Application of LaPUCHI1 gene and encoded protein thereof in regulating and controlling root formation of lupin albicans under phosphorus deficiency condition

Abstract

The invention discloses an application of a LaPUCHI1 gene and an encoding protein thereof in regulating and controlling root formation of lupinus albus under a phosphorus deficiency condition. The nucleotide sequence of the LaPUCHI1 gene is as shown in SEQ ID NO. 1, and the amino acid sequence of the protein coded by the LaPUCHI1 gene is as shown in SEQ ID NO. 2. Researches find that the LaPUCHI1 gene positively regulates and controls the formation of lupin root formation under a phosphorus deficiency condition, the overexpression of the lupin root formation of the lupin is promoted, and the silencing or knockout of the lupin root formation of the lupin is inhibited under the phosphorus deficiency condition. The LaPUCHI1 gene is a key regulation and control gene for formation of root discharge of lupin in a phosphorus deficiency environment, and a valuable gene resource is provided for formation of root discharge of lupin in the phosphorus deficiency environment.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and particularly relates to the application of LaPUCHI1 gene and its encoded protein in regulating the root formation of white lupin under phosphorus deficiency conditions. Background Art

[0002] White lupine (Lupinus albus), also known as "lupine", is an annual or perennial herbaceous plant of the genus Lupinus in the subfamily Fabaceae, with ornamental and edible value. White lupine has a long flowering period and can be used as a good cut flower material. Its seeds are rich in protein and can be used as green manure feed. It is an important leguminous food crop.

[0003] Row roots refer to a group of brush-like, dense and limited root clusters formed on the lateral roots. White lupin can grow row roots in a phosphorus-deficient environment, releasing a large amount of organic acid to activate the insoluble phosphorus in the soil and achieve efficient utilization of phosphorus in the soil. Even without the application of phosphorus fertilizer, it can still grow normally, improve soil type and enhance soil fertility. It is a model plant for plant nutrition research. The morphological and physiological factors of white lupin in adapting to phosphorus deficiency have been widely studied. However, the molecular regulatory mechanism of efficient phosphorus utilization in white lupin remains unclear. Summary of the invention

[0004] The object of the present invention is to provide an application of the LaPUCHI1 gene and its encoded protein in regulating the root formation of white lupin under phosphorus deficiency conditions.

[0005] To achieve the above object, the present invention adopts the following technical solution: A first aspect of the present invention provides a LaPUCHI1 gene, the nucleotide sequence of the LaPUCHI1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by the LaPUCHI1 gene is shown as SEQ ID NO.2.

[0006] The second aspect of the present invention provides the use of the above-mentioned LaPUCHI1 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 root formation of white lupin under phosphorus deficiency conditions includes: overexpressing the LaPUCHI1 gene of white lupin to promote root formation of white lupin under phosphorus deficiency conditions; silencing or knocking out the LaPUCHI1 gene of white lupin to inhibit root formation of white lupin under phosphorus deficiency conditions.

[0007] The third aspect of the present invention provides a method for promoting root formation of white lupin under phosphorus deficiency conditions, comprising the steps of transforming a biological material overexpressing the LaPUCHI1 gene into white lupin to construct a transgenic white lupin plant overexpressing the LaPUCHI1 gene; the nucleotide sequence of the LaPUCHI1 gene is shown in SEQ ID NO.1; Furthermore, the promoting of white lupine root formation under phosphorus deficiency conditions refers to increasing the number of white lupine roots under phosphorus deficiency conditions.

[0008] The fourth aspect of the present invention provides a method for inhibiting root formation of white lupin under phosphorus deficiency conditions, comprising the steps of transforming a biological material with a silenced LaPUCHI1 gene into white lupin to construct a transgenic white lupin plant with a silenced LaPUCHI1 gene; the nucleotide sequence of the LaPUCHI1 gene is shown in SEQ ID NO.1; Furthermore, the inhibiting of white lupine root formation under phosphorus deficiency conditions refers to reducing the number of white lupine roots under phosphorus deficiency conditions.

