is:

[0026] MGNPESNLPPGFRFHPTDEELILHYLSKKVASIPLPVSIIAEVDIYKLDPWDLPAKATFGEKEWYFFSPRDRKYPNGARPNRAAASGYWKATGTDKTIVTSLQGGAQESVGVKKALVFYKGRPPKGVKTNWIMHEYRLVDNNKPIKLKDSSMRLDDWVLCRIYKKSKHPLTSTEAS IGVGDVDQAEEHVFKETLLPISKSPTPPPQNTLISQKSVSFSNLLDAMDYSILSSFLSENHTTHPPGVGSSTGFNTGNLDQQPSPIDNSNGYMFQKNNPQLNPSVSNMENMMRPKRQVSNMDEETLYPSKKYLSSSCNFPTNINNQYENPQWNYLMKQSLLNQRLMLGPHLQFQG.

[0027] In the present invention, the nucleotide sequences of the primer pairs for amplifying the GmNAC181 gene are shown in SEQ ID NOs. 3 to 4;

[0028] The SEQ ID NO.3 is: AGCGGCCGAATTCCCCGGGATGGGAAACCCAGAAT;

[0029] The SEQ ID NO.4 is: CGAGATCAGTTATCTAGATCCGGTGGATCCTCCTTGAAATTGAAGATGAGGACC.

[0030] The present invention also provides a recombinant expression vector, which comprises the GmNAC181 gene and an empty vector;

[0031] The empty vector is pTF101 vector.

[0032] The present invention also provides a method for overexpressing the recombinant expression vector in regulating crop K + Application in absorption and / or transport.

[0033] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the GmNAC181 gene and an empty vector bacterium;

[0034] The empty-load bacteria are Agrobacterium rhizogenes.

[0035] The present invention also provides the recombinant bacteria in regulating crop K + Application in absorption and / or transport.

[0036] The present invention also provides a kit, which comprises the GmNAC181 gene.

[0037] The present invention also provides the kit for regulating crop K + Application in absorption and / or transport.

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] In the embodiments and experimental examples of the present invention, the soybean variety is Williams 82 (W82), provided by Huazhong Agricultural University; the Escherichia coli DH5α strain was purchased from Beijing Quanshijin Biotechnology Co., Ltd., and the Agrobacterium tumefaciens EHA101 strain was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0040] Example 1 Construction of GmNAC181 gene overexpression vector

[0041] The nucleotide sequence of the GmNAC181 gene (SEQ ID NO.1) was obtained by PCR amplification using the KOD amplification method. The sequence size was 1056 bp. The amplified GmNAC181 gene was connected to the T vector ( -Blunt3 CloningVector, purchased from Beijing Quanshijin Biotechnology Co., Ltd.) to obtain the target vector plasmid; restriction endonucleases (SmaI and BamH I) were used to digest the target vector plasmid, and the digested vector plasmid was excised and recovered and purified; the recovered target gene fragment was connected between Sma I and BamH I of the pTF101 vector multi-cloning site driven by the 35S promoter using the homologous recombination method to obtain a connection product; the connection product was heat-shocked and transformed into a competent Escherichia coli DH5α strain, cultured at 37°C overnight, and positive clones were picked to obtain an overexpression vector carrying the target gene;

[0042] The specific steps of the KOD amplification method are as follows: KOD DNA polymerase from TOYOBO Company was used. Soybean genomic cDNA was used as a template, and nucleotide sequences SEQ ID NO.3 (AGCGGCCGAATTCCCCGGGATGGGAAACCCAGAAT) and SEQ ID NO.4 (CGAGATCAGTTATCTAGATCCGGTGGATCCTCCTTGAAATTGAAGATGAGGACC) were used as primers for PCR amplification to obtain the nucleotide sequence of the GmNAC181 gene (SEQ ID NO.1);

[0043] The reaction system and reaction procedure of PCR amplification are shown in Tables 1-2;

[0044] Table 1 PCR amplification reaction system

[0045] Components Usage KOD buffer 5μL dNTP (2.5mmol / L) 4μL <![CDATA[MgSO4]]> 3.75μL Primer F 1.25μL Primer R 1.25μL DNA template 1.25μL KOD polymerase 1μL <![CDATA[ddH2O]]> 32.5μL

