White lupin root growth key gene LaLBD29 and application thereof

Abstract

The invention discloses a lupinus albus root growth key gene LaLBD29 and application of the lupinus albus root growth key gene LaLBD29. The nucleotide sequence of the LaLBD29 gene is as shown in SEQ ID NO. 1, and the amino acid sequence of the protein coded by the LaLBD29 gene is as shown in SEQ ID NO. 2. Wild lupinus albus is taken as a background material, and the transgenic technology is utilized to prove that the silent LaLBD29 gene can inhibit the root formation and phosphorus deficiency stress resistance of the lupinus albus under the phosphorus deficiency condition. The invention provides important theoretical and practical basis for breeding and genetic improvement of phosphorus deficiency-resistant crop varieties.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a key gene LaLBD29 for root growth of white lupin and an application thereof. Background Art

[0002] Lack of phosphorus in the environment can lead to a significant decrease in the photosynthesis efficiency of plants. Studies have shown that when plants are under phosphorus deficiency stress, their root morphology will undergo adaptive changes to alleviate the adverse effects of phosphorus deficiency on themselves. For example, when the concentration of inorganic phosphorus in the aboveground part decreases, the root system will respond to this signal and induce changes in the root structure to form rows of roots to cope with phosphorus deficiency stress.

[0003] White lupin (Lupinus albus) produces special root morphology and physiological responses under low-phosphorus / deficient conditions, so it is used as a model plant to study root responses to phosphorus starvation. Row roots increase the surface area and volume of the root system, and can activate the phosphorus fixed in the soil, which is beneficial for the root system to absorb the phosphorus fixed in the soil. Row roots activate the phosphorus fixed in the soil by secreting organic acids and protons, thereby improving the efficiency of phosphorus utilization. Therefore, identifying the key genes that regulate the growth of row roots in white lupin is crucial for studying the mechanism of row root growth. Summary of the invention

[0004] The purpose of the present invention is to provide a key gene LaLBD29 for root growth of white lupin and application thereof.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention provides a LaLBD29 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] The second aspect of the present invention provides a protein encoded by the LaLBD29 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] The third aspect of the present invention provides the use of the LaLBD29 gene in regulating the root formation of white lupin under phosphorus deficiency conditions; Furthermore, the regulation of the root formation of white lupine under phosphorus deficiency conditions refers to regulating the number of white lupine roots under phosphorus deficiency conditions; Furthermore, the regulation of the number of roots of white lupin under phosphorus deficiency conditions refers to: silencing the LaLBD29 gene of white lupin, reducing the total number of roots and the number of roots per unit root length of white lupin under phosphorus deficiency conditions.

[0008] The fourth aspect of the present invention provides a method for regulating the formation of row roots of white lupin under phosphorus deficiency conditions, which comprises transforming a biological material with a silenced LaLBD29 gene into a recipient white lupin to construct a transgenic white lupin with a silenced LaLBD29 gene, wherein the number of row roots of the transgenic white lupin under phosphorus deficiency conditions is reduced compared with that of the recipient white lupin; the nucleotide sequence of the LaLBD29 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the LaLBD29 gene is shown in SEQ ID NO.2; Furthermore, the reduction in the number of row roots refers to a reduction in the total number of row roots and the number of row roots per unit root length. The fifth aspect of the present invention provides the use of the LaLBD29 gene in regulating the ability of white lupin to tolerate phosphorus deficiency stress; Furthermore, the regulation of the ability of white lupin to tolerate phosphorus deficiency stress refers to: silencing the LaLBD29 gene of white lupin to reduce the ability of white lupin to tolerate phosphorus deficiency stress.

[0009] The sixth aspect of the present invention provides a method for regulating the ability of white lupin to tolerate phosphorus deficiency stress, which comprises transforming a biological material with a silenced LaLBD29 gene into a recipient white lupin to construct a transgenic white lupin with a silenced LaLBD29 gene, wherein the transgenic white lupin has a reduced ability to tolerate phosphorus deficiency stress compared to the recipient white lupin; the nucleotide sequence of the LaLBD29 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded thereby is shown in SEQ ID NO.2.

[0010] Compared with the prior art, the present invention has the following beneficial effects: The present invention first reveals the function of the LaLBD29 gene in improving the phosphorus deficiency tolerance of white lupin. The present invention uses wild-type white lupin as the background material and uses transgenic technology to confirm that silencing the LaLBD29 gene will inhibit the rooting growth and phosphorus deficiency tolerance of white lupin under phosphorus deficiency conditions, providing important theoretical and practical basis for the breeding and genetic improvement of phosphorus deficiency-tolerant crop varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 :Expression of LaLBD29 gene in root segments of white lupin root row.

[0012] Figure 2 : A: Detection of the expression level of LaLBD29 gene in the control group white lupine and TRV:LaLBD29 white lupine (transgenic white lupine with LaLBD29 gene silenced).