[0009] The significant advantages of the present invention are: The present invention has found that the LaPUCHI1 gene is a key regulatory gene for the root formation of white lupin under a phosphorus-deficient environment. Overexpression of the LaPUCHI1 gene of white lupin promotes the root formation of white lupin under phosphorus-deficient conditions; silencing or knocking out the LaPUCHI1 gene of white lupin inhibits the root formation of white lupin under phosphorus-deficient conditions. The present invention provides valuable gene resources for the root formation of white lupin under a phosphorus-deficient environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 : a shows the dynamic changes of the expression level of LaPUCHI1 gene in the lateral roots of white lupin under normal conditions (NP) and phosphorus deficiency conditions (LP); b shows the schematic diagram of the rooting growth stages of white lupin under phosphorus deficiency conditions, where MPR represents lateral roots without lateral roots, PE represents the root segment before rooting, YCR represents the young rooting root segment, GAP represents the root segment without lateral roots between young and mature rooting roots, MCR represents the mature rooting root segment, and OCR represents the senescent rooting root segment; c shows the expression level of LaPUCHI1 gene in different root segments of lateral roots with lateral roots of white lupin under phosphorus deficiency conditions.

[0011] Figure 2 : a, phenotypic diagram of transgenic white lupin plants overexpressing the LaPUCHI1 gene; b, expression level of the LaPUCHI1 gene in transgenic white lupin plants overexpressing the LaPUCHI1 gene; c, number of roots in transgenic white lupin plants overexpressing the LaPUCHI1 gene.

[0012] Figure 3: a, phenotype of transgenic white lupin plants with LaPUCHI1 gene silenced; b, expression level of LaPUCHI1 gene in transgenic white lupin plants with LaPUCHI1 gene silenced; c, number of roots in transgenic white lupin plants with LaPUCHI1 gene silenced.

[0013] Note: 35S::PUCHI1 indicates the transgenic white lupin plant overexpressing the LaPUCHI1 gene, and TRV::PUCHI1 indicates the transgenic white lupin plant silencing the LaPUCHI1 gene. DETAILED DESCRIPTION

[0014] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0015] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0016] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0017] The nucleotide sequence of the white lupin LaPUCHI1 gene described 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.

[0018] The formula of the phosphorus-deficient nutrient solution described in the following embodiment is: MgSO4 54μM, Ca(NO3)2 400μM, K2SO4 200μM, Na-Fe-EDTA 10μM, H3BO3 2.4μM, MnSO4 0.24μM, ZnSO4 0.1μM, CuSO4 0.018μM, Na2MoO4 0.03μM.

[0019] The formula of the normal nutrient solution described in the following examples is to add KH2PO4 with a final concentration of 250 μM to the phosphorus-deficient nutrient solution.

[0020] The primer sequences used in the following examples are as follows: Example 1: Expression of the LaPUCHI1 gene in white lupin is induced by phosphorus deficiency In order to explore whether the LaPUCHI1 gene plays a role in the root formation of white lupin under phosphorus deficiency conditions, different phosphorus treatment experiments were set up in this example, and the culture environment was divided into phosphorus-deficient nutrient solution and normal nutrient solution. First, the seeds of white lupin were surface disinfected with 1.5% sodium hypochlorite for 7 minutes, and then rinsed with distilled water 5 times to wash off the disinfectant on the surface; then, a small amount of water and seeds were placed in a culture dish, and the seeds were allowed to germinate at room temperature for about 7 days; thereafter, the evenly growing plants were placed in a 10L bucket filled with nutrient solution for culture, and the nutrient solution was replaced every 5 days to ensure an adequate supply of nutrients. All lateral roots of white lupin plants under the two treatment conditions were collected after 2, 3, 4, 5, and 6 weeks of culture, respectively, for total RNA extraction, reverse transcription, and detection of LaPUCHI1 gene expression using primers LaPUCHI1-F and LaPUCHI1-R. The results are shown in the figure. Figure 1 As shown in a, the expression level of LaPUCHI1 gene in white lupin was significantly increased under phosphorus deficiency conditions, and the expression level of LaPUCHI1 gene increased with the extension of phosphorus deficiency treatment time, indicating that LaPUCHI1 gene was induced by phosphorus deficiency; Figure 1 As shown in b, under phosphorus deficiency conditions, white lupin will form row roots on the lateral roots, and about 30 to 40 clusters of row roots can be formed after about 10 days of cultivation. After a large number of row roots are formed on the lateral roots of white lupin plants in the phosphorus deficiency group, the lateral roots with row roots are collected and divided into different root segments for total RNA extraction and reverse transcription. The expression level of the LaPUCHI1 gene is detected using primers LaPUCHI1-F and LaPUCHI1-R. The results are shown in Figure 1 As shown in c, the expression level of LaPUCHI1 gene was highest in the root segment before root germination.

