CN119979570B

CN119979570B - Pop_A17G020368 gene and application thereof in promoting plant growth

Info

Publication number: CN119979570B
Application number: CN202510472598.2A
Authority: CN
Inventors: 崔亚宁; 徐昌文; 刘艳; 林金星; 路笃贤; 王笑
Original assignee: Weishan County Natural Resources And Planning Bureau; Beijing Forestry University
Current assignee: Weishan County Natural Resources And Planning Bureau; Beijing Forestry University
Priority date: 2025-04-16
Filing date: 2025-04-16
Publication date: 2025-06-24
Anticipated expiration: 2045-04-16
Also published as: CN119979570A

Abstract

The invention discloses a Pop_A17G020368 gene and application thereof in promoting plant growth, and relates to the technical field of plant genetic engineering, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 1. The function of the Pop_A17G020368 gene is verified by cloning and analyzing the Pop_A17G020368 gene and combining agrobacterium-mediated genetic transformation technology to overexpress the Pop_A17G020368 gene in 84K poplar. Compared with the wild type, the plant with the over-expression Pop_A17G020368 gene can obviously promote the growth of poplar with high plant height and thick stem. The important role of Pop_A17G020368 gene in promoting the growth of poplar plant height and secondary growth is demonstrated. The invention has important practical and theoretical guiding significance for researching the growth stability of the tree and cultivating new varieties of the tree.

Description

Pop_A17G020368 gene and application thereof in promoting plant growth

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a Pop_A17G020368 gene and application thereof in promoting plant growth.

Background

Poplar is an important forest resource, and has the characteristics of fast growth, strong adaptability, wide application and the like, and is widely applied to forestation, urban greening and wood production. However, traditional poplar breeding methods have long cycle and low efficiency, and are difficult to meet the increasing wood demand. Along with the development of molecular biology and genetic engineering technology, improving the growth traits of poplar by genetic engineering means has become an effective breeding strategy. However, the number of functional genes associated with poplar secondary growth is currently limited.

Therefore, the mining of more genes for promoting growth is a problem that the skilled person is urgent to solve.

Disclosure of Invention

In view of this, the present invention provides the pop_a17g020368 gene and its use in promoting plant growth.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the nucleotide sequence of the Pop_A17G020368 gene is shown as SEQ ID NO. 1.

Another object of the present invention is to provide pop_A17G020368 protein, the amino acid sequence of which is shown in SEQ ID NO. 2.

It is another object of the present invention to provide a biomaterial, which is any one of the following:

an expression cassette capable of over-expressing a gene with a nucleotide sequence shown as SEQ ID NO. 1;

B, a recombinant vector containing the expression cassette A;

c, a recombinant microorganism containing the expression cassette A or the recombinant vector B;

and D, non-regenerable plant parts containing the expression cassette A or the recombinant vector B or the recombinant microorganism C.

It is another object of the present invention to provide the use of the above gene, or the above protein, or the above biological material for promoting plant growth.

Preferably, the use is to increase plant height and/or increase plant stem base diameter.

The invention also aims to provide a breeding method for promoting plant growth, which utilizes a transgenic means to improve the expression quantity of the Pop_A17G020368 gene, wherein the nucleotide sequence of the Pop_A17G020368 gene is shown as SEQ ID NO. 1.

Compared with the prior art, the invention verifies the function of the Pop_A17G020368 gene by cloning and analyzing the Pop_A17G020368 gene and combining with agrobacterium-mediated genetic transformation technology to overexpress the Pop_A17G020368 gene in 84K poplar. Compared with the wild type, the plant with the over-expression Pop_A17G020368 gene can obviously promote the growth of poplar with high plant height and thick stem. The important role of Pop_A17G020368 gene in promoting the growth of poplar plant height and secondary growth is demonstrated. The invention has important practical and theoretical guiding significance for researching the growth stability of the tree and cultivating new varieties of the tree.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the phenotype of wild type and transgenic plants.

FIG. 2 is an electrophoresis chart of positive over-expression plant detection.

FIG. 3 shows the expression of the pop_A17G020368 gene in wild type and transgenic plants.

FIG. 4 shows the expression of the Pop_A17G020368 gene in different tissues of wild type.

FIG. 5 shows the quantitative results of the plant heights of wild type and transgenic plants.