[0013] Figure 3:A: The total number of roots in the control group white lupine and TRV:LaLBD29 white lupine; B: The number of roots per unit root length in the control group white lupine and TRV:LaLBD29 white lupine.

[0014] Figure 4 : A: Phosphorus content in the aboveground parts of white lupine in the control group and TRV:LaLBD29 white lupine; B: Phosphorus content in the roots of white lupine in the control group and TRV:LaLBD29 white lupine.

[0015] Note: *, P<0.05; **, P<0.01. DETAILED DESCRIPTION

[0016] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0018] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0019] The formula of the normal phosphorus nutrient solution used in the following examples is: 0.525M potassium sulfate, 0.375M magnesium sulfate heptahydrate, 0.5M calcium nitrate tetrahydrate, 20mM sodium ferric ethylenediaminetetraacetate, 25mM boric acid, 1.5mM manganese sulfate monohydrate, 1.5mM zinc sulfate heptahydrate, 0.5mM cupric sulfate pentahydrate, 0.025mM ammonium molybdate tetrahydrate, and 0.25mM potassium dihydrogen phosphate.

[0020] The formula of the phosphorus-deficient nutrient solution used in the following examples is: 0.525M potassium sulfate, 0.375M magnesium sulfate heptahydrate, 0.5M calcium nitrate tetrahydrate, 20mM sodium ferric ethylenediaminetetraacetate, 25mM boric acid, 1.5mM manganese sulfate monohydrate, 1.5mM zinc sulfate heptahydrate, 0.5mM copper sulfate pentahydrate, and 0.025mM ammonium molybdate tetrahydrate.

[0021] The nucleotide sequence of the LaLBD29 gene involved in the following examples is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2.

[0022] Embodiment 1: Select white lupine seeds with full grains, good appearance, no obvious mildew spots and no damage from the seed bank, place them in a triangular bottle, add pure water to cover the surface of the seeds, and place them on a shaker at 30°C and 180rpm overnight to awaken the seed activity. The seeds can be obviously seen to absorb water and swell the next day. Rinse the seeds 3 times with pure water; cut yellow parchment paper into 5cm×15cm rectangular papers, soak them with pure water, wrap the white lupine seeds with the yellow parchment paper, and all the seed germination holes are facing downward to ensure that the white lupine seedlings remain straight; put the yellow parchment paper wrapped with the white lupine seeds into a hydroponic box, add pure water to a position of about 1 / 2 the volume of the hydroponic box, and germinate for 2 to 3 days at room temperature away from light, during which time the water is changed once a day; germination for 2 to 3 days After the white lupine grows roots about 2 to 5 cm long, rinse with pure water for 3 times; prepare two 25cm (width) × 35cm (length) × 10cm (height) rectangular hydroponic pots, add 1-fold diluted normal phosphorus nutrient solution, and cut out foam boards of the same size and shape as the hydroponic pots. Cut small holes on the foam boards at intervals of 2 cm, fix the white lupine seedlings with true leaves in the board holes with sponges in the same direction, immerse the roots in the nutrient solution, and move to the greenhouse for 3 days to allow the white lupine seedlings to initially adapt to the environment of the nutrient solution. Then select the seedlings with the same growth trend and transplant them into the phosphorus-deficient nutrient solution for treatment; after 4 weeks, take out the white lupine seedlings with complete row roots. The entire row root on a lateral root can be divided into: MPR (minus phosphorus root, phosphorus-deficient non-rooting segment), RT (Roottip), YCR (juvenile cluster root), GAP (root segment without roots between juvenile and mature cluster roots), MCR (mature cluster root), OCR (old cluster root). Total RNA of the samples was extracted, and the extracted total RNA was reverse transcribed using a kit to obtain cDNA. Real-time fluorescence quantitative PCR instrument (BIO-RAD, CFX96) was used for qRT-PCR detection of LaLBD29 gene. 2 -ΔΔCt The relative quantitative data were analyzed by the method of qLaLBD29 F (3'-ATACTCCCTCAAC AACACAACACTATGG-3') and qLaLBD29 R (3'-GAGGTGCTTTCATCAAGGGTAGTTCC-3').

[0023] like Figure 1 As shown, the LaLBD29 gene is highly expressed in the root tip of white lupin, i.e., at the beginning stage of root development. This gene is likely to play an important role in the response of white lupin to phosphorus deficiency stress.

[0024] Embodiment 2: Using white lupin root cDNA as template, PCR amplification was performed with primers pTRV2-LaLBD29 F (3'-ATGGAGGCCTTCTAGCTCCCTGTGAGTGATCGTTGC-3') and pTRV2-LaLBD29 R (3'-TACCGAATTCTCTAGCCAGAGAGATTGAAACAACTTTGGC-3') to obtain the silencing fragment used to silence the target gene LaLBD29.