[0021] Example 2: Function of the LaPUCHI1 gene in white lupin To verify the effect of the LaPUCHI1 gene on the root formation of white lupin, this example created a transgenic white lupin root system by overexpressing the gene in a rooting experiment and virus-induced gene silencing (VIGS).

[0022] Primers pFGC5941-LaPUCHI1-F and pFGC5941-LaPUCHI1-R were designed according to the nucleotide sequence of the target gene to introduce AscI restriction sites at both ends of the gene, and the LaPUCHI1 gene in white lupin was amplified by PCR to obtain the PCR amplification product of the LaPUCHI1 gene; the pFGC5941 plasmid containing the CaMV 35S promoter was digested with restriction endonuclease AscI to obtain the pFGC5941 plasmid after digestion; the PCR amplification product of the LaPUCHI1 gene was connected with the pFGC5941 plasmid after digestion by seamless cloning to obtain the LaPUCHI1 gene overexpression vector. The LaPUCHI1 gene overexpression vector contains the nucleotide fragment shown in SEQ ID NO.3; the LaPUCHI1 gene overexpression vector was transformed into Agrobacterium K599 strain by heat shock method to obtain Agrobacterium containing the LaPUCHI1 gene overexpression vector. After disinfection, the seeds of white lupin were placed on moist filter paper for germination until a 2 cm embryo was grown, and then transferred to deionized water. After culturing in the dark for 3 days, the top of the main root (1 cm) was cut off, and the experimental group was infected with Agrobacterium containing the LaPUCHI1 gene overexpression vector, and the control group was infected with Agrobacterium containing an empty vector. They were cultured in the dark at room temperature on 0.5MS medium containing 100 mM acetobutyric acid for 3 days, and then cultured in phosphorus-deficient nutrient solution for 3 weeks. After the cultivation, the positive seedlings were screened by PCR amplification using primers eGFP-F and eGFP-R, and the relative expression of the target gene in the hairy roots was detected by qPCR using primers 35S::LaPUCHI1-F and 35S::LaPUCHI1-R to further verify the positive seedlings; the number of rows of roots formed by the white lupin plants was recorded. Figure 2 a is the phenotype of the control white lupine plant and the white lupine plant with overexpression of LaPUCHI1 gene. Figure 2 As shown in b, compared with the control white lupine plants, the relative expression level of the LaPUCHI1 gene in the hairy roots of the white lupine plants with overexpression of the LaPUCHI1 gene was significantly upregulated. Figure 2 As shown in c, compared with the control white lupine plants, the number of rafting roots of the white lupine plants overexpressing the LaPUCHI1 gene increased by 52.4%, indicating that the significant upregulation of the LaPUCHI1 gene promoted the formation of rafting roots of white lupine under phosphorus deficiency conditions.

