Phosphatase-encoding gene ecpap19 and application thereof

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to a phosphatase coding gene EcPAP19 and application thereof. The nucleotide sequence of the phosphatase coding gene EcPAP19 is shown as SEQ ID NO:1. In the examples, the acid phosphatase gene EcPAP19 is cloned from a phoenix eye blue, and it is found that, under the condition of organic phosphorus, the control rice has poor utilization of organic phosphorus, thus leading to growth limitation due to lack of phosphorus; and the transgenic rice can utilize organic phosphorus, and has no obvious change in growth compared with normal culture medium, which indicates that the EcPAP19 gene is a key gene for promoting absorption of phosphorus elements by plants. The application has important application value in enhancing absorption of phosphorus by plants and breeding of new varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a phosphatase-encoding gene EcPAP19 and its applications. Background Technology

[0002] Phosphorus is an essential nutrient for all life on Earth. In plants, it is a key element involved in photosynthesis and respiration, and a major element in the synthesis of nucleic acids and plasma membranes. However, plants can only obtain phosphorus passively from the environment through their roots by absorbing inorganic phosphates. Therefore, the utilization rate of phosphate fertilizers in crops is generally less than 25%, making it crucial to improve phosphate fertilizer utilization efficiency for crops.

[0003] Therefore, in-depth exploration of the key genes regulating phosphorus absorption in plants is of great significance for understanding the molecular mechanisms of phosphorus absorption in plants and for cultivating new plant varieties that utilize phosphorus efficiently. Summary of the Invention

[0004] The purpose of this invention is to provide a phosphatase-encoding gene, EcPAP19, and its applications. By overexpressing the EcPAP19 gene in rice, it was found that transgenic rice can utilize organic phosphorus, indicating that the EcPAP19 gene is a key gene promoting phosphorus absorption in plants.

[0005] The present invention provides a phosphatase-encoding gene EcPAP19, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0006] This invention also provides the application of the phosphatase-encoding gene EcPAP19 described above in enhancing phosphorus absorption in plants and / or creating new germplasm that enhances phosphorus absorption.

[0007] The present invention also provides primer pairs for amplifying the phosphatase-encoding gene EcPAP19 described above, wherein the primer pairs include a forward primer and a reverse primer;

[0008] The nucleotide sequence of the forward primer is shown in SEQ ID NO:2;

[0009] The nucleotide sequence of the reverse primer is shown in SEQ ID NO:3.

[0010] The present invention also provides a biological material overexpressing the phosphatase-encoding gene EcPAP19 described above, the biological material comprising one or more of the following: a recombinant expression vector containing the phosphatase-encoding gene EcPAP19, a recombinant microorganism containing the phosphatase-encoding gene EcPAP19, a transgenic cell line containing the phosphatase-encoding gene EcPAP19, a recombinant microorganism containing the recombinant expression vector, and a transgenic cell line containing the recombinant expression vector.

[0011] As a preferred embodiment, the basic framework of the recombinant expression vector containing the phosphatase encoding gene EcPAP19 includes the pBWA(V)HS vector.

[0012] This invention also provides the application of the primer pairs or biomaterials described above in enhancing the absorption of phosphorus by plants.

[0013] This invention also provides the application of the primer pairs or biological materials described above in the creation of new germplasm with enhanced phosphorus absorption.

[0014] The present invention also provides a method for enhancing the absorption of phosphorus by plants, comprising the following steps: overexpressing the phosphatase-encoding gene EcPAP19 in the genome of a target plant to obtain a plant with enhanced phosphorus absorption; the nucleotide sequence of the phosphatase-encoding gene EcPAP19 is shown in SEQ ID NO:1.

[0015] This invention also provides a method for creating new germplasm that enhances phosphorus absorption, characterized by comprising the following steps: overexpressing the phosphatase-encoding gene EcPAP19 in the genome of a target plant to obtain the new germplasm that enhances phosphorus absorption; the nucleotide sequence of the phosphatase-encoding gene EcPAP19 is shown in SEQ ID NO:1.

