PagPB gene for regulating and controlling woody plant root system to absorb and utilize nitrogen and application of PagPB gene

Abstract

The invention discloses a PagPB gene for regulating and controlling a woody plant root system to absorb and utilize nitrogen and application of the PagPB gene, and relates to the technical field of genetic engineering breeding, and the nucleotide sequence of the PagPB gene is as shown in SEQ ID NO.1. It is found that the nitrogen absorption and utilization efficiency of the poplar root system can be improved through the silent PagPB gene, the nitrogen absorption and utilization efficiency of the poplar root system is reduced through overexpression of the PagPB gene, important gene resources are provided for mining of woody plant nitrogen absorption and utilization key regulatory genes, and the PagPB gene has important application value in the fields of forest genetic engineering and clonal forestry.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering breeding, and in particular relates to a PagPB gene for regulating the absorption and utilization of nitrogen by the root system of woody plants and an application thereof. Background Art

[0002] Nitrogen is one of the macroelements necessary for plant growth and development. The efficiency of plant roots in absorbing and utilizing soil nitrogen is the key to restricting plant growth and development. Plant roots adapt to the complex and changeable soil nitrogen environment, and the regulation of nitrogen absorption and utilization efficiency depends on changes in the expression patterns of related genes, such as the expression of key genes such as nitrate nitrogen or ammonium nitrogen transporter genes, protein-coding genes and small RNAs. As an important component of the ecosystem, woody plants have made important contributions to ecosystem stability, agricultural production, biodiversity, carbon sequestration, stress resistance, molecular breeding and germplasm innovation, as well as sustainable agriculture and forestry. They maintain the soil nitrogen cycle through nitrogen fixation and nitrogen absorption, reduce the application of chemical fertilizers, increase the yield and quality of forest products, provide habitats for organisms, enhance carbon sequestration functions, improve stress resistance, cultivate excellent varieties, and achieve sustainable development of agriculture and forestry. The prior art has not found key regulatory genes for nitrogen absorption and utilization by woody plant roots. Summary of the invention

[0003] Based on this, the present invention provides a PagPB gene and application for regulating the absorption and utilization of nitrogen by the roots of woody plants, in order to provide excellent germplasm for the breeding of new resource-efficient forest tree varieties.

[0004] The present invention is achieved through the following technical solutions:

[0005] The present invention provides a PagPB gene for regulating the absorption and utilization of nitrogen by roots of woody plants. The nucleotide sequence of the PagPB gene is shown in SEQ ID NO.1.

[0006] The PagPB gene is used in regulating the absorption and utilization of nitrogen by the roots of woody plants.

[0007] The application of the PagPB gene in regulating the absorption and utilization of nitrogen by the roots of woody plants is to increase the absorption and utilization efficiency of nitrogen by the roots and to increase the root biomass by silencing the PagPB gene.

[0008] Improving the absorption and utilization efficiency of nitrogen by the root system refers to increasing the root NO3 - and NH4 + Net absorption rate, root nitrate-nitrogen, ammonium-nitrogen content and nitrate reductase activity.

[0009] The application of the PagPB gene in regulating the absorption and utilization of nitrogen by the roots of woody plants includes constructing a PagPB gene silencing vector, transforming woody plants, and obtaining PagPB gene-silenced transgenic plants to improve the absorption and utilization efficiency of nitrogen by the roots and increase the root biomass.

[0010] Preferably, the specific method for constructing the PagPB gene silencing vector is as follows:

[0011] (1) Using primers specific to the PagPB gene sequence, the full-length sequence of the PagPB gene was amplified, the first PCR product was recovered, and ligated into the T plasmid to obtain the plasmid pM-PagPB.

[0012] (2) The pFGC5941 RNAi vector was double-digested, and the plasmid pM-PagPB was amplified using PagPB-cis-F and PagPB-cis-R as primers. The second PCR product was recovered and ligated with the double-digested pFGC5941 RNAi vector; the ligated product was introduced into bacteria to obtain the recombinant vector pM-PagPB-C.

[0013] (3) Double-enzyme-cut recombinant vector pM-PagPB-C, amplify plasmid pM-PagPB using PagPB-anti-F and PagPB-anti-R as primers, recover the third PCR product and connect it with the double-enzyme-cut recombinant vector pM-PagPB-C to obtain the PagPB gene silencing vector.