[0023] Primers pTRV2-LaPUCHI1-F and pTRV2-LaPUCHI1-R were designed according to the nucleotide sequence of the target gene, and PCR amplification was performed using the cDNA of the lateral root of white lupin as a template to obtain a LaPUCHI1 gene silencing fragment with an XbaI restriction site; the pTRV2 vector was digested with the restriction endonuclease XbaI to obtain a pTRV2 vector after digestion; the LaPUCHI1 gene silencing fragment with the XbaI restriction site was connected with the pTRV2 vector after digestion through seamless cloning to obtain a pTRV2-LaPUCHI1 vector, and the pTRV2-LaPUCHI1 vector contained a nucleotide fragment as shown in SEQ ID NO.4; the pTRV2-LaPUCHI1 vector was transferred into GV3101 Agrobacterium to obtain a pTRV2-LaPUCHI1 strain; the pTRV1 vector and the pTRV2 vector were respectively transferred into GV3101 Agrobacterium to obtain a pTRV1 strain and a pTRV2 strain, respectively. pTRV2-LaPUCHI1 strain, pTRV1 strain, and pTRV2 strain were grown in YEB medium containing corresponding antibiotics, and the growth environment temperature was set at 28°C. After overnight culture, the cells were collected and resuspended in an infiltration buffer containing 10 mM 2-N-morpholineethanesulfonic acid, 0.1 mM acetobutyric acid, and 10 mM MgCl2 to a final OD 600 =0.5, and cultured at room temperature for 2 hours. After disinfection, the seeds of white lupin were placed on moist filter paper for germination until a 2 cm embryo grew out, and then transferred to deionized water. After culturing in the dark for 3 days, the top of the main root (1 cm) was cut off. The experimental group mixed the pTRV1 resuspension with the pTRV2-LaPUCHI1 resuspension in a volume ratio of 1:1 and used it to soak the roots of white lupin plants for 16 hours. The control group mixed the pTRV1 resuspension with the pTRV2 resuspension in a volume ratio of 1:1 and used it to soak the roots of white lupin plants for 16 hours. After the soaking, the roots of the white lupin plants were rinsed with sterile water and then transferred to a phosphorus-deficient nutrient solution for 3 weeks. After the cultivation, the positive seedlings were screened by PCR amplification using primers positive-LaPUCHI1-F and positive-LaPUCHI1-R, and the relative expression level of the target gene in the hairy roots was detected by qPCR using primers TRV-LaPUCHI1-F and TRV-LaPUCHI1-R to further verify the positive seedlings; the number of rows of roots formed by the white lupin plants was recorded. Figure 3 a is the phenotype of the control white lupine plant and the LaPUCHI1 gene silenced white lupine plant. Figure 3 As shown in b, compared with the control white lupine plants, the relative expression level of the LaPUCHI1 gene in the LaPUCHI1 gene silenced white lupine plants was significantly downregulated. Figure 3As shown in c, compared with the control white lupine plants, the number of rafting roots of the LaPUCHI1 gene-silenced white lupine plants decreased by 71.5%, indicating that the silencing of the LaPUCHI1 gene inhibited the formation of rafting roots of white lupine under phosphorus deficiency conditions.

[0024] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A LaPUCHI1 A gene characterized by: Said LaPUCHI1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. According to claim 1 LaPUCHI1 A gene characterized by: Said LaPUCHI1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. The method according to claim 1 LaPUCHI1 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 3, characterized in that: The method for regulating the root formation of white lupine under phosphorus deficiency conditions comprises: overexpressing LaPUCHI1 Gene, promotes root formation in white lupine under phosphorus deficiency; silences or knocks out the LaPUCHI1 Gene, inhibiting root formation in white lupin under phosphorus deficiency conditions.

6. A method for promoting root formation of white lupin under phosphorus deficiency conditions, characterized in that: Including overexpression LaPUCHI1 The biological material of the gene was transformed into white lupin, and the overexpression was obtained LaPUCHI1 The steps of producing transgenic white lupin plants with the gene; LaPUCHI1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

7. The method according to claim 6, characterized in that: The promoting of white lupine root formation under phosphorus deficiency conditions refers to increasing the number of white lupine roots under phosphorus deficiency conditions.

8. A method for inhibiting root formation of white lupin under phosphorus deficiency conditions, characterized in that: Including silence LaPUCHI1 The biological material of the gene was transformed into white lupin, and the construct was silenced LaPUCHI1 The steps of producing transgenic white lupin plants with the gene; LaPUCHI1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

9. The method according to claim 8, characterized in that: The inhibition of white lupine root formation under phosphorus deficiency conditions refers to reducing the number of white lupine roots under phosphorus deficiency conditions.

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