[0016] As a preferred option, the plant includes rice.

[0017] Beneficial Effects: This invention provides a phosphatase-encoding gene, EcPAP19, the nucleotide sequence of which is shown in SEQ ID NO:1. In one embodiment, the acid phosphatase gene EcPAP19 was cloned from water hyacinth. By overexpressing the EcPAP19 gene in rice, it was found that under organic phosphorus conditions, control rice had poor utilization of organic phosphorus, resulting in phosphorus deficiency and limited growth; while transgenic rice could utilize organic phosphorus, and its growth was not significantly different from that in normal culture medium, indicating that the EcPAP19 gene is a key gene for promoting phosphorus absorption in plants. This invention has important application value in enhancing phosphorus absorption in plants and in the breeding of new varieties. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 Phylogenetic tree of the EcPAP19 protein;

[0020] Figure 2 Subcellular localization of EcPAP19 protein;

[0021] Figure 3 This study describes the utilization of organic and inorganic phosphorus by genetically modified rice and wild-type rice. Detailed Implementation

[0022]

[0023] In one specific embodiment, the phosphatase encoding gene EcPAP19 is cloned from the water hyacinth plant and is the purple acid phosphatase encoding gene EcPAP19 for water hyacinth. In another specific embodiment, acid phosphatase is a family of hydrolytic enzymes universally present in organisms, capable of hydrolyzing organic phosphorus compounds under acidic conditions. When plants are in low inorganic phosphate conditions, their roots can secrete acid phosphatase to degrade organic phosphorus compounds into inorganic phosphates for plant absorption. Purple acid phosphatase is an important component of acid phosphatases, named for the purple color of its aqueous solution. It has relatively wide pH adaptability and temperature stability, and can act on a wide range of substrates. Under weakly acidic conditions with a pH of 4.0 to 7.0, purple acid phosphatase can catalyze the hydrolysis of phosphate monoesters and acid anhydrides, releasing inorganic phosphates that plants can absorb, thereby improving phosphorus utilization efficiency in soil or water and playing a key role in plant phosphorus absorption and utilization.

[0024] This invention also provides the application of the aforementioned phosphatase-encoding gene EcPAP19 in enhancing phosphorus uptake in plants and / or creating new germplasm with enhanced phosphorus uptake. As a specific embodiment, this invention cloned an acid phosphatase EcPAP19 from the plant water hyacinth. Transcriptome and quantitative fluorescence expression analysis showed that it plays an important role in the phosphorus uptake process of water hyacinth.

[0025] The present invention also provides primer pairs for amplifying the phosphatase-encoding gene EcPAP19 described above, wherein the primer pairs include a forward primer and a reverse primer;

[0026] The nucleotide sequence of the forward primer is shown in SEQ ID NO:2;

[0027] The nucleotide sequence of the reverse primer is shown in SEQ ID NO:3.

[0028] The sequences of the primer pairs described in this invention are as follows:

[0029] Forward primer (SEQ ID NO:2): 5'-ATGGCCGGGAGGTCCTCAGG-3';

[0030] Reverse primer (SEQ ID NO:3): 5'-TACAGAGCTGGGTTCAGGTGAAG-3'.

[0031] This invention also provides a biological material overexpressing the aforementioned phosphatase-encoding gene EcPAP19. The biological material includes one or more of the following: a recombinant expression vector containing the phosphatase-encoding gene EcPAP19; a recombinant microorganism containing the phosphatase-encoding gene EcPAP19; a transgenic cell line containing the phosphatase-encoding gene EcPAP19; a recombinant microorganism containing the recombinant expression vector; and a transgenic cell line containing the recombinant expression vector. The basic framework of the recombinant expression vector containing the phosphatase-encoding gene EcPAP19 of this invention includes the pBWA(V)HS vector. As one embodiment, the recombinant expression vector containing the phosphatase-encoding gene EcPAP19 includes inserting the EcPAP19 gene into the pBWA(V)HS vector to obtain the pBWA(V)HS-EcPAP19 overexpression vector. As one embodiment, the recombinant microorganism containing the phosphatase-encoding gene EcPAP19 includes Agrobacterium GV3101 containing the EcPAP19 gene.

