Application of PsAAAP21 gene in regulation and control of nitrogen utilization efficiency of poplar

Abstract

The invention discloses application of a PsAAAP21 gene in regulation and control of nitrogen utilization efficiency of poplar, and belongs to the technical field of molecular biology. The method comprises the following steps: constructing a plant inhibition expression vector containing a gene PsAAAP21, carrying out heterologous transformation on the plant inhibition expression vector to poplar 84K, screening to obtain a transgenic positive plant, and carrying out phenotypic analysis on the positive plant and a wild plant under different nitrogen treatment conditions to obtain a transgenic plant capable of efficiently utilizing nitrogen. The invention provides an important nitrogen efficient utilization gene resource possibly having universality, materials are provided for subsequent related research, and a foundation is laid for efficient utilization research of forest resources.

Description

TECHNICAL FIELD

[0001] The present application relates to the application of PsAAAP21 gene in regulating nitrogen utilization efficiency of poplar, and belongs to the technical field of plant genetic engineering. BACKGROUND

[0002] Nitrogen is an essential nutrient element for plant growth, which is absorbed from rhizosphere in the form of nitrate and ammonium, and stored in the form of amino acids and nitrogen-containing compounds. Amino acids are important organic substances, which maintain the normal growth and development of eukaryotes, and regulate the metabolism, structure and biosynthesis of various compounds. Amino acids can be directly absorbed by root system as nitrogen source, and also can be used as neurotransmitters and hormones for communication between cells and tissues. The implementation of these functions requires a specific transport system to transport amino acids, and amino acid / auxin permease (AAAP) is a protein that performs this function.

[0003] AtAAP1 / NAT2 is the first identified plant amino acid transporter, which was found in Arabidopsis thaliana in 1993. The AAAP gene family is one of the largest amino acid transporter families, which includes members of almost all eukaryotes. Aa_trans is a specific domain of AAAP gene. So far, the functions of various AAAPs have been identified in several species such as Arabidopsis thaliana and Camellia sinensis.

[0004] Although AAAP genes have been identified and characterized in several plants, there is no analysis and demonstration of AAAP genes in nitrogen absorption and utilization of poplar. Poplar is an important energy tree species, and improving nitrogen utilization efficiency of poplar will help to reduce the use of fossil fuels, protect the global environment, and provide a reference for the research of other woody plants.

[0005] Therefore, it is of important theoretical and practical significance to study the role of AAAP gene in the response of poplar to low nitrogen, and the application of PsAAAP21 gene in regulating nitrogen utilization efficiency of poplar provided by the present application will help to lay a good foundation for the subsequent breeding of nitrogen-efficient poplar, and provide theoretical support and technical exploration for improving wood yield under the condition of poor site. SUMMARY

[0006] The present application aims to provide the application of PsAAAP21 gene in regulating nitrogen utilization efficiency of poplar, so as to promote the development of breeding technology for nitrogen-efficient forest trees, and provide technical means for the cultivation or screening of excellent tree species.

[0007] The above-mentioned purpose of the present application is achieved by the following technical solution: The present application is based on a poplar amino acid transport gene PsAAAP21, the coding region nucleotide sequence and amino acid sequence of which are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

[0008] Preferably, the CDS of the Populus nitrogen utilization efficiency regulating gene PsAAAP21 is 1383 bp in length, encodes 460 amino acids and 1 stop codon.

[0009] Another object of the present application is to provide application of the Populus nitrogen utilization efficiency regulating gene PsAAAP21 in regulating plant height and ground diameter of Populus under low nitrogen conditions.

[0010] The above object of the present application is achieved by the following technical scheme: The application of the Populus nitrogen utilization efficiency regulating gene PsAAAP21 in regulating plant height and ground diameter of Populus under low nitrogen conditions is characterized in that the expression of the Populus inhibiting gene PsAAAP21 is inhibited.