FIG. 6 shows the quantitative results of the diameter of the stem base of wild type and transgenic plants.

Detailed Description

In order that the invention may be understood more fully, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended claims. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It will be appreciated that the experimental procedures described in the examples below, without specifying the specific conditions, are generally followed by conventional conditions, such as those described in the Sambrook et al molecular cloning, A laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations. The various reagents commonly used in the examples are all commercially available products.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The expression cassette of the invention refers to a DNA capable of expressing the Pop_A17G020368 gene in a host cell, wherein the DNA not only can comprise a promoter for starting the transcription of the coding gene, but also can comprise a terminator for stopping the transcription of the coding gene. Further, the expression cassette may also include an enhancer sequence.

Example 1

Cloning and vector construction of Pop_A17G020368 gene

1. Total RNA extraction wild type silver gland Yang Moxing series 84K poplar total RNA was extracted using RNA extraction kit (purchased from Beijing Edley Biotechnology Co., ltd.).

CDNA synthesis, namely, reversely transcribing the total RNA extracted in the previous step into single-stranded cDNA by using a cDNA synthesis kit EASYSCRIPT First-STRAND CDNA SYNTHETIC Supermix (purchased from Beijing full-scale gold biotechnology Co., ltd.) through a one-step method.

3. Cloning the gene, namely amplifying the gene fragment by adopting high-fidelity enzyme.

The forward and reverse primers of pCAMBIA2300:35s: pop_A17G020368-GFP were designed by referring to CDS sequence of Pop_A17G020368 gene with BamHI and KpnI double cleavage sites, and were entrusted with the synthesis primers of biosystems for amplification of Pop_A17G020368 gene.

Pop_A17G020368 gene CDS sequence:

ATGCCTTCCCAGACCAACTCCCACTTCTCCCTTCCAAGGTGGATTGGATTACTGTCTCACAAGAACAGCCAAAGCGAAACTGGTGACAATAATGACAACAGCAACCTTAACAGTACCAACAACATTGACGACAGCAACCCTATCTCTAACTCCATAGAATGCTACGCTTGCACACAAGTAGGAGTTCCTGTCTTCCACTCCACAAGCTGTGACCAGGCCCACCAGCCTGAATGGGAGGCCTCCGCGGGCTCATCATTAGTCCCCATCAAAAACCGGCTAGGTTCAAGAAAGAGCCCAGCAAGCCGAGCCCAATCTCGCCGGCCGGCCGGTCCATTCGGAACCATTCTCGATCCACGTAGCAAGCGGGTACAGAAATGGAACCGGGCTTTCCTGCTAGCACGTGGCATGGCTTTGGCAGTCGATCCCTTGTTTTTCTACGCTCTGTCGATAGGGAGAAACGGGGCCCCTTGCTTGTACATGGACGGTGGGTTGGCCGCAATCGTGACCGTCCTTCGCACGAGTGTGGATGCTATACACTTGTGCCATCTGTGGTTACAATTCAGGTTGGCTTATGTGTCAAGAGAGTCTCTTGTCGTGGGGTGCGGGAAACTGGTGTGGGACGCACGTGCCATCGCATCTCATTACGTGAGGTCTCTTAAGGGCTTCTGGTTTGATGCTTTTGTCATTCTTCCCGTTCCTCAGGCTGTATTTTGGTTACTTGTACCCAGATTAATCAGAGAAGAGCAGATTAAGCTCATAATGACAATACTTTTGCTGATCTTCTTGTTCCAATTCCTCCCTAAGGTGTACCACTGCATATGTTTGATGAAAAGAATGCAAAAGGTCACAGGTTATATCTTTGGCACCATTTGGTGGGGCTTCGGCCTTAATCTCATTGCCTACTTCATTGCCTCTCATGTTGCTGGTGGATGTTGGTATGTCCTCGCAATACAACGCGTCGCTTCATGTCTCAGGCAGAGTTGTGAGAGGAGGCCTAATTGTGATCTCTCTTTGGCTTGCTCGGAGGAGGTTTGCTATCAGTTCCTGTTACGATCAGGCACAATTGGAAATCCATGCGTTGGAAACACAACACATACTGTTAGAAAGCCTATGTGCCTGGATGTTAATGGAGCTTTCAATTATGGGATTTACAAGTGGGCTCTTCCAGTCATTTCTAGCAATTCTTTGTCTGTGAAGATTCTTTATCCCATTTTCTGGGGTTTAATGACCCTCAGCACCTTTGGCAATGATCTTGAACCAACAAGTCACTGGCTAGAAGTAATCTTCAGTATATGTATCGTTCTCAGTGGTTTAATGCTCTTCACTCTTCTGATTGGGAACATCCAGGTGTTCTTGCACGCGGTCATGGCAAAGAAGAGAAAAATGCAGCTGAGATGCGGAGATATGGAATGGTGGATGAGGAGGAGACAGTTGCCTTCTCGTTTGAGACAGAGAGTTCGCCATTATGAACGGCAGAGATGGGCAACCATGGGAGGTGAAGATGAGATAGAACTAATCACAGACTTACCTGAAGGACTCCGGAGAGATATCAAGCGCTATCTTTGCCTAGATCTTATCAAGAAGGTGACACAAATGAACACTTAG,SEQ ID NO.1.