[0032] This invention also provides the application of the primer pairs or biomaterials described above in enhancing the absorption of phosphorus by plants.

[0033] This invention also provides the application of the primer pairs or biological materials described above in the creation of new germplasm with enhanced phosphorus absorption.

[0034] This invention also provides a method for enhancing phosphorus absorption in plants, comprising the following steps: overexpressing the phosphatase-encoding gene EcPAP19 in the genome of a target plant to obtain plants with enhanced phosphorus absorption; the nucleotide sequence of the phosphatase-encoding gene EcPAP19 is shown in SEQ ID NO:1. In an embodiment of this invention, the pBWA(V)HS-EcPAP19 overexpression vector is transformed into Agrobacterium GV3101, and the overexpression vector is transformed into the genome of the target plant using an Agrobacterium-mediated transgenic method to obtain plants with enhanced phosphorus absorption.

[0035] This invention also provides a method for creating new germplasm with enhanced phosphorus absorption, characterized by the following steps: overexpressing the phosphatase-encoding gene EcPAP19 in the genome of a target plant to obtain the new germplasm with enhanced phosphorus absorption; the nucleotide sequence of the phosphatase-encoding gene EcPAP19 is shown in SEQ ID NO:1. In an embodiment of this invention, the pBWA(V)HS-EcPAP19 overexpression vector is transformed into Agrobacterium GV3101, and the overexpression vector is transformed into the genome of the target plant using an Agrobacterium-mediated transgenic method to obtain the new germplasm with enhanced phosphorus absorption. The plant mentioned in this invention includes rice.

[0036] Specific embodiments of the present invention show that after overexpressing the phosphatase-encoding gene EcPAP19 in Nipponbare rice, under organic phosphorus conditions, the control rice had poor utilization of organic phosphorus, resulting in phosphorus deficiency and restricted growth; while the transgenic rice could utilize organic phosphorus, and its growth was not significantly different from that in the normal culture medium. This indicates that the transgenic rice can secrete the EcPAP19 protein and deliver it outside the root system, hydrolyzing organic phosphorus in the water into inorganic phosphate, thus promoting the plant's absorption of phosphorus.

[0037] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a phosphatase-encoding gene EcPAP19 and its applications provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0038] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, and commercially available products well known to those skilled in the art can be used.

[0039] Example

[0040] 1. Extraction of total RNA and synthesis of cDNA from water hyacinth: Water hyacinth samples were flash-frozen in liquid nitrogen and ground into powder. Total RNA was extracted using the RNA Simple Total RNA Kit (Beijing Tiangen Biotech Co., Ltd.). The total RNA was reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novizan Biotechnology Co., Ltd.).

[0041] 2. Cloning of the water hyacinth blue acid phosphatase gene EcPAP19: The sequence of the water hyacinth blue EcPAP19 gene is shown in SEQ ID NO:1. A pair of primers were designed based on this sequence: forward primer: 5'-ATGGCCGG GAGGTCCTCAGG-3' (SEQ ID NO:2), and reverse primer: 5'-TACAGAGCTGGGTTC AGGTGAAG-3' (SEQ ID NO:3). PCR amplification was performed using the cDNA reverse transcribed in step 1 as a template. The PCR reaction conditions were: 94℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 55℃ annealing for 5 s, 72℃ extension for 30 s, for a total of 30 cycles; and 72℃ extension for 10 min. PCR products were subjected to 1% agarose gel electrophoresis. Bands of the correct length were excised under a gel imaging system and recovered using FastPure Gel DNA Extraction Mini Kit-DC301 (Nanjing Novizan Biotechnology Co., Ltd.). PCR was then performed using primers with homologous arms (CS554-EcPAP19-F: 5'-catttggagaggacacgcATGGCCGG GAGGTCCTCAGG-3' (SEQ ID NO:4); CS554-EcPAP19-R: 5'-tcgcccttgctcac catgaaTACAGAGCTGGGTTCAGGTGAAG-3' (SEQ ID NO:5), where lowercase letters represent homologous arms). After length detection by electrophoresis, the gel was excised and recovered using the same procedure as above. The recovered fragments were stored at -20℃.