[0011] The application of the Populus nitrogen utilization efficiency regulating gene PsAAAP21 in regulating plant height and ground diameter of Populus under low nitrogen conditions is characterized in that a plant inhibiting expression vector containing the PsAAAP21 gene is constructed, and the vector is heterologously transformed into Populus alba x P. glandulosa 84K, transgenic positive plants are obtained through screening, and through phenotype analysis of the positive plants and wild type plants under low nitrogen conditions, transgenic plants with increased plant height and ground diameter under low nitrogen conditions are obtained.

[0012] The application of the Populus nitrogen utilization efficiency regulating gene PsAAAP21 in regulating plant height and ground diameter of Populus under low nitrogen conditions is characterized in that the following steps are included: 1) Populus simonii 'Tongliao1' cutting seedlings cultured in a greenhouse of the Chinese Academy of Forestry in Haidian District, Beijing are collected, RNA is extracted, cDNA is reverse transcribed, the CDS sequence of PsAAAP21 is cloned, the sequence is connected to a pMD19-T vector, sequencing is performed, and after correct identification, an inhibiting expression vector is constructed and heterologously transformed into Populus alba x P. glandulosa 84K; 2) hygromycin resistance and PCR technology are used to screen positive plants of PsAAAP21 gene heterologously transformed Populus alba x P. glandulosa 84K, transgenic positive plants are obtained, and RNA extraction is performed to obtain transgenic plants with significantly inhibited expression; 3) The PsAAAP21 transgenic Populus alba x P. glandulosa 84K and non-transgenic Populus alba x P. glandulosa 84K were subjected to low nitrogen treatment, and it was found that PsAAAP21 promoted the growth of poplar height and ground diameter under low nitrogen conditions.

[0013] The above research results prove that PsAAAP21 has a promoting effect on the nitrogen utilization efficiency of poplar under low nitrogen conditions, and has important application value in forest molecular breeding and excellent variety breeding.

[0014] Compared with the prior art, the main beneficial technical effects of the present application are: The present application takes P. simonii 'Tongliao1' as the material, and screens and identifies PsAAAP21 gene. The phenotype identification of the inhibition expression Populus alba x P. glandulosa based on the same shows that it has the ability to promote the growth of poplar under low nitrogen conditions, which shows that PsAAAP21 gene can positively regulate the nitrogen utilization efficiency of plants, and provides a new selection for screening the dominant nitrogen utilization efficiency gene, and has important application value in the field of forest genetic engineering.

[0015] The present application will be further described below through specific embodiments and drawings, but it does not mean the limitation of the protection scope of the present application. DETAILED DESCRIPTION

[0016] Figure 1 The present application will be further described below through specific embodiments and drawings, but it does not mean the limitation of the protection scope of the present application. Figure 2 The present application will be further described below through specific embodiments and drawings, but it does not mean the limitation of the protection scope of the present application. Figure 3 The present application will be further described below through specific embodiments and drawings, but it does not mean the limitation of the protection scope of the present application. DETAILED DESCRIPTION

[0017] The present application will be further described below through specific embodiments and drawings, but it does not mean the limitation of the protection scope of the present application.

[0018] Unless otherwise specified, the reagents involved in the following examples are commercially available conventional reagents, and the methods used are commonly used methods in the technical field. EMBODIMENT