[0014] The specific primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0015] The PagPB-cis-F primer sequence is shown in SEQ ID NO.5, and the PagPB-cis-R primer sequence is shown in SEQ ID NO.6.

[0016] The PagPB-anti-F primer sequence is shown in SEQ ID NO.7, and the PagPB-anti-R primer sequence is shown in SEQ ID NO.8.

[0017] Preferably, the transformation method is Agrobacterium-mediated transformation.

[0018] Preferably, the woody plant is poplar.

[0019] Preferably, the variety of the poplar is Populus alba xP. glandulosa 84K.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) Discovery of new genes: Through screening and identification, the present invention successfully discovered a new PagPB gene using 84k silver gland poplar as material. This discovery enriched the gene resources of poplar and provided a new direction for subsequent research.

[0022] (2) Revealing a new mechanism of nitrogen absorption and utilization: The present invention found that silencing the PagPB gene can improve the efficiency of nitrogen absorption and utilization by poplar roots, while overexpressing the PagPB gene reduces the efficiency of nitrogen absorption and utilization by poplar roots. The present invention reveals the key role of the PagPB gene in regulating the absorption and utilization of nitrogen by poplar roots. This discovery provides a new strategy for in-depth analysis of the molecular mechanism of nitrogen absorption and utilization by woody plants.

[0023] (3) Providing high-quality new germplasm: The present invention uses the nucleotide sequence of the PagPB gene to design primers, respectively construct PagPB gene silencing and overexpression vectors, and transfer the PagPB gene into 84K poplar through GV3101 Agrobacterium-mediated transgenic poplar plants. The PagPB gene is successfully silenced and overexpressed, thus providing high-quality new germplasm for the breeding of new resource-efficient poplar varieties in my country.

[0024] (4) Possessing important application value: The present invention has important application value in the field of forest genetic engineering and can provide new technical support for forest breeding, sustainable utilization of forest resources and improvement of the ecological environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 The DNA identification results of the PagPB gene silencing and overexpression strains of the present invention;

[0027] Figure 1 In the figure, (a) PagPB-RNAi1, 2, 3, 4, 16, 20, 21 and 22 are transgenic silencing lines; (b) PagPB-OE1-21 are transgenic overexpression lines; 84K: non-transgenic poplar negative control; P: vector positive control;

[0028] Figure 2 The relative expression patterns of PagPB genes in the roots of PagPB gene silenced and overexpressed transgenic plants and wild-type plants of the present invention;

[0029] Figure 2In the figure, (a) is the relative expression level of PagPB gene in the root system of PagPB gene silenced transgenic plants and wild-type plants; (b) is the relative expression level of PagPB gene in the root system of PagPB gene overexpressed transgenic plants and wild-type plants;

[0030] Figure 3 The NO3 in the roots of the PagPB gene-silenced and over-expressed transgenic poplar plants and wild-type plants of the present invention - and NH4 + Net absorption rate determination results;

[0031] Figure 3 (a) shows the NO3 in the roots of PagPB gene-silenced transgenic poplar plants and wild-type plants. - Net absorption rate measurement results; (b) NH4 in the roots of PagPB gene-silenced transgenic poplar plants and wild-type plants + Net absorption rate measurement results; (c) NO3 in the roots of PagPB-overexpressing transgenic poplar plants and wild-type plants - Net absorption rate measurement results; (d) NH4 in the roots of PagPB-overexpressing transgenic poplar plants and wild-type plants + Net absorption rate determination results;

[0032] Figure 4 The statistical results of root biomass of PagPB gene silenced and overexpressed transgenic poplar plants and wild-type plants of the present invention;

[0033] Figure 4 In the figure, (a) is the statistical result of biomass in the root system of PagPB gene silenced transgenic plants and wild-type plants; (b) is the statistical result of biomass in the root system of PagPB gene overexpressed transgenic plants and wild-type plants;

[0034] Figure 5 The statistical results of nitrate nitrogen and ammonium nitrogen contents in the roots of PagPB gene silenced and overexpressed transgenic poplar plants and wild-type plants of the present invention;