[0042] 3. Sequence alignment and phylogenetic tree construction: Protein sequences of all PAP gene family members in water hyacinth, pickerelweed, Arabidopsis thaliana and rice were aligned using MAFFT, and then the aligned sequence files were used to construct a tree using IQ-TREE. The parameters were modified to make the tree construction method based on the most suitable model after calculation, and the Bootstrap value was 1000.

[0043] 4. Real-time quantitative PCR reaction: Total RNA from different parts of *Eichhornia crassipes* (strawberries, roots, and leaves) under different phosphorus concentrations (DP: 0 mg / L, LP: 5.0 mg / L, MP: 50 mg / L, HP: 100 mg / L) was used as a template (RNA extraction was performed according to step 1). Forward primer: 5'-AGCACAGATTCCATACGCCTTAC-3' (SEQ ID NO: 6), reverse primer: 5'-CGTCTCGCTTCGGTTCACTT-3' (SEQ ID NO: 7) were used as primers. Real-time quantitative PCR was performed using ChamQ SYBR qPCR Master Mix (Nanjing Novizan Biotechnology Co., Ltd.) with EcUBC as an internal control gene. Genomic DNA was removed from the extracted total RNA using the following mixture: RNase-free ddH2O 16 μL, 4×g DNAwiperMix 4 μL, RNA 1 pg. -1 μg. The reverse transcription reaction system consisted of 4 μL of 5×HiScriptⅢqRT SuperMix and 16 μL of the first step reaction solution. The reaction temperature was 37℃ for 15 min, followed by 85℃ for 5 s.

[0044] 5. Subcellular localization: pART-CAM-EGFP-CS544 (Nanjing Jisi Huiyuan Biotechnology Co., Ltd.) was double-digested using two fast digestion enzymes, EcoRI and XhoI (Thermo Fisher Scientific). The digestion regimen was as follows: 1 μg plasmid, 2 μL 10×FastDigest green buffer, 1 μL XhoI, 1 μL EcoRI, and ddH2O to 20 μL. The mixture was placed in a PCR instrument and digested at 37℃ for 15 min. The digestion products were subjected to 1% agarose gel electrophoresis and recovered by gel imaging (same as step 2). The pART-CAM-EGFP-CS544-EcPAP19 recombinant plasmid was constructed using homologous recombination. The system used was 300 ng linearized vector, 60 ng insert fragment, 5 μL 2×CE Mix, and ddH2O to a final volume of 10 μL. The plasmid was cloned using DH5α and transformed into Agrobacterium GV3101 (Nanjing Enzyme-Proly Biotechnology Co., Ltd.). The steps were as follows: 1) The GV3101 competent cells were removed from -80℃ and quickly thawed on ice for 5 min. 2) The pART-CAM-EGFP-CS544-EcPAP19 recombinant plasmid was added and mixed well. The mixture was then incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and in an ice bath for 5 min. 3) 900 μL of antibiotic-free YEB liquid medium was added, mixed well, and cultured at 28℃ with shaking at 200 rpm for 3 h. 4) The culture was centrifuged at 6000 rpm for 1 min, 900 μL of supernatant was discarded, and 100 μL of supernatant was used to gently resuspend the bacterial cells. The cells were then spread onto YEB plates containing 1‰ Kan and incubated upside down at 28℃ for 3 days. The transformed bacterial suspension was evenly spread on YEB solid medium containing 1‰ Rif and 1‰ Kan, and incubated at 28°C for 60 h. A single colony was picked with a sterile pipette tip and placed in a 2 mL sterile centrifuge tube. 1 mL of YEB liquid medium containing 1‰ Rif and 1‰ Kan was added, and the mixture was incubated at 28°C and 200 rpm until the suspension became turbid. 100 μL of the bacterial suspension was transferred to a sterile 150 mL Erlenmeyer flask, and 50 mL of YEB liquid medium containing 1‰ Rif and 1‰ Kan was added. The mixture was incubated at 28°C and 200 rpm until OD reached [value missing]. 600 To a concentration of 0.8, transfer the bacterial culture to a 50 mL sterile centrifuge tube, centrifuge at 3000 rpm for 10 min, and discard the supernatant. Resuspend the culture in a resuspension solution containing 10 mM MgCl2, 10 mM MES, and 100 μM acetylsylcholine to an OD value of 0.8. 600 The concentration was 1.0. After the resuspension was left to stand in the dark for 3 hours, it was injected into the tobacco leaves from the underside using a sterile syringe. After overnight incubation for 12 hours, it was incubated under normal light conditions for 48 hours and observed under a confocal microscope.