[0019] I. Cloning of P. simonii PsAAAP21 gene With small leaf poplar (P. simonii 'Tongliao1') as the material, the total RNA reagent kit of Tian Gen polysaccharide and polyphenol was used to extract the RNA of the leaf of small leaf poplar (total RNA of small leaf poplar), and the method was as follows: (1) 0.1 g of small leaf poplar leaf tissue was frozen with liquid nitrogen, ground in a mortar pre-cooled with liquid nitrogen, and the leaf tissue sample was kept in a frozen state during the grinding process. After the tissue sample was ground into powder, it was transferred to a 1.5 ml centrifuge tube, then 500 μl of lysis solution and 10 μl of β-mercaptoethanol were added, and then the tissue and reagent were fully mixed with a vortex oscillator; (2) Centrifuge at 12000 rpm for 2 minutes, collect the supernatant; (3) Transfer the supernatant to a filter column, centrifuge at 12000 rpm for 2 minutes, and carefully pipette the supernatant into another centrifuge tube; (4) Add 0.4 times (200 μl) of anhydrous ethanol in the centrifuge tube, mix well, and move to the adsorption column. The adsorption column was transferred to the centrifuge tube, centrifuged at 12000 rpm for 15 seconds, and the liquid was discarded; (5) Add 80 μl of DNA enzyme working solution (10 μl of DNA enzyme storage solution + 70 μl of buffer) to the adsorption column, and stand at room temperature for 15 minutes; (6) Add 350 μl of deproteinizing solution to the adsorption column, centrifuge at 12000 rpm for 15 seconds, and discard the liquid; (7) Add 500 μl of rinsing solution to the adsorption column, centrifuge at 12000 rpm for 15 seconds, and discard the liquid; (8) Repeat (7); (9) Centrifuge at 12000 rpm for 2 minutes, move to a new centrifuge tube, dry, add 30 μl of RNA-free ultrapure water, dissolve, precipitate, stand at room temperature for 2 minutes, centrifuge at 12000 rpm for 1 minute, and the plant total RNA extract is obtained; Take 2.0 μg of RNA from each sample, reverse transcribe it into cDNA by using Tian Gen reverse transcription reagent, and the experimental consumables are free of RNA contamination. The reaction is carried out on ice; the reverse transcription steps are as follows: 1) gDNA removal reaction (10 μl system) is shown in Table 1: Table 1 Components Volume 5x gDNA buffer 2 μl Total RNA 0.2~0.5 μg RNAse free water up to 10 μl Reaction conditions: 42°C, reaction for 3 minutes; 2) The reaction product of the previous step is used as a template for reaction (20 μl system) as shown in Table 2: Table 2 Components Volume 10x King RT buffer 2 μl Fast King RT Enzyme mix 1 μl FQ-RT Primer mix 2 μl RNAse free water up to 10 μl 3) 1) is added to 2), reaction conditions: 42°C, reaction 15 minutes; 95°C, reaction 3 minutes, dilution for use, 10 microliters of cDNA + 190 microliters of ultrapure water, which is the template cDNA for use; With reference to the published Populus simoniiii genome sequence, the primers (amplifon include start codon and stop codon) are designed using Primer3 software for full-length amplification of the gene; wherein the PsAAAP21 ORF forward primer PsAAAP21-F is as shown in SEQ ID NO. 1 of the sequence listing, the reverse primer PsAAAP21-R is as shown in SEQ ID NO. 2, the PsAAAP21 inhibitory forward primer PsAAAP21-RNAi-F is as shown in SEQ ID NO. 5 of the sequence listing, and the reverse primer PsAAAP21-RNAi-R is as shown in SEQ ID NO. 6; The cDNA of Populus simonii is used as a template for PCR amplification with the corresponding primers. The PCR reaction system (20 microliter system) is shown in Table 3 below: Table 3 Components Volume 2 x Ex Taq 10 μl -RNAi-F 1.0 μl -RNAi-R 1.0 μl cDNA 1.0 μl ddH2O 7.0 μl The reaction conditions are shown in Table 4 below: Table 4 Reaction temperature Reaction time 94℃ 5 min 94℃ 30 sec 55℃ 30 sec 35 cycles 68℃ 90 sec 68℃ 7 min The target fragment is recovered by gel cutting, and the PCR amplified target fragment is recovered by using the gel recovery kit of the American OMEGA biotechnology company. PsAAAP21 The target fragment is recovered by gel cutting, and the PCR amplified target fragment is recovered by using the gel recovery kit of the American OMEGA biotechnology company. 