amino acid sequence:

MPSQTNSHFSLPRWIGLLSHKNSQSETGDNNDNSNLNSTNNIDDSNPISNSIECYACTQVGVPVFHSTSCDQAHQPEWEASAGSSLVPIKNRLGSRKSPASRAQSRRPAGPFGTILDPRSKRVQKWNRAFLLARGMALAVDPLFFYALSIGRNGAPCLYMDGGLAAIVTVLRTSVDAIHLCHLWLQFRLAYVSRESLVVGCGKLVWDARAIASHYVRSLKGFWFDAFVILPVPQAVFWLLVPRLIREEQIKLIMTILLLIFLFQFLPKVYHCICLMKRMQKVTGYIFGTIWWGFGLNLIAYFIASHVAGGCWYVLAIQRVASCLRQSCERRPNCDLSLACSEEVCYQFLLRSGTIGNPCVGNTTHTVRKPMCLDVNGAFNYGIYKWALPVISSNSLSVKILYPIFWGLMTLSTFGNDLEPTSHWLEVIFSICIVLSGLMLFTLLIGNIQVFLHAVMAKKRKMQLRCGDMEWWMRRRQLPSRLRQRVRHYERQRWATMGGEDEIELITDLPEGLRRDIKRYLCLDLIKKVTQMNT,SEQ ID NO.2.

The primer sequences were as follows:

2300-0368-F:5’-CATTTGGAGAGGACAGGGTACCATGCCTTCCCAGACCAACTCC-3’,SEQ ID NO.3;

2300-0368-R:5’-CAGAAAATTTCTAGAGGATCCAGTGTTCATTTGTGTCACCTTC-3’,SEQ ID NO.4。

4. Vector linearization, namely carrying out double enzyme digestion on a vector plasmid of pCAMBIA2300-35S-GFP by using restriction enzymes BamHI and KpnI to obtain a linearized vector fragment.

5. Agarose gel electrophoresis detection and purification recovery, namely, electrophoresis detection is carried out by using agarose gel with the concentration of 1%, the size of the Pop_A17G020368 band of the target gene and the size of the band of the linearization vector are respectively about 1602bp and 12000bp, and the target gene fragment and the linearization vector fragment are purified and recovered by using an agarose gel recovery kit (purchased from Beijing Edley Biotechnology Co., ltd.).

6. Vector construction the amplified Pop_A17G020368 gene fragment was inserted between BamHI and KpnI sites of pCAMBIA2300-35S-GFP vector and recombinant vector pCAMBIA2300:35S: pop_A17G020368-GFP was constructed using a premix Gibson Assembly Master Mix (available from NEB Co.) of seamless cloning.

7. Transformation of E.coli competent cells TOP 10 (available from Beijing Edley Biotech Co., ltd., specific methods and amounts refer to the instructions) the reaction product of step 6 was added to E.coli competent cells for transformation, and then uniformly spread on LB plates containing kanamycin. Inverted cultures were incubated overnight at 37 ℃.

