CN116218868A - Populus deltoides low-phosphorus-resistant gene PdPHT1-2 and encoding protein and application thereof - Google Patents

Abstract

The invention discloses a low phosphorus resistant gene PdPHT1-2 of aspen, and a coding protein and application thereof, belonging to the field of plant molecular biology. The invention clones the gene of the mountain new poplar pdPHT1 family into the gene of the pdPHT1-2, constructs a plant expression vector pBI121-pdPHT1-2-3HA, transfers the plant expression vector into arabidopsis through an inflorescence dip-dyeing method, finally obtains an arabidopsis transgenic plant over-expressing the gene of the mountain new poplar pdPHT1-2, carries out iterative screening to obtain a homozygous strain, carries out phenotypic observation, and discovers that the transgenic plant is compared with a wild type control under each phosphorus concentration treatment gradient: the overground parts grow higher than the wild parts, the root system is longer, the density is higher, the fresh weight is obviously increased, and a theoretical basis is provided for the efficient molecular improvement breeding of poplar phosphorus.

Description

Populus deltoides low-phosphorus-resistant gene PdPHT1-2 and encoding protein and application thereof

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to a low phosphorus resistance gene PdPHT1-2 of mountain new poplar, and a coding protein and application thereof.

Background

The mountain new poplar is an excellent tree species obtained by hybridization with the mountain poplar (P.davidiana) as a female parent and the Xinjiang poplar (P.alba var. Pyramida) as a male parent, has the advantages of high growth speed, strong adaptability, good stress resistance, natural falling of the fruit sequence without flying flock, and high economic value and ornamental value.

Plant PHT1 phosphotransporter family members play an important role in soil Pi (inorganic phosphorus) uptake and Pi mobilization in plants. Since most of the soil has an effective phosphorus concentration of 1-10uM, which is far lower than that required for normal growth and development of plants, the phosphorus absorption capacity of plants under the condition of low effective phosphorus concentration affects the tolerance of the plants to low phosphorus stress. The acquisition and transportation of phosphate have a great Pi concentration difference inside and outside the membrane, even if the intracellular Pi concentration is more than one thousand times of the extracellular concentration, the plant can still obtain Pi through the high-affinity phosphorus transporter, so that the condition of plant phosphorus deficiency caused by low effective phosphorus concentration of soil is relieved, and the PHT1 family gene is less explored in mountain new poplar at present.

Disclosure of Invention

Aiming at the defects existing in the prior art, the first technical problem to be solved by the invention is to provide a low phosphorus resistant gene PdPHT1-2 of mountain new poplar; the second technical problem to be solved by the invention is to provide the protein encoded by the low phosphorus resistance gene PdPHT1-2 of mountain new poplar; the third technical problem to be solved by the invention is to provide the application of the shanxin poplar low phosphorus resistance gene PdPHT1-2 in improving the low phosphorus resistance of plants.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the low phosphorus resistance gene PdPHT1-2 of the populus tremulosa has a nucleotide sequence shown as SEQ ID NO.1 or a DNA sequence encoding SEQ ID NO. 2.

The amino acid sequence of the protein coded by the low phosphorus resistance gene PdPHT1-2 of the populus tremuloides is shown as SEQ ID NO. 2.

Fusion proteins obtained by ligating a tag at the N-terminus or/and the C-terminus of the protein shown in SEQ ID NO.2 are also within the scope of the present invention.

A recombinant expression vector or a recombinant strain containing the low phosphorus resistant gene PdPHT1-2 of the aspen.

The application of the shanxin poplar low phosphorus resistance gene PdPHT1-2 in improving the low phosphorus resistance of plants.

Further, the low phosphorus resistant gene PdPHT1-2 of the populus euphratica is sequentially constructed on an entry vector and a target vector by using a GATEWAY technology to obtain a plant expression vector, then a plant is transformed by using an agrobacterium-mediated genetic transformation method, and finally the transgenic plant with the low phosphorus resistant performance is obtained through screening and identification.

Further, the plants include arabidopsis thaliana and aspen.

Further, the plant expression vector is pBI121-PdPHT1-2-3HA.

Compared with the prior art, the invention has the beneficial effects that:

the invention clones the gene of the mountain new poplar pdPHT1 family into the gene of the pdPHT1-2, constructs a plant expression vector pBI121-pdPHT1-2-3HA, transfers the plant expression vector into arabidopsis through an inflorescence dip-dyeing method, finally obtains an arabidopsis transgenic plant over-expressing the gene of the mountain new poplar pdPHT1-2, carries out iterative screening to obtain a homozygous strain, carries out phenotypic observation, and discovers that the transgenic plant is compared with a wild type control under each phosphorus concentration treatment gradient: the overground parts grow higher than the wild parts, the root system is longer, the density is higher, the fresh weight is obviously increased, and a theoretical basis is provided for the efficient molecular improvement breeding of poplar phosphorus.