1) Cut the target fragment band into a 1.5 milliliter sterile centrifuge tube, add 600 microliters of binding liquid to melt the gel at 50°C; 2) Move the melted liquid into a filter column, install the filter column into a centrifuge tube, centrifuge at 12500 rpm for 2 minutes, and discard the waste liquid; 3) Add 600 microliters of eluent, centrifuge at 12500 rpm for 30 minutes, and discard the waste liquid; 4) Repeat (3); 5) Empty the filter column and centrifuge at 12500 rpm for 2 minutes; 6) Transfer the filter column into another 1.5 milliliter centrifuge tube, add 30 microliters of sterile deionized water, stand at room temperature for 5 minutes, centrifuge at 12500 rpm for 2 minutes, and repeat the elution once; 7) After detection by 0.1% agarose gel electrophoresis, store at -20°C; After ligation to pMD19-T, sequencing is performed, and the full-length cDNA sequence of the gene is 1383 bp, which is named PsAAAP21The gene, with its sequence shown in SEQ ID NO.5, and the protein sequence it expresses, shown in SEQ ID NO.6, were obtained. PsAAAP21 -The RNAi sequence is 311 bp, as shown in SEQ ID NO.7 of the sequence listing; two, PsAAAP21 Construction of plant gene expression vectors The Gateway method was used to construct a repressive expression vector, and the vector was amplified by PCR to obtain a Gateway-tagged vector. PsAAAP21 Gel recovery product of RNAi target fragment, forward primer PsAAAP21 -RNAi-F is shown in SEQ ID NO.3 of the sequence listing, reverse primer PsAAAP21 - RNAi-R, as shown in SEQ ID NO.4, was first constructed into the pDONR222 intermediate vector via a BP reaction (Thermo Fisher Scientific, Shanghai, China). The BP reaction system is shown in Table 5, yielding the recombinant plasmid pDNOR222- PsAAAP21 -RNAi, subsequently, using LR reaction (Thermo Fisher Scientific, Shanghai, China) PsAAAP21 The RNAi target fragment was constructed into the pH7GWIWG2 vector, resulting in pH7GWIWG2- PsAAAP21 ; The BP reaction system (5 μL system) is shown in Table 5 below: Table 5 Components Volume pDONR222 75 ng RNAi target fragment 150 ng BP enzyme 0.8 μl Water up to 5 μl Reaction conditions: 16℃, reaction time 1 hour; The ligation product was transformed into E. coli DH5α. The specific transformation steps are as follows: 1. Take 5 μL of ligation product from the ice box and add it to 50 μL of commercially available E. coli competent cells DH5α. Mix gently with a pipette and incubate on ice for 30 minutes. 2. Place the transformed bacterial culture in a 42℃ heat shock bath for 90 seconds, then remove and place in an ice bath for 5 minutes; 3. Add 300 μL of LB liquid culture medium, incubate at 37°C and 180 rpm for 1 hour; 4. Centrifuge at room temperature, 4000 rpm for 5 minutes, discard the supernatant, and resuspend the remaining bacterial culture; 5. Spread the mixed transformed cells onto LB agar plates containing 50 mg / L Kan, air dry, seal, and invert in a 37°C incubator for 12-14 hours. Randomly select several single colonies that have grown on the resistance plate, add 300 μL of LB liquid medium containing Kan (50 mg / L), and incubate at 37°C, 180 rpm for 4-5 hours with shaking. Then, use the bacterial culture as a template for PCR detection of positive clones. The PCR reaction system (20 μL system) is shown in Table 6 below: Table 6 Components Volume 10 x PCR buffer 2.5 μl M13F 1.0 μl -RNAi-R 1.0 μl cDNA 0.1~0.2 μg Water up to 20 μl The reaction conditions are shown in Table 7 below: Table 7 Reaction temperature Reaction time 94℃ 5 min 94℃ 30 sec 55℃ 30 sec 35 cycles 68℃ 90 sec 68℃ 7 min After the reaction, 5 μL of PCR amplification product was taken and detected by 0.1% agarose gel electrophoresis and photographed under UV light using a gel imaging system. Clones with bands the same size as the amplification primers were considered positive clones. 3-5 PCR positive clones were selected and sequenced by Beijing Qingke Biotechnology Co., Ltd. to confirm successful construction into the intermediate vector. Based on the sequencing results, the target bacterial strain was added to an equal volume of 50% sterile glycerol, vortexed, pre-frozen in liquid nitrogen, and then stored at -80°C. Alternatively, pDNOR222- could be extracted and preserved through propagation culture. PsAAAP21 -RNAi plasmid; pDNOR222- PsAAAP21 - RNAi plasmid gene fragments were digested and LR ligated; The enzyme digestion reaction system (10 μL system) is shown in Table 8 below: Table 8 Components Volume MluI enzyme 0.5 μl NEBuffer 3.1 1 μl Plasmid of interest 0.2~0.5 μg Water up to 10 μl Reaction conditions: 37℃, 3 hours; pDNOR222- PsAAAP21 - RNAi plasmids were ligated using the LR reaction. The LR reaction system (5 μL system) is shown in Table 9 below: Table 9 Components Volume pH7GWIWG2 