[0045] 6. Transgenic Validation: Transgenic Vector Construction and Rice Transformation: The EcPAP19 gene was amplified using specific primers (forward primer: 5'-ATGGCCGGGAGGTCCTCAGG-3'(SEQ ID NO:2), reverse primer: 5'-TACAGAGCTGGGTTCAGGTGAAG-3'(SEQ ID NO:3)), and then constructed into the pBWA(V)HS vector (Li Q, Xu J, Yang L, Zhou X, Cai Y and Zhang Y (2020) Transcriptome Analysis of Different Tissues Reveals Key Genes Associated With Galanthamine Biosynthesis in Lycoris longituba. Front. Plant Sci. 11:519752. doi:10.3389 / fpls.2020.519752), forming the 35S overexpression vector pBWA(V)HS-EcPAP19. The recombinant vector pBWA(V)HS-EcPAP19 was transformed into Agrobacterium strain GV3101 via electroporation (method as in step 5), and then transformed into the rice variety Nipponbare using the callus method (Hiei, Y., Ohta, S., Komari, T., and Kumashiro, T. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6:271-282.). Nipponbare rice containing the empty vector pBWA(V)HS-gbd was used as a control. Control and transgenic plants were cultured in Yoshida medium (Coolaber) for complete nutrient rice cultivation and in Yoshida medium with organic phosphorus replacing inorganic phosphorus, respectively. Rice growth was observed after 7 days of cultivation.

[0046] 7. Experimental Results

[0047] 1) The protein sequence encoded by the EcPAP19 gene cloned in this invention was compared with the protein sequences of other PAPs from water hyacinth, rice, pickerelweed, and Arabidopsis, and a phylogenetic tree was constructed. The results showed that EcPAP19 is most closely related to PcPAP24 of pickerelweed and OsPAP10 of rice. Figure 1 ).

[0048] 2) Under phosphorus-deficient conditions, EcPAP19 was expressed in large quantities in the roots of water hyacinth, indicating that it plays an important role in the process of phosphorus absorption in the roots (Table 1).

[0049] 3) Subcellular localization: The EcPAP19 protein is located extracellularly, indicating that it can be secreted outside the root system. Figure 2 ).

[0050] 4) EcPAP19 can be expressed in the rice variety Nipponbare (Table 2). Under organophosphorus conditions, the control rice showed poor utilization of organophosphorus, leading to phosphorus deficiency and restricted growth; while the transgenic rice could utilize organophosphorus, and its growth was not significantly different from that in the normal culture medium (Table 3 and 4). Figure 3 This indicates that transgenic rice can secrete the EcPAP19 protein and deliver it outside the root system, hydrolyzing organic phosphorus in the water into inorganic phosphate, thus promoting the plant's absorption of phosphorus.

[0051] Table 1. Expression level analysis of EcPAP19 gene in different tissues of water hyacinth.