[0046] Table 2 PCR amplification reaction program

[0047]

[0048] Example 2

[0049] (1) Take out 200 μL of the frozen competent Agrobacterium EHA101 strain, add 10 μL of the overexpression vector carrying the target gene described in Example 1 after thawing, gently tap the tube wall to mix, and place on ice for 30 minutes; put it in liquid nitrogen for 5 minutes, take it out, transfer it to 37°C for 5 minutes, add 800 μL of LB non-resistant liquid culture medium, and culture it at 28°C, 150 r / min for 4 hours; centrifuge at 4000 r / min for 30 seconds, discard the supernatant, add 100 μL of LB liquid culture medium, suspend the bacteria and apply it to the culture medium containing 50 mg / mL Kan; culture it at 28°C until white transformants grow, and obtain Agrobacterium transformants;

[0050] (2) The transgenic plants GmNAC181 overexpression lines GmNAC181OE-23# and GmNAC181OE-37# were obtained using the soybean stable genetic transformation system (Paz MM, Martinez JC, Kalvig AB, Foger TM, Wang K. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Rep, 2006, 25: 206-213.).

[0051] Example 3 Expression detection of tissue materials

[0052] (1) Take 0.2 g of the root material of GmNAC181OE-23# and GmNAC181OE-37# described in Example 2 and put them into a 2 mL centrifuge tube; put a 5 mm steel ball, grind the quick-frozen root material in liquid nitrogen with a high-throughput tissue grinder, add 1 mL of Trizol total RNA extraction reagent, shake and mix, and place on ice for 10 min; add 200 μL of chloroform, shake vigorously on a vortexer and let stand at 25°C for 10 min; centrifuge at 4°C, 12000 r / min for 10 min; transfer the supernatant into another RNase Free centrifuge tube with an RNase free pipette tip, add an equal volume of isopropanol, mix by inversion, and precipitate at -20°C for 30 min; centrifuge at 4°C, 12000 r / min for 10 min; discard the supernatant, wash the precipitate twice with 75% ethanol (prepared with DEPC-treated sterile water); centrifuge at 12000 r / min for 5 min; Carefully remove the supernatant with a free pipette tip, and let the RNA stand at room temperature in a fume hood to dry; add 30 μL of DEPC-treated sterile water to dissolve the RNA, and heat the metal heating block at 50°C for 5 minutes to obtain the RNA to be tested;

[0053] (2) using RT supermix (purchased from Beijing Novo Biotechnology Co., Ltd., catalog number R223-01) kit to reverse transcribe the RNA to be detected in step (1) into cDNA;

[0054] The specific steps are as follows: use the following reaction system: 3 μg RNA, 2 μL 5×gDNA digester Buffer, 1 μL gDNA digester, and DEPC-ddH2O to supplement the entire reaction system to 10 μL; react at 42°C for 2 min using a PCR instrument to obtain an initial reaction product; add 10 μL of the reaction solution to the initial reaction product, ⅡSuperMix plus 10μL; PCR instrument reaction 50℃, 15min→85℃, 5s, obtain cDNA;

[0055] (3) The cDNA in step (2) was detected and analyzed using the reagent SYBR Green Mix (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) and the instrument Bio-Rad CFX96 / 384. W82 was used as the control group. The expression level of the GmNAC181 gene was detected as follows: Figure 1 As shown;

[0056] The primers used in the detection process are as follows:

[0057] Upstream primer: GAGGAGCACAAGAGAGCGTT (SEQ ID NO. 5);

[0058] Downstream primer: CATGGAGGAGTCTTTGAGCTT (SEQ ID NO. 6);

[0059] The reaction system was: 2×SuperReal PreMix Plus 10 μL, upstream primer 0.4 μL, downstream primer 0.4 μL, cDNA template 0.2 μL, ddH2O 9 μL;

[0060] The reaction program was as follows: 95°C, 15 min→(95°C, 10 s→60°C, 34 s)×40 cycles→65°C, 5 s→95°C, 5 s.

[0061] from Figure 1 It can be seen that compared with the GmNAC181 expression level in the control group W82, the overexpression folds of GmNAC181OE-23# and GmNAC181OE-37# were 457.6 and 241.4 times.