75 ng pDNOR222--RNAi plasmid 150 ng LR enzyme 0.8 μl Water up to 5 μl Reaction conditions: 25℃, reaction time 2.5 hours; The ligation product was transformed into E. coli, single colonies were picked and sequenced, and the correct single colony was identified as pH7GWIWG2- PsAAAP21 Plasmid, ultimately cloned amino acid transport gene PsAAAP21 The inhibitory expression vector pH7GWIWG2- PsAAAP21 ; three, PsAAAP21 Genetic transformation and detection 1. PsAAAP21 Genetic transformation The constructed inhibitory expression vector pH7GWIWG2- was implanted via electroporation. PsAAAP21 The bacteria were transferred into Agrobacterium GV3101 and then transformed into *Populus alba* 84K via Agrobacterium-mediated genetic transformation. Populus alba x P. glandulosa The transformation steps are as follows: Silver poplar 84K (used for genetic transformation) Populus alba x P. glandulosaCallus was cultured at a temperature of 23-25℃, with a light exposure of 16 / 8 hours (day / night) and a light intensity of 50 μMm. -2 s -1 Agrobacterium containing the target expression vector was cultured under specific conditions at OD2000. 600 =0.6-0.8% infect callus. Infected callus is placed on adventitious shoot induction medium (L&M, Lloyd & McCown Woody Plant Basal Medium with Vitamins) and co-cultured for 3 days in the dark at 22±2℃. Leaves after co-culture are then transferred to L&M medium supplemented with 0.5 mg / L 6-benzyl aminopurine (6-BA), 0.05 mg / L naphthaleneacetic acid (NAA), 3 mg / L hygromycin B, and 200 mg / L timentin. Culture is then carried out at 23-25℃, with a light cycle of 16 / 8 h (day / night) and a light intensity of 50 μMm. -2 s -1 Under these conditions, resistant adventitious shoots were induced and screened. After 30-45 days of induction culture, the resistant adventitious shoots were transferred to rooting medium (1 / 2 Murashige and Skoog (MS) medium supplemented with 0.05 mg / L IBA and 0.02 mg / L NAA) containing 3 mg / L hygromycin B and 200 mg / L timentin until rooting was induced. DNA was extracted from the leaves of the rooted plants for PCR detection. 2. Detection of transgenic plants with suppressed expression Take the above-mentioned growth on the resistant medium PsAAAP21 Gene suppression of silver gland 84K expression ( Populus alba x P. glandulosa Genomic DNA is extracted, and the vector sequence and inserted gene fragment are amplified by PCR. Clear bands are obtained, indicating a transgenic plant. Figure 1 As shown, this illustrates the expression of non-transgenic *Sapindus mukorossi* 84K and its inhibition in Example 1 of this invention. PsAAAP21 Image of transgenic *Populus alba* 84K positive plants; simultaneously, RNA was extracted and RT-PCR was performed, and a total of 9 plants with suppressed expression were screened, with expression levels lower than the non-transgenic wild-type WT. The nomenclature is as follows. Figure 2 As shown; the quantitative primers are PsAAAP21- RT - F, as shown in SEQ ID NO.9; PsAAAP21- RT - R is shown in SEQ ID NO.10; Four, PsAAAP21 Phenotype observation of transgenic plant To PsAAA21 Inhibit expression of transgenic aspen 84K two strains (R11, R7) and non-transgenic aspen 84K (CK) to carry out low nitrogen treatment, the culture place is the aspen group culture room of China Forestry Science Research Institute, select the growth state consistent of inhibiting expression of transgenic aspen 84K, plant in vermiculite and perlite, grow for a month, then, use the Hoagland's nutrient solution containing 0.5mM, 4mM nitrogen, irrigate once every 3 days, culture for 45 days, on the 45th day, observe the growth state of each transgenic poplar and control, count the plant height and ground diameter of transgenic poplar under each treatment condition;As Figure 3 shown, the non-transgenic aspen 84K of embodiment 1 of the present application and PsAAA21 Inhibit expression of transgenic poplar plant height, ground diameter comparison chart, under the condition of 4mM nitrogen, PsAAA21 The plant height and ground diameter of transgenic poplar are slightly higher than CK, but the significance is not strong;Under the condition of 0.5mM nitrogen, PsAAA21 The plant height and ground diameter of transgenic poplar are significantly higher than CK;Therefore, according to the phenotype and statistical data, it is preliminarily speculated that PsAAA21 PsAAA21 The gene has the effect of improving the growth of poplar under low nitrogen condition to some extent.