Drawings

FIG. 1 is a graph showing the LR reaction result of PdPHT 1;

FIG. 2 is a graph showing the result of detection of PdPHT1 transgene;

FIG. 3 is a graph showing the results of iterative resistance screening of progeny of transgenic homozygotes of PdPHT1-2 Arabidopsis;

FIG. 4 is a graph showing the results of identifying the transgenic phenotype of the PdPHT1-2 gene; in the figure, A is the growth phenotype of the overground part of the plant under different phosphorus concentration gradient treatments; b is the phenotype of plant root systems under different phosphorus concentration gradient treatments.

FIG. 5 is a graph showing the results of identifying the transgenic phenotype of the PdPHT1-2 gene; in the graph, A is the fresh plant quality under different phosphorus concentration gradient treatments; b is the increasing trend of the fresh quality of plants under the gradient treatment of different phosphorus concentrations;

FIG. 6 is a graph showing the results of seed germination differences under different phosphorus concentration gradient treatments; in the figure, a: sowing for 3 days; b: sowing for 5 days; c: germination rate; d: sowing for 10 days.

Detailed Description

The invention is further described below in connection with specific embodiments. The molecular biology experimental methods not specifically described in the following examples can be carried out by referring to the methods listed in the "molecular cloning Experimental guidelines (third edition) J.Sam Brookfield or the methods conventional in the art, or according to the kit and the product instructions. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The plant materials adopted by the invention are as follows: arabidopsis thaliana (Arabidopsis thaliana), populus deltoides (P.davidiana. Times.P.bolleana) tissue culture seedlings were irradiated at 25℃for 16h.

The carrier and the strain adopted by the invention are as follows: the vector pMD 19-T vector was purchased from Dalianbao bioengineering company (Takara); pBI121-des-3HA was saved by the university of Nanjing forestry, and E.coli strain Top10 and Agrobacterium GV3101 were purchased from the department of Optimago, inc.

Example 1

1. Cloning of Populus aspen PdPHT1 Gene

(1) Primer design

And (3) taking the related gene sequences screened by the populus tomentosa genome database as a reference, carrying out Primer design by using an online sequence Primer3, and verifying the designed primers by using NCBI-Primer Blast.

PHT1-2-F：5’-ATGGCTAGGGACCAATTGGTCGTC-3’；

PHT1-2-R：5’-CACTGAAGCCATCCTAGCTGAAGC-3’。

(2) Gene cloning and recovery of target fragment

Using a cDNA mixture sample synthesized by reverse transcription as a template, cloning of a gene was performed using KOD high-fidelity enzyme. The reaction system is as follows: eDNA 2. Mu.l, PHT1-2-F Primer (10. Mu.M) 1.5. Mu.l, PHT1-2-R Primer (10. Mu.M) 1.5. Mu.l, 10 XKOD Buffer 5. Mu.l, mgSO ₄ 3μl、dNTP Mixture(10mM each)5μl、KOD-Plus 1μl、ddH ₂ O31. Mu.l, total volume 50. Mu.l. The reaction procedure is: 94 ℃ for 3min;94 ℃ for 30s, 58-62 ℃ for 30s and 68 ℃ for 2min, and 38 cycles are carried out; 20min at 68 ℃; preserving at 4 ℃.

2. Construction of plant expression vectors

The overexpression vector was constructed using the GATEWAY technique, i.e.the fragment of interest in the entry vector (linker-7) was transferred to the protoplast expression vector pBI121-des-3HA by LR reaction.

The LR reaction system is as follows: entry vector 50ng, expression vector 75ng, LR close ^TM II Plus enzyme mix 0.5μl、ddH ₂ O2. Mu.l, total volume was 2.5. Mu.l. The reaction procedure is: and 1.5h at 25 ℃.

3. Transgenic detection and homozygote acquisition

PCR detection was performed using a 35S promoter using an upstream primer (35S-unitary-F) and a downstream specific primer (PdPHT 1-2-R) for the PdPHT1-2 gene, and the 1% gel electrophoresis detection result is shown in FIG. 2, and the band length matches the target fragment length, thus preliminarily proving that the PdPHT1 gene has been transferred into Arabidopsis.

Screening out the positive arabidopsis seedlings by using the screening pressure of antibiotic Km, and performing iterative screening after transplanting to finally obtain the homozygous transgenic strain of which the offspring does not have character separation. The present invention provides a transgenic homozygous material of PdPHT1-2, designated PdPHT1-2-OV, and further phenotypically observed (as shown in FIG. 3).