[0062] Example 4 Soybean low potassium tolerance experiment

[0063] (1) After germination of GmNAC181OE-23#, GmNAC181OE-37#, and W82 in vermiculite with water for 7 days, they were moved to 1 / 2 concentration Hoagland nutrient solution for seedling acclimatization to ensure that the materials adapted to the environment;

[0064] (2) After 3 days of seedling acclimatization, the cotyledons are removed to prevent the potassium in the cotyledons from affecting the phenotype, thereby obtaining the treated material;

[0065] (3) The treated material was divided into two equal parts and then moved to a potash chamber with a potassium concentration of 0.05 mmol / LK. + (Low potassium stress, LK) and 2.5mmol / LK + The plants were cultured in Hoagland nutrient solution with a concentration of 1.5 K (normal potassium treatment, NK) and the symptoms of the plants were observed after 14 and 18 days of cultivation, such as Figure 2 As shown;

[0066] from Figure 2 It can be seen that there are obvious differences in potassium deficiency symptoms among GmNAC181OE-23#, GmNAC181OE-37# and W82 at 14d and 18d (obvious yellowing of leaf margins under low potassium conditions).

[0067] Example 5 Determination and calculation of leaf potassium accumulation

[0068] (1) Take leaf samples of the plants cultivated in Example 4, rinse with water, then rinse with distilled water, put them into kraft paper envelopes, inactivate at 105° C. for 30 min, and then dry at 80° C. to constant weight. Weigh the leaves on a 1 / 10,000 balance, which is the dry weight of the leaves;

[0069] (2) Leaf samples of the plants cultivated in Example 4 were respectively taken, washed with distilled water, sterilized at 105° C. for 30 min, dried at 65° C. for 48 h, and crushed through a 60-mesh sieve to obtain dry leaf samples.

[0070] (3) Weigh 0.3 g of dry leaf sample and place it in a 50 mL plastic tube. Add 25 mL of 1 mol / L hydrochloric acid and shake until mixed. Oscillate at 28 °C and 200 r / min for 90 min. Filter with quantitative filter paper, extract 1 mL of filtrate, dilute to 5 mL with distilled water, and measure the potassium content with a flame photometer.

[0071] (4) Calculate potassium accumulation, where potassium accumulation = leaf dry weight × potassium content. The calculation result is shown in 3.

[0072] from Figure 3 It can be seen that under normal potassium treatment conditions, there was no significant difference in leaf potassium accumulation among GmNAC181OE-23#, GmNAC181OE-37# and W82 at either 14 or 18 days; under low potassium stress conditions, the leaf potassium accumulation of GmNAC181OE-23# and GmNAC181OE-37# lines was significantly higher than that of W82 at 14 and 18 days.

[0073] As can be seen from the above examples, the present invention provides a method for overexpressing the GmNAC181 gene in regulating crop K + Overexpression of GmNAC181 gene can improve K absorption and / or transport in soybean crops. + absorb.

[0074] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Overexpression of the GmNAC181 gene shown in SEQ ID NO.1 regulates crop K + Application in absorption and / or transport.

2. The use according to claim 1, characterized in that: The amino acid sequence encoded by SEQ ID NO.1 is shown in SEQ ID NO.

2.

3. The use according to claim 2, characterized in that: The nucleotide sequences of the primer pair for amplifying the GmNAC181 gene are shown in SEQ ID NOs. 3-4.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the GmNAC181 gene according to claim 1 and an empty vector; The empty vector is pTF101 vector.

5. Overexpression of the recombinant expression vector of claim 4 in regulating crop K + Application in absorption and / or transport.

6. A recombinant bacterium, characterized in that: The recombinant bacteria include the GmNAC181 gene of claim 1 and an empty vector bacteria; The empty-load bacteria are Agrobacterium rhizogenes.

7. The recombinant bacteria according to claim 6 are used to regulate crop K + Application in absorption and / or transport.

8. A kit, characterized in that: The kit comprises the GmNAC181 gene according to claim 1.

9. The kit according to claim 8 is used to regulate crop K + Application in absorption and / or transport.

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