[0035] Figure 5 In the figure, (a) is the statistical result of nitrate nitrogen content in the root system of PagPB gene-silenced transgenic poplar plants and wild-type plants; (b) is the statistical result of ammonium nitrogen content in the root system of PagPB gene-silenced transgenic poplar plants and wild-type plants; (c) is the statistical result of nitrate nitrogen content in the root system of PagPB overexpressed transgenic poplar plants and wild-type plants; (d) is the statistical result of ammonium nitrogen content in the root system of PagPB overexpressed transgenic poplar plants and wild-type plants;

[0036] Figure 6The results of measuring the activities of nitrate reductase NR and glutamine synthetase GS in the roots of the PagPB gene-silenced and overexpressed transgenic poplar plants and wild-type plants of the present invention;

[0037] Figure 6 In the figure, (a) is the result of determination of nitrate reductase NR activity in the roots of PagPB gene-silenced transgenic poplar plants and wild-type plants; (b) is the result of determination of glutamine synthetase GS activity in the roots of PagPB gene-silenced transgenic poplar plants and wild-type plants; (c) is the result of determination of nitrate reductase NR activity in the roots of PagPB overexpressed transgenic poplar plants and wild-type plants; (d) is the result of determination of glutamine synthetase GS activity in the roots of PagPB overexpressed transgenic poplar plants and wild-type plants. DETAILED DESCRIPTION

[0038] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0040] The terms "first", "second" and "third" are used for descriptive purposes only and should not be understood as indicating or suggesting relative importance or implicitly indicating the number of the indicated technical features. They are only used to distinguish different PCR products.

[0041] The sources of the reagents and instruments used in the present invention are shown in Table 1:

[0042] Table 1

[0043]

[0044]

[0045] The present invention uses 84k silver gland poplar as material and successfully discovers a new PagPB gene, enriching the poplar gene resources. The nucleotide sequence of the PagPB gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the PagPB gene is shown in SEQ ID NO.2.

[0046] SEQ ID NO.1:

[0047] ATGAAGAGCTGCATTTATACAAAATTAGTTGGCGAGAGGACTTCTCTAGGAGCAACTCGAAGAAAACTTCCGCAGAAACGAGGAAATAATGACAGGAAATTAAGCAGGAGCGTCAAAACGATAAGAGCTGACATGGTTGAGATCAGCGAGGGACAAAAACGCATAAGAGAGGGACAGAAGGAAATCAGGAAAAGATTT CAAGAAATAAGCGAAGAGACCGCCAAACTGAGAGAGGAAACTAACGTAATCTCCAAGCAGAGCTCTGAAAATCAACTCAGGCTTGATCTAATGTTTCAAATCGTCAAAGCAAGAGCAGAGAATGATTATGCCAAAGACGCCTTGCTCACTCAAACCTTACGTTAGTCATCCTCCAAATCTTTGTGCCTTGTGTTTAA

[0048] SEQ ID NO.2:

[0049] MKSCIYTKLVGERTSLGATRRKLPQKRGNNDRKLSRSVKTIRADMVEISEGQKRIREGQKEIRKRFQEISEETAKLREETNVISKQSSENQLRLDLMFQIVKARAENDYAKDALLTQTLRLVILQIFVPCV

[0050] Furthermore, the present invention utilizes homologous recombination methods to construct PagPB gene silencing and overexpression vectors, respectively, and transfers the PagPB gene silencing and overexpression vectors into 84k silver gland poplar through Agrobacterium-mediated method, and creates PagPB gene silenced and overexpression transgenic poplar plants, respectively, to explore the regulatory effect of PagPB gene on the absorption and utilization of nitrogen by poplar, in order to provide excellent new germplasm for the breeding of new resource-efficient poplar varieties in my country.

[0051] In order to better illustrate the present invention, the present invention is further described below in conjunction with the embodiments. The following are specific embodiments.

[0052] Example 1. Construction of PagPB gene silencing and overexpression vector

[0053] (1) Primer5 software was used to design PagPB gene sequence-specific primers for full-length amplification of the PagPB gene; wherein the forward primer for full-length amplification of the PagPB gene was as shown in SEQ ID NO.3, and the reverse primer was as shown in SEQ ID NO.4.