35S-universal-F：5’-CGCACAATCCCACTATCCTT-3’；

PdPHT1-2-R：5’-CACTGAAGCCATCCTAGCTGAAGC-3’。

(1) PCR detection of transgenic plants

The assay was performed using the Plant Direct PCR Kit kit from nuezan biotechnology Co., ltd, as follows:

sample treatment: a small young leaf (about 1-3mm in diameter) was taken, placed in 20. Mu. lPlant Direct Lysis Buffer A, and ground as much as possible. Heating at 95 ℃ for 5-10min, centrifuging briefly after heating, and taking 1 μl of supernatant as a template of a PCR reaction system.

The reaction system: plant leaves/crude samples 1. Mu.l, 2X Plant Direct Master Mix. Mu.l, primer1 (10. Mu.M) 1. Mu.l, primer 2 (10. Mu.M) 1. Mu.l, ddH2O 7. Mu.l, and the total system was 20. Mu.l. The reaction procedure is: 98 ℃ for 5min;95 ℃ for 10s, 58-72 ℃ for 15s, 72 ℃ for 1min/kb, and 35 cycles are total; 72 ℃ for 5min; preserving at 4 ℃.

Gel electrophoresis detection: and adding bromophenol blue into the PCR product, mixing uniformly, spotting on 1% agarose gel, and detecting by electrophoresis.

(2) Screening of homozygotes

To obtain transgenic offspring that can be stably inherited, positive homozygous plants are screened by antibiotic Km screening pressure, as follows:

preparing an arabidopsis culture medium, placing the arabidopsis culture medium at about 50 ℃ after sterilization, adding Km (50 mg/L) and Cef (400 mg/L), uniformly mixing, and pouring the mixture into a flat plate; placing the seeds into 1/4-1/6 of the joint part of the bottom of the tube, cleaning with 1ml of sterile water, and removing floating seeds; adding 1ml of 75% alcohol, mixing for 30s, standing for 15s, and sucking ethanol with a gun; adding 1m110%84, swirling for 3min, standing for 15s, sucking the supernatant with a gun, washing with sterilized water for 4 times, standing each time, and sucking the supernatant; 1ml of 0.05% agaros (agarose) solution suspending the seeds; the seeds were beaten onto a flat plate of medium (500 ul each) and were beaten with a scissors head to disperse the seeds as much as possible. Standing for 40 minutes, and waiting for water evaporation; sealing the culture dish, wrapping with newspaper, and culturing in dark at 4deg.C for 3 days (inducing seed germination); culturing in a light incubator for 16h with attention to pollution. After about 10 days, positive seeds should be significantly larger than non-transformed plants and green (non-transgenic plants are smaller and leaves are relatively whitish in color); after the culture medium plate is germinated and grows to 4 leaves (one week to two weeks), large green plants are selected and transplanted to a small flowerpot; when more leaves are available, taking the leaves for transgene detection; collecting seeds, and repeating until the culture medium is large green plantlet, i.e. the screened seeds are homozygote without character separation.

4. Transgenic arabidopsis thaliana low-phosphorus stress treatment

The Arabidopsis related treatment is mainly carried out on a solid culture medium; adopting MS solid culture medium as a base; by adjusting KH ₂ PO ₄ The adding amount of the mother solution controls the concentration of the P element, and simultaneously, KCl mother solution is correspondingly added to complement the K element to the original standard level.

KH is not added in the preparation ₂ PO ₄ MS macroelement mother liquor of (2);

preparing KH ₂ PO ₄ Mother liquor: 0 mM Pi (phosphorus deficiency), 0.0625 mM Pi (low phosphorus; 1/20 of normal phosphorus content of MS), 0.625 mM Pi (medium phosphorus; 1/2 of normal phosphorus content of MS), 1.25 mM Pi (high phosphorus; normal phosphorus content of MS);

preparation and KH ₂ PO ₄ KCl mother liquor with the same molar concentration as the mother liquor;

preparing corresponding culture mediums according to the four treatment gradients of phosphorus, and sub-packaging the square culture dishes;

the first treatment mode is as follows: wild type and transgenic seedlings, which were plated for about 10 days, were transferred into 4 different treatment gradients of medium, respectively. 2 plates of each treatment seedling transplanting, one plate is horizontally cultivated for observing the growth size; and the other plate is used for obliquely standing (inclined standing) culture and observing the growth difference of root systems. And (5) irradiating for 16 hours, and treating for one week.

The second treatment mode is as follows: the sterilized seeds were directly sown into the medium of each gradient, and the seed germination difference was observed. 16 hours of light, treatment for 10 days.