[0052] creeping stem DP 3.245 1.214 a creeping stem LP 0.552 0.217 b creeping stem MP 0.373 0.156 b creeping stem HP 0.410 0.260 b root DP 16.955 6.507 a root LP 2.604 0.563 b root MP 3.249 1.387 b root HP 0.945 0.607 b leaf DP 4.272 2.081 a leaf LP 0.364 0.087 b leaf MP 0.141 0.217 b leaf HP 0.136 0.130 b

[0053] Table 2. Expression of the EcPAP19 gene in transgenic rice.

[0054] CK Complete Nutrition 1 0.000 0.000 CK Complete Nutrition 2 0.000 0.000 CK Complete Nutrition 3 0.000 0.000 CK Organophosphorus 1 0.000 0.000 CK Organophosphorus 2 0.000 0.000 CK Organophosphorus 3 0.000 0.000 Genetically Modified Complete Nutrition 1 9316.174 962.421 Genetically Modified Complete Nutrition 2 34606.265 3054.709 Genetically Modified Complete Nutrition 3 76261.748 1977.358 Genetically modified organophosphate 1 39497.078 4569.282 Genetically modified organophosphate 2 45710.420 6823.594 Genetically modified organophosphate 3 5537.109 420.801

[0055] Table 3. Utilization of organic and inorganic phosphorus by transgenic rice and wild-type rice.

[0056] CK Organophosphorus 24.7 1.99833 b CK Complete Nutrition 29.125 1.98053 a Genetically modified organophosphates 30.6 1.57692 a Genetically modified complete nutrition 29.15 0.91469 a

[0057] Therefore, this invention cloned the acid phosphatase gene EcPAP19 from water hyacinth. By overexpressing the EcPAP19 gene in rice, it was found that under organic phosphorus conditions, control rice utilized organic phosphorus poorly, leading to phosphorus deficiency and restricted growth; while transgenic rice could utilize organic phosphorus, and its growth was not significantly different from that in normal culture medium. This indicates that the EcPAP19 gene is a key gene for promoting phosphorus absorption in plants. This invention has significant application value in enhancing phosphorus absorption in plants and in the breeding of new varieties.

[0058] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A phosphatase-encoding gene EcPAP19 Its characteristics are, The phosphatase encoding gene EcPAP19 The nucleotide sequence is shown in SEQ ID NO:

1.

2. The phosphatase-encoding gene according to claim 1 EcPAP19 Its application in enhancing rice's absorption of organophosphorus compounds and / or preparing new rice germplasm with enhanced organophosphorus absorption is characterized by... The application involves overexpressing the phosphatase-encoding gene in rice. EcPAP19 .

3. An overexpression method for the phosphatase-encoding gene of claim 1 EcPAP19 The biomaterial is characterized by, The biological material contains the phosphatase encoding gene. EcPAP19 Recombinant expression vectors or containing the phosphatase-encoding gene EcPAP19 Transgenic cell lines.

4. The biomaterial according to claim 3, characterized in that, Contains the phosphatase encoding gene EcPAP19 The basic framework of the recombinant expression vector includes the pBWA(V)HS vector.

5. The application of the biomaterial described in claim 3 or 4 in enhancing the absorption of organophosphorus compounds in rice.

6. The application of the biomaterial described in claim 3 or 4 in the preparation of new rice germplasm with enhanced organophosphorus absorption.

7. A method for enhancing the absorption of organophosphorus compounds in rice, characterized in that, Includes the following steps: Overexpression of phosphatase-encoding genes in the rice genome EcPAP19 This yields rice with enhanced organophosphate absorption; the phosphatase encoding gene... EcPAP19 The nucleotide sequence is shown in SEQ ID NO:

1.

8. A method for preparing new rice germplasm with enhanced organophosphorus absorption, characterized in that, Includes the following steps: Overexpression of phosphatase-encoding genes in the rice genome EcPAP19 The new rice germplasm with enhanced organophosphorus uptake was obtained; the phosphatase encoding gene EcPAP19 The nucleotide sequence is shown in SEQ ID NO:1.

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