[0054] SEQ ID NO.3: 5'-atttggagagaacacgggggactttgcaacatgaagagctgcatttatacaaaat-3'

[0055] SEQ ID NO.4: 5'-tttgtagtctccgtcgtggtctttgtaatcaacacaaggcacaaagatttggagg-3'

[0056] The high-fidelity PCR reaction system is as follows: TaKaRa high-fidelity amplification enzyme PrimeSTAR, forward primer, reverse primer, and template is 84K poplar root total RNA; after full-length cDNA reverse transcription, full-length sequence amplification is performed, and the reaction procedure is: pre-denaturation at 94°C, 7min; 94°C, 30s; 50°C, 45s; 72°C, 24s, 30 cycles; 72°C for 10min, 16°C for 30min.

[0057] The final obtained full-length sequence of the PagPB gene was 396 bp, named PagPB gene, as shown in the sequence SEQ ID NO.1.

[0058] (2) Use 1.5% agarose gel electrophoresis at 5 v / cm for 20 minutes to cut out the 396 bp electrophoresis fragment of the PagPB gene under ultraviolet light, recover the first PCR product, connect it to the T plasmid, and name the sequencing-positive plasmid pM-PagPB.

[0059] (3) The pFGC5941 RNAi vector was double-digested with endonucleases AscI and Swa I. The digestion reaction temperature was 37°C, the system was 10 μL, and the reaction time was 1 h. The plasmid pM-PagPB was amplified with PagPB-cis-F and PagPB-cis-R as primers. After the second PCR product was recovered, it was connected with the double-digested pFGC5941 RNAi vector. The primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6. The ligation product was further introduced into DH5α Escherichia coli by heat shock transformation, and a single colony was picked for sequencing. The plasmid of the positive colony was retained, and the sequencing positive plasmid was named pM-PagPB-C.

[0060] PagPB-cis-F: as shown in SEQ ID NO.5, is:

[0061] 5'-atttacaattaccatggggcgcgccAACGAGGAAAATAATGACAGG-3'

[0062] PagPB-cis-R: as shown in SEQ ID NO.6, is:

[0063] 5'-acataagaaattcttacacatttaaatGTGAGCAAGGCGTCTTTGGCA-3'

[0064] (4) The recombinant vector pM-PagPB-C was double-digested with endonucleases BamHI and XbaI, the digestion reaction temperature was 37°C, the system was 10 μL, the reaction time was 1 h, and the plasmid pM-PagPB was amplified with PagPB-anti-F and PagPB-anti-R as primers. After the third PCR product was recovered, it was connected with the double-digested recombinant vector pM-PagPB-C to obtain the PagPB gene silencing vector. The primer sequences are shown in SEQ ID NO.7 and SEQ ID NO.8. The obtained recombinant plasmid was further transferred into Agrobacterium GV3010 using the freeze-thaw method to obtain the PagPB silenced recombinant Agrobacterium.

[0065] PagPB-anti-F: as shown in SEQ ID NO.7, is:

[0066] 5'-gtcaatttgcaggtatttggatccGTGAGCAAGGCGTCTTTGGCA-3'

[0067] PagPB-anti-R: as shown in SEQ ID NO.8, is:

[0068] 5'-cgggtcttaattaactctctagaAACGAGGAAAATAATGACAGG-3'

[0069] (5) The full-length sequence of the PagPB gene obtained in step (1) was subjected to 1.5% agarose gel electrophoresis at 5 v / cm for 20 minutes. The 396 bp electrophoresis fragment of the PagPB gene was cut out under ultraviolet light, and the gel was recovered to detect the sequence correctness.

[0070] (6) The pBWA(V)HS-ccdB-3xflag overexpression vector was digested with BsaI and Eco31I endonucleases. The digestion reaction system was 20 μL, the reaction temperature was 37°C, and the reaction was performed for 1 h to obtain the linearized pBWA(V)HS-ccdB-3xflag vector.

[0071] (7) Using homologous recombination method, the full-length sequence of the PagPB gene detected correctly in step (5) is connected with the linearized pBWA(V)HS-ccdB-3xflag vector obtained in step (6).

[0072] (8) The ligation product was transformed into DH5α competent E. coli by heat shock transformation, and then transformed into a kanamycin-resistant plate, cultured at 37°C for 12 hours, and then identified by colony PCR. The positive plasmid was transformed into GV3101 Agrobacterium to obtain pBWA(V)HS-PB overexpression recombinant Agrobacterium.