[0020] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not the limitation of the embodiments of the present application, for ordinary skilled in the art, on the basis of the above description, other different forms of changes or variations can also be made, here it is impossible to enumerate all the embodiments, any changes or variations derived from the technical scheme of the present application still falls within the protection scope of the present application.

Claims

1. Application of PsAAAP21 gene in regulating nitrogen utilization efficiency of poplar.

2. The application of PsAAAP21 gene in regulating nitrogen utilization efficiency of poplar according to claim 1, in particular, the application of PsAAAP21 gene in improving the growth of poplar under low nitrogen condition.

3. The use of the PsAAAP21 gene to improve the growth of poplar under low nitrogen conditions according to claim 2, characterized in that: Making poplar contain PsAAAP21 gene.

4. The use of the PsAAAP21 gene to improve the growth of poplar under low nitrogen conditions according to claim 2, characterized in that: Making poplar inhibit expression of PsAAAP21 gene.

5. The use of the PsAAAP21 gene to improve the growth of poplar under low nitrogen conditions according to claim 4, characterized in that: Constructing poplar inhibition expression vector containing PsAAAP21 gene, heterologous transformation of the vector into Populus alba var. pyramidalis 84K, screening of transgenic positive plants, phenotype analysis of the positive plants and wild type plants under different nitrogen treatment conditions, and obtaining of transgenic plants with high nitrogen utilization efficiency.

6. The use of the PsAAAP21 gene to improve the growth of poplar under low nitrogen conditions according to claim 5, characterized in that: Specifically comprising the following steps: (1) Collecting tissue culture seedlings of Populus simonii, extracting RNA, reverse transcribing into cDNA, cloning RNAi sequence of PsAAAP21, connecting the sequence with pH7GWIWG2 vector for sequencing, identifying the sequence, constructing inhibition expression vector, and heterologous transformation of the vector into Populus alba var. pyramidalis 84K; (2) Screening of positive plants by using hygromycin resistance and PCR technology, obtaining of transgenic positive plants, RNA extraction, phenotype statistics, different nitrogen treatment of the transgenic plants, analysis of the growth of the plants, and obtaining of transgenic plants with high nitrogen utilization efficiency.

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