8. And (3) positive clone identification, namely selecting a single clone to carry out colony PCR identification by using JP-0368-F, JP-GFP-R as a primer, selecting a plurality of positive clones, marking out and preserving seeds on a new kanamycin plate, simultaneously inoculating into LB liquid culture medium containing kanamycin, and carrying out shaking culture at 37 ℃ and 180-230 rpm for overnight. The next day the bacterial fluid was sent to the company for sequencing. The sequencing primer sequences were as follows:

JP-0368-F:5’-GAAAGCCTATGTGCCTGGATG-3’,SEQ ID NO.5;

P-GFP-R:5’- TGTTGACGAGGGTGTCTCCCT -3’,SEQ ID NO.6。

9. Plasmid extraction correctly edited vector plasmids were extracted using a plasmid miniprep kit (kit purchased from the company beijing, biochemical technology, diurnal root), the procedure being referred to the kit instructions.

10. Agrobacterium competent cells GV3101 (purchased from Shanghai Biotechnology Co., ltd., specific procedures are described) were transformed, and finally resuspended cells were plated on solid LB medium supplemented with 50 mg/L kanamycin and 50 mg/L rifampicin, and cultured at 28℃for 36-48 h. The primers JP-0368-F and JP-GFP-R bacterial liquid PCR are used for detecting positive clones, and the agrobacterium containing the correct over-expression vector is obtained.

Example 2

Agrobacterium-mediated genetic transformation

1. The over-expression vector of Pop_A17G020368 is guided into poplar leaves through an agrobacterium-mediated genetic transformation system, and is subjected to leaf pre-culture, agrobacterium dip-dyeing, dark culture, induction of germination, induction of rooting by a tissue culture bottle, seedling hardening and transplanting in a greenhouse for soil culture.

(1) Blade preculture, namely, opening a high-temperature sterilizer in advance to sterilize scissors, forceps and a tissue culture scalpel at high temperature in a sterile super clean bench, cooling, cutting the blades of the wild 84K poplar, vertically cutting veins by the scalpel to cut 3-4 wounds in the middle of the blades, placing the blades on a preculture medium configured in advance, and culturing under illumination conditions (16 h/dark 8 h) for 2-3 days.

(2) Agrobacteria is immersed and dark cultured, wherein the agrobacteria are immersed and dark cultured for 12-16 hours in advance, 500 mu L-1mL of agrobacteria containing the correctly edited pop_A17G020368 vector are added into 100mL of LB liquid medium containing antibiotics (50 mg/L kanamycin and 50 mg/L rifampicin), and after being mixed evenly, the agrobacteria are put into 28 ℃ and cultured for overnight under shaking at 200 rpm/min. The next day, the OD ₆₀₀ of the agrobacterium is measured to be 0.6-1.0. In a sterile super clean bench, sterilizing tweezers in advance by ultraviolet sterilization and opening a high-temperature sterilizer, clamping the blades on the preculture medium into an agrobacterium conical flask after the tweezers are cooled, and uniformly shaking the conical flask for 15-20min and 2-3 min. After timing, the leaves are clamped onto filter paper after high-pressure steam sterilization, redundant bacterial liquid on the surfaces of the leaves is sucked, and the leaves are placed into a dark culture medium at 25 ℃ for 2-3 days in the dark.

(3) Inducing bud differentiation, namely transferring the dark cultured leaves to a differentiation culture medium with the kanamycin screening pressure of 20-30 mg/L for inducing differentiation, changing the culture medium once every 6-8 days, and culturing under the condition of illumination (16 h/8 h of illumination) at 25 ℃ until adventitious buds are differentiated.

(4) The tissue culture bottle induces rooting, namely, adventitious buds growing to the length of 1-2cm are separated from leaves by sterilized forceps and transferred to rooting culture medium with kanamycin screening pressure of 15-20 mg/L, cultured under the condition of illumination (16 h/dark 8h culture) at 25 ℃ until rooting, and propagated after about 4-5 weeks until normal plants grow to the size.

(5) And (3) after seedling hardening, transplanting the seedlings in a greenhouse for soil cultivation, wherein when the tissue culture seedlings grow for about 45 days and the root system is developed, the seedlings are hardened for 3-5 days and then transplanted. Washing off the culture medium of poplar root with clear water, transplanting to sterilized nutrient soil, and culturing in a greenhouse.

The formulation of the medium used in this example is shown in Table 1:

TABLE 1

Example 3

1. Identification of transgenic overexpressing plants

The transformed poplar leaves of different strains were cut in an ultra clean bench, each strain was labeled at the same time, and genomic DNA was extracted using a genomic DNA extraction kit (available from Jiangsu kang, century Biotech Co., ltd., for specific procedures, reference instructions).