[0073] Example 2: Creation of transgenic poplar plants with PagPB gene silencing and overexpression

[0074] (1) 84K tissue culture seedlings were cultured under the conditions of 16 h of light during the day and 25°C. The PagPB-silenced recombinant Agrobacterium and the pBWA(V)HS-PB-overexpressing recombinant Agrobacterium were cultured at OD 600 When the pH value was 0.6-0.8, 10 μM acetosyringone As was added and the culture was continued on a shaker at 28°C for 30 min for later use.

[0075] (2) Infection was carried out using the leaf disc method, and the leaves were cultured in the dark for 4 days after infection. After cleaning, the leaf discs were transferred to culture dishes containing PagPB gene silencing and overexpression poplar selection medium, respectively, and PagPB gene silencing and overexpression resistant adventitious buds were induced and screened under the conditions of 16 h of light during the day and a culture temperature of 24°C;

[0076] (3) The explants carrying PagPB gene silenced and overexpressed adventitious buds were transferred to PagPB gene silenced and overexpressed poplar rooting medium for further culture.

[0077] (4) The CTAB method was used to extract leaf DNA from gene-silenced and overexpressed transgenic poplars and wild-type plants, respectively. Using leaf DNA as a template, PCR was performed using primers pFGC5941-F, with a primer sequence as shown in SEQ ID NO.9, and primers pFGC5941-R, with a primer sequence as shown in SEQ ID NO.10, to identify PagPB silenced transgenic plants. In overexpressed transgenic poplars, PCR was performed using a forward primer for the hygromycin resistance gene carried on the vector, with a primer sequence as shown in SEQ ID NO.11, and a reverse primer, with a primer sequence as shown in SEQ ID NO.12, to identify PagPB gene overexpressing transgenic plants. The positive control was a PagPB gene silencing or overexpression recombinant vector plasmid, and the negative control was a wild-type poplar plant. The results are shown in Figure 2. Figure 1As shown, PagPB-RNAi1, 2, 3, 4, 16, 20, 21 and 22 are the electrophoresis results of transgenic silencing lines, and OE1-21 are the electrophoresis results of transgenic overexpression lines; P is the positive control of the PagPB gene silencing or overexpression recombinant vector plasmid; 84K is the negative control of wild-type poplar plants.

[0078] Wherein, the pFGC5941 forward primer is as shown in the sequence list as SEQ ID NO.9, see Table 2 below;

[0079] Table 2

[0080] name SEQ ID NO.9 pFGC5941 forward primer 5'-CTCCTTTGCCCCGGAGATTACA-3'

[0081] The reverse primer of pFGC5941 is SEQ ID NO.10 in the sequence list, see Table 3 below;

[0082] Table 3

[0083] name SEQ ID NO.10 pFGC5941 reverse primer 5'-GGGGTAATGTTGTTTGTTGTTTGTT-3'

[0084] The forward primer of hygromycin resistance gene is as shown in the sequence list, SEQ ID NO.11, see Table 4 below;

[0085] Table 4

[0086] name SEQ ID NO.11 Hygromycin resistance gene forward primer 5'-gagcatatacgcccggagtc-3'

[0087] The reverse primer of the hygromycin resistance gene is SEQ ID NO.12 in the sequence list, see Table 5 below;

[0088] Table 5

[0089] name SEQ ID NO.12 Hygromycin resistance gene reverse primer 5'-caagacctgcctgaaaccga-3'

[0090] (5) Using root samples of gene-silenced, overexpressed transgenic poplar plants and wild-type poplar plants as materials, total RNAs of the above samples were extracted using a total RNA extraction kit and a reverse transcription kit, and reverse transcription was performed. RT-qPCR was performed using a TBGreen qRT-PCR kit.