(1) Growth phenotype of PdPHT1-2 transgenic plants under different phosphorus concentration gradient treatments

Wild-type and transgenic Arabidopsis seedlings, which were relatively consistent in status at the initial stage of germination of the seed plates, were transplanted into four phosphorus concentrations (0 mM Pi, 0.0625 mM Pi, 0.625 mM Pi, 1.25 mM Pi) medium to culture Arabidopsis seedlings (in this way, the influence of non-consistent wild-type and transgenic seed germination rates was circumvented). Culture for 10 days after germination and transplantation, the results show that: under the treatment gradient of each phosphorus concentration, the overground part growth vigor of the transgenic arabidopsis is higher than that of the wild type, and the over-expression of the PdPHT1-2 gene is presumed to promote the absorption and utilization of phosphorus nutrition of plants. The higher the phosphorus concentration, the more pronounced the growth advantage of the aerial parts of the transgenic plants (as indicated by a in fig. 4).

The root phenotype shows that under the treatment condition of each phosphorus concentration gradient, the transgenic plant has more obvious advantages compared with the root phenotype of a wild type control: the root system is longer and the density is greater (as shown by B in fig. 4). Along with the decrease of the phosphorus concentration, the root system of the transgenic plant is healed to show the root system configuration with increased density and increased lateral roots, and the assumption is that the transgenic plant is easier to adjust and change the root system configuration and increase the lateral roots under the condition of lower phosphorus concentration so as to improve the contact area of the root system and the growth substrate, thereby obtaining more phosphorus.

(2) Statistical analysis of growth of PdPHT1-2 transgenic plants under different phosphorus concentration gradient treatments

6 wild-type and transgenic Arabidopsis thaliana with consistent growth states were individually selected and weighed (6 biological replicates), 3 times each to reduce errors (3 technical replicates). Comparing the fresh weights of the transgenic plants and the wild plants under different phosphorus concentration gradients, respectively, the method can find that: fresh weight data for both transgenic and wild-type arabidopsis appear to have an increasing trend with increasing phosphorus concentration, i.e. peak at full phosphorus concentration conditions (1.25 mMPi) (a in fig. 5). At low phosphorus concentrations (0.0625 mM Pi), the fresh weight of the wild type increased less significantly than that of the phosphorus deficiency (0 mM Pi), whereas at medium phosphorus concentrations (0.625 mM Pi), the fresh weight increased significantly than that of the low phosphorus concentration (0.0625 mM Pi). Whereas fresh weight data of transgenic Arabidopsis thaliana overexpressing PdPHT1-2 increased significantly at low phosphorus (0.0625 mM Pi) relative to phosphorus deficiency (0 mM Pi), the most significant increase was in medium phosphorus (0.625 mM Pi) (B in FIG. 5).

(3) Germination and growth phenotypes of PdPHT1-2 transgenic plants under different phosphorus concentration gradient treatments

Transgenic seeds were sown directly into media of different phosphorus concentration treatment gradients. After 3 days of culture, it was observed that the germination rate of transgenic arabidopsis seeds was greater than that of wild type under phosphorus deficiency (0 mM Pi), but that of wild type arabidopsis seeds was higher under low phosphorus (0.0625 mM Pi); the germination rates were the same at medium phosphorus (0.625 mM Pi) and total phosphorus (1.25 mM Pi) concentrations (A, C in FIG. 6). After 5 days, the germination rate of both transgenic and wild type arabidopsis seeds increased, and the germination trend was the same as 3 days. However, the difference between the germination rates was still large under phosphorus deficiency conditions, whereas the germination rate of wild type seeds was reduced compared to that of transgenic seeds at low phosphorus (0.0625 mM Pi) (B, C in FIG. 6). After 10 days of treatment, the growth performance of the germinated seedlings was observed, and transgenic plants were slightly larger than wild-type growth under each gradient condition (D in FIG. 6), presumably over-expressing PdPHT1-2 so that the plants could acquire more phosphorus nutrition from the medium.

Claims (7)

1. The nucleotide sequence of the low phosphorus resistant gene PdPHT1-2 of the aspen is shown as SEQ ID NO. 1.

2. The protein encoded by the low phosphorus resistance gene PdPHT1-2 of mountain new poplar as claimed in claim 1, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 2.

3. A recombinant expression vector or recombinant strain comprising the aspen low phosphorus resistance gene PdPHT1-2 of claim 1.

4. The use of the shanxin poplar low phosphorus resistance gene PdPHT1-2 as claimed in claim 1 for improving the low phosphorus resistance of plants.

5. The use according to claim 4, wherein the low phosphorus resistant gene PdPHT1-2 of aspen is constructed on an entry vector and a target vector in sequence by using the GATEWAY technology to obtain a plant expression vector, and then the plant is transformed by agrobacterium-mediated genetic transformation, and the transgenic plant with low phosphorus resistance is finally obtained by screening and identifying.

6. The use according to claim 5, wherein the plants comprise arabidopsis thaliana and aspen.

7. The use according to claim 5, wherein the plant expression vector is pBI121-PdPHT1-2-3HA.

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