Using primers JP-0368-F and JP-GFP-R, positive plants were identified that were successfully transgenic by PCR cloning of a fragment 800bp containing Pop_A17G020368 expressed in fusion with GFP. The OE-1 and OE-2 were identified as Pop_A17G020368 gene over-expression successful strains (see FIG. 1-FIG. 3).

Action of Pop_A17G020368 gene in regulating tree height and secondary growth process

(1) The fluorescent real-time quantitative PCR method detects the expression quantity of Pop_A17G0203688 in different tissue sites:

84K Yang Shuzong RNA extraction, namely cutting rhizome and leaf parts of wild 84K plants to detect the expression quantity of target genes in different tissue parts, and cutting over-expression plants OE-1/-2 of different strains and wild 84K tissue culture seedling leaves to identify the target gene expression condition of the over-expression plants. Total RNA solution was obtained by extraction using liquid nitrogen milling according to EASYspin Plus plant RNA rapid extraction kit (available from Eddley Biotech Co., ltd. Beijing, specific procedures are described).

CDNA Synthesis Total RNA from the different strains was subjected to reverse transcription using a one-step cDNA synthesis kit (available from Beijing full gold Biotechnology Co., ltd., see the instructions for specific procedures).

The fluorescent real-time quantitative PCR method is to quantitatively determine the expression of different parts of the Pop_A17G020368 gene by using SYBRGreen qPCRMix of Beijing Aidelai biotechnology, and refer to a kit instruction book, a reaction system and a PCR program. The PCR primers of the Pop_A17G020368 genes are qPCR-0368-F1 and qPCR-0368-R1, the internal reference genes are Actin, the primers are Actin-F and Actin-R, and repeated results of three biological experiments show that the tissue expression quantity of the Pop_A17G020368 genes in 84K is highest, the tissues are stems and roots, and the detection results of the expression quantity of different tissue parts are shown in figure 4. The expression level of Pop_A17G020368 in OE-1 and OE-2 plants is higher than that of wild type, and the detection result of the transcription level expression level of transgenic poplar is shown in figure 3.

qPCR-0368-F1:5’-GAGACAGAGAGTTCGCCATT-3’,SEQ ID NO.7;

qPCR-0368-R1:5’-GATAAGATCTAGGCAAAGAT-3’,SEQ ID NO.8。

Actin-F:5’-AAACTGTAATGGTCCTCCCTCCG-3’,SEQ ID NO.9;

Actin-R:5’-GCATCATCACAATCACTCTCCGA-3’,SEQ ID NO.10。

(2) Phenotype observation of transgenic plants Wild Type (WT) strains with consistent vigour after rooting, over-expressed (OE-1/-2) strains were transferred to soil for growth. Phenotypic observation and statistical measurement show that the plant height of the over-expressed strain in the same growth period is significantly higher than that of the wild type strain (see figure 5), and the stem base diameters of the wild type strain and the OE-1/-2 strain are counted, so that the stem base diameters of the OE-1/-2 strain are significantly larger than that of the wild type strain (see figure 6).

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The Pop_A17G020368 gene, characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO: 1.

2. Pop_A17G020368 protein, characterized in that the amino acid sequence of the protein is shown in SEQ ID NO.2.

3. A biomaterial, characterized in that the biomaterial is any one of the following:

A: an expression cassette comprising an expression vector pCAMBIA2300-35S-GFP into which a gene with a nucleotide sequence as shown in SEQ ID NO.1 is inserted;

B: a recombinant vector containing the expression cassette described in A;

C: a recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B;

D: A non-regenerable plant part containing the expression cassette described in A, the recombinant vector described in B, or the recombinant microorganism described in C.

4. Use of the gene according to claim 1, or the protein according to claim 2, or the biomaterial according to claim 3 in promoting the growth of poplar.

5. The use according to claim 4, characterized in that the use is to increase the height of poplar trees and/or increase the diameter of the base of poplar stems.

6. A breeding method for promoting poplar growth, characterized in that: using genetic modification means,

Pop_A17G020368Pop_A17G020368

The gene was inserted into the expression vector pCAMBIA2300-35S-GFP to improve

Pop_A17G020368Pop_A17G020368

The expression level of the gene

Pop_A17G020368Pop_A17G020368

The nucleotide sequence of the gene is shown in SEQ ID NO:1.