[0091] Wherein, the quantitative forward primer of PagPB gene is as shown in the sequence list as SEQ ID NO.13, see Table 6 below;

[0092] Table 6

[0093] name SEQ ID NO.13 PagPB gene quantitative forward primer 5'-CCGCAGAAACGAGGAAATAA-3'

[0094] The quantitative reverse primer of PagPB gene is SEQ ID NO.14 in the sequence list, see Table 7 below;

[0095] Table 7

[0096] name SEQ ID NO.14 PagPB gene quantitative reverse primer 5'-CTCAGTTTGGCGGTCTCTTC-3'

[0097] Actin was used as an internal reference gene, wherein the Actin quantitative forward primer was as shown in the sequence list as SEQ ID NO.15, see Table 8 below;

[0098] Table 8

[0099] name SEQ ID NO.15 Actin quantitative forward primer 5'-CCCATTGAGCACGGTATTGT-3'

[0100] Actin quantitative reverse primer is SEQ ID NO.16 in the sequence list, see Table 9 below;

[0101] Table 9

[0102] name SEQ ID NO.16 Actin quantitative reverse primer 5'-TACGACCACTGGCATACAGG-3'

[0103] The PCR program was: 98°C, 10 s; 94°C, 15 s, 60°C, 30 s, 40 cycles; the expression level of PagPB gene was measured using 2 -△△Ct The expression patterns of transgenic and wild-type PagPB genes were analyzed by t-test. Log2(FC)≥1 or ≤-1 and P<0.05 were defined as statistically significant.

[0104] The results are as follows Figure 2 As shown, the relative expression levels of PagPB gene in the roots of PagPB gene silencing transgenic poplars RNAi16 and RNAi22, overexpressing transgenic poplars OE3 and OE4 and wild-type 84K poplar. Compared with the wild-type poplar 84K, the PB gene expression levels of PagPB gene silencing transgenic poplars RNAi16 and RNAi22 were significantly lower than those of the wild-type control, while the expression level of PagPB gene in the roots of transgenic poplars OE3 and OE4 was significantly increased.

[0105] Example 3: Functional analysis of PagPB gene regulating nitrogen absorption and utilization in poplar roots

[0106] In this example, the PagPB gene silencing, overexpression transgenic strains and wild-type root NO3 - and NH4 + The net absorption rate, root biomass, root nitrate and ammonium nitrogen content, and the activity of nitrate reductase (NR) and glutamine synthetase (GS) are measured in the following steps:

[0107] (1) Transgenic poplars were obtained according to the method of Example 2. Three-month-old PagPB gene-silenced, overexpressed transgenic strains and wild-type poplar seedlings were used as experimental materials. The NO3 concentration at 10 mm from the root tip of the PagPB gene-silenced, overexpressed transgenic strains and wild-type strains was measured using a non-invasive micro-measurement system based on scanning ion selective electrode technology. - and NH4 + Net absorption rate, the results are as follows Figure 3 As shown, compared with the wild-type poplar 84K, the root NO3 - The net absorption rate is significantly increased, NH4 + The net absorption rate was also increased, while the root NO3 of the overexpressing transgenic lines OE3 and OE4 - The net absorption rate is reduced, NH4 + The net absorption rate was significantly reduced.

[0108] (2) Root samples of PagPB gene-silenced and overexpressed transgenic lines and wild-type lines were harvested and the root biomass of transgenic and wild-type plants was measured. The results were as follows: Figure 4 As shown, compared with the wild-type poplar 84K, the biomass of the PagPB gene-silenced transgenic poplars RNAi16 and RNAi22 was increased, while the root biomass of the overexpressed transgenic poplars OE3 and OE4 was reduced.

[0109] (3) Root samples of PagPB gene silenced, overexpressed transgenic lines and wild-type lines were harvested and the contents of nitrate nitrogen and ammonium nitrogen were determined. The results were as follows: Figure 5 As shown, compared with the wild-type poplar 84K, the nitrate nitrogen and ammonium nitrogen contents in the roots of PagPB gene-silenced transgenic poplars RNAi16 and RNAi22 were significantly increased, while the nitrate nitrogen and ammonium nitrogen contents in the roots of overexpressing transgenic poplars OE3 and OE4 were decreased.

[0110] (4) Root samples of PagPB gene silenced, overexpressed transgenic lines and wild-type lines were harvested and the activities of nitrate reductase and glutathione reductase were determined using a nitrate reductase activity detection kit and a glutamine synthetase test kit. The results are as follows: Figure 6 As shown in the figure, compared with the wild-type poplar 84K, the root nitrate reductase activity of the PagPB gene silenced transgenic poplars RNAi16 and RNAi22 was significantly increased, while the root nitrate reductase activity of the overexpressed transgenic poplars OE3 and OE4 was reduced.