[0025] Prepare white lupin materials, and when the radicle elongates to 2-3 cm, move the seedlings to a culture bottle filled with sand and phosphorus-deficient nutrient solution for cultivation. When the first round of true leaves unfold, you can start virus transfection. Pick the K599 Agrobacterium-positive clones carrying the corresponding viral plasmids and place them in 5 mL of LB liquid culture medium containing 50 μg / mL kanamycin and 25 μg / mL rifampicin. Cultivate overnight at 28°C and 200 rpm / min until the culture solution becomes turbid but not whitish; then transfer to 50 mL of LB liquid culture medium containing 50 μg / mL kanamycin and 25 μg / mL rifampicin, and culture at 28°C and 200 rpm / min. When the bacterial solution concentration reaches the predetermined OD 600 The cells were collected by centrifugation and suspended in 25 ml of infection buffer (MES 10 mM; AS 100 μM; MgCl2 10 mM; sterile water as solvent) to adjust the OD 600 to 1.0-1.5, and stand at room temperature for 3 hours; mix equal concentrations of Agrobacterium with pTRV1 plasmid and Agrobacterium with pTRV2 plasmid inserted with target gene silencing fragment in a volume ratio of 1:1 to obtain the Agrobacterium infection solution of the experimental group. Mix equal concentrations of Agrobacterium with pTRV1 plasmid and Agrobacterium with pTRV2 empty vector in a volume ratio of 1:1 to obtain the Agrobacterium infection solution of the control group. Pour the Agrobacterium infection solution around the root system of white lupin, and irrigate each white lupin with about 20 ml of infection solution. After that, irrigate once every five days, for a total of 3 times. Take samples one week after the last irrigation, and use primers qLaLBD29 F (3'-ATACTCCCTCAACAACACAACACT ATGG-3') and qLaLBD29 R (3'-GAGGTGCTTTCATCAAGGGTAGTTCC-3') to detect the LaLBD29 gene silencing efficiency.

[0026] like Figure 2 As shown in the figure, under phosphorus deficiency conditions, the expression level of the LaLBD29 gene in the LaLBD29 silenced strain was significantly reduced compared to the control white lupin. The phenotypic photos show that the number of lateral roots and row roots in the LaLBD29 silenced strain was reduced. Figure 3As shown in the figure, under phosphorus deficiency conditions, the number of roots in the control group of white lupine was about 15 clusters per plant, with a root length of about 0.019 clusters per centimeter, and the number of roots in the LaLBD29 silenced strain was about 9 clusters per plant, with a root length of about 0.009 clusters per centimeter. Compared with the control group of white lupine, the number of roots in the LaLBD29 silenced strain was significantly reduced, indicating that the silencing of the LaLBD29 gene has a significant effect on the number of roots in white lupine under phosphorus deficiency conditions. Figure 4 As shown in the figure, under phosphorus deficiency conditions, the phosphorus content in the aboveground and root of the white lupin in the control group was about 0.3 mg / g and 1.03 mg / g, respectively, while the phosphorus content in the aboveground and root of the LaLBD29 gene silenced strain was about 0.24 mg / g and 1.11 mg / g, respectively. -1 , there is no significant difference between the two.

[0027] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. LaLBD29 A gene characterized by: Said LaLBD29 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1 LaLBD29 A gene encoding a protein characterized by: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. The method according to claim 1 LaLBD29 Application of genes in regulating root formation in white lupin under phosphorus deficiency conditions.

4. The use according to claim 3, characterized in that: The regulation of white lupine root formation under phosphorus deficiency conditions refers to regulating the number of white lupine roots under phosphorus deficiency conditions.

5. The use according to claim 4, characterized in that: The regulation of the number of roots of white lupin under phosphorus deficiency conditions refers to: LaLBD29 Gene, reduces the total number of roots and the number of roots per unit root length of white lupin under phosphorus deficiency conditions.

6. A method for regulating root formation of white lupin under phosphorus deficiency conditions, characterized in that: Will be silent LaLBD29 The biomaterial of the gene was transformed into the recipient white lupin, and the construct was silenced LaLBD29 A transgenic white lupine having a gene, wherein the number of roots of the transgenic white lupine under phosphorus deficiency conditions is reduced compared with that of the recipient white lupine; LaLBD29 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

7. The method according to claim 1, characterized in that: The reduction in the number of rows of roots refers to a reduction in the total number of rows of roots and the number of rows of roots per unit root length.

8. The method according to claim 1 LaLBD29 Application of genes in regulating the tolerance of white lupin to phosphorus deficiency stress.

9. The use according to claim 8, characterized in that: The regulation of the ability of white lupin to tolerate phosphorus deficiency stress refers to: silencing the LaLBD29 Gene, reducing the ability of white lupin to tolerate phosphorus deficiency stress.

10. A method for regulating the ability of white lupin to tolerate phosphorus deficiency stress, characterized in that: Will be silent LaLBD29 The biomaterial of the gene was transformed into the recipient white lupin, and the construct was silenced LaLBD29 A transgenic white lupine having a gene, wherein the transgenic white lupine has a reduced ability to tolerate phosphorus deficiency stress compared to the recipient white lupine; LaLBD29 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

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