[0111] In summary, the present invention transfers the PagPB gene into 84k poplars, and the root NO3 - and NH4+ The net absorption rate increased, the root biomass increased, the root nitrate nitrogen and ammonium nitrogen contents increased significantly, and the nitrate reductase activity also increased. Compared with the wild type, the transgenic poplars overexpressing PagPB had a lower NO3 - and NH4 + The net absorption rate decreased, the nitrate nitrogen content in the roots decreased significantly, and the ammonium nitrogen content and nitrate reductase activity decreased. This indicates that silencing the PagPB gene can improve the absorption and utilization efficiency of nitrogen by the poplar root system, and overexpressing the PagPB gene reduces the absorption and utilization efficiency of nitrogen by the poplar root system, proving that the PagPB gene is a key regulatory gene for regulating nitrogen in the poplar root system, especially the absorption and utilization of nitrate nitrogen, and has important application value in the fields of forest genetic engineering and asexual forestry.

[0112] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0113] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A PagPB gene for regulating nitrogen absorption and utilization by roots of woody plants, characterized in that: The nucleotide sequence of the PagPB gene is shown in SEQ ID NO.

1.

2. Use of the PagPB gene as claimed in claim 1 in regulating nitrogen absorption and utilization by roots of woody plants.

3. The use of the PagPB gene in regulating nitrogen absorption and utilization by roots of woody plants as claimed in claim 2, characterized in that: By silencing the PagPB gene, the root nitrogen absorption and utilization efficiency is improved and the root biomass is increased.

4. The use of the PagPB gene in regulating nitrogen absorption and utilization by roots of woody plants as claimed in claim 3, characterized in that: Improving the efficiency of nitrogen absorption and utilization by roots means: increasing the root NO3 - and NH4 + Net absorption rate, root nitrate-nitrogen, ammonium-nitrogen content and nitrate reductase activity.

5. The use of the PagPB gene in regulating nitrogen absorption and utilization by roots of woody plants as claimed in claim 3, characterized in that: Construct a PagPB gene silencing vector, transform woody plants, and obtain PagPB gene-silenced transgenic plants to improve the root system's nitrogen absorption and utilization efficiency and increase root biomass.

6. The use of the PagPB gene in regulating nitrogen absorption and utilization by roots of woody plants as claimed in claim 5, characterized in that: The specific method for constructing the PagPB gene silencing vector is as follows: (1) using primers specific to the PagPB gene sequence to amplify the full-length sequence of the PagPB gene, recovering the first PCR product, and ligating it into the T plasmid to obtain the plasmid pM-PagPB; (2) double-enzyme digestion of pFGC5941 RNAi vector, using PagPB-cis-F and PagPB-cis-R as primers to amplify plasmid pM-PagPB, recovering the second PCR product and connecting it with the double-enzyme digestion of pFGC5941 RNAi vector; introducing the ligation product into bacteria to obtain the recombinant vector pM-PagPB-C; (3) Double-enzyme digestion of the recombinant vector pM-PagPB-C, using PagPB-anti-F and PagPB-anti-R as primers to amplify the plasmid pM-PagPB, recovering the third PCR product and connecting it with the double-enzyme digestion recombinant vector pM-PagPB-C to obtain the PagPB gene silencing vector; The specific primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4; The PagPB-cis-F primer sequence is shown in SEQ ID NO.5, and the PagPB-cis-R primer sequence is shown in SEQ ID NO.6; The PagPB-anti-F primer sequence is shown in SEQ ID NO.7, and the PagPB-anti-R primer sequence is shown in SEQ ID NO.

8.

7. The use of the PagPB gene in regulating nitrogen absorption and utilization by roots of woody plants as claimed in claim 5, characterized in that: The transformation method is Agrobacterium-mediated method.

8. The use of the PagPB gene in regulating nitrogen absorption and utilization by roots of woody plants as claimed in claim 5, characterized in that: The woody plant is poplar.

9. The use of the PagPB gene in regulating nitrogen absorption and utilization by roots of woody plants according to claim 8, characterized in that: The variety of the poplar is P. alba x P. glandulosa 84K.

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