CN118581135A - Application of a Poplar CDPK6 Gene in Forest Tree Breeding - Google Patents

Abstract

The present invention provides an application of a poplar CDPK6 gene in forest breeding. Poplar plants with excellent material quality can be obtained by overexpressing the CDPK6 gene in poplars. The development status of poplar plants can be predicted by detecting the expression level of the CDPK6 gene in the plants. The present invention also provides a poplar CDPK6 gene, the gene sequence is shown in SEQ ID NO: 1 or the encoded protein sequence is shown in SEQ ID NO: 2. The present invention also provides a method for regulating the thickness of the xylem cell wall and/or the number of xylem cell layers in the stem of a poplar, as well as the transformation vector, primers and strains used in the method. The present invention reveals for the first time the application characteristics of the CDPK6 gene in promoting the thickening of the xylem cell wall of poplars, which is of great significance in the genetic improvement of poplar wood properties.

Description

Application of a Poplar CDPK6 Gene in Forest Tree Breeding

Technical Field

The invention belongs to the field of genetic engineering technology, and in particular relates to an application of a poplar CDPK6 gene in forest breeding.

Background Art

As an important renewable biomass energy source in nature, wood is a potential natural resource to replace fossil fuels such as oil and natural gas. At the same time, wood also has important applications and economic value in the fields of pulp and paper, plywood and packaging materials. Therefore, increasing the volume of wood per unit area and improving wood quality have become important goals of forest breeding research. Wood formation is a complex biological process, involving the continuous division and differentiation of apical meristem and vascular cambium cells, followed by cell wall thickening, programmed cell death and heartwood formation. Functional genomics studies have shown that wood formation is a quantitative trait controlled by multiple genes, which is the result of the synergistic action of multiple genes, multiple levels and multiple pathways, and has a very complex genetic regulation mechanism.

Among them, protein kinases form complex genetic interaction networks in systems such as plant secondary growth and cell signal transduction. In plants, one of the most studied protein kinases is the Ca ²⁺ /CaM-dependent protein kinase, which contains four highly conserved domains, namely the N-terminal variable domain, the kinase domain, the autoinhibitory linker domain, and the C-terminal calmodulin-like regulatory domain with an EF-hand structure. In addition, Ca ²⁺ /CaM-dependent protein kinases mainly transfer and covalently bind the phosphate group of ATP in the kinase domain to the hydroxyl group of specific serine, threonine or tyrosine residues in specific protein molecules, thereby causing changes in the conformation and activity of proteins and enzymes, further affecting the regulatory function of proteins on downstream genes.

Poplar (Populus L.) is the main afforestation tree species in northern my country. Its rapid growth characteristics make it an ideal choice for observing forest growth, development, and physiological and biochemical processes. As a model tree, poplar has a completely sequenced genome sequence and genetic background, which provides strong support for gene function research. Compared with other tree species, poplar has a smaller genome, which simplifies research steps such as gene localization, cloning, and expression. Therefore, it is of great application significance to use molecular biology and genetic engineering and other technical means to study the excellent genes that affect the quality of poplar wood.

Summary of the invention

The present invention provides an application of a poplar CDPK6 gene in forest tree breeding.

In an optional application, the development of tree xylem is regulated by controlling the expression level of the gene.

Another optional application is to predict the development of tree xylem by detecting the expression level of the CDPK6 gene in plants.

The present invention also provides a method for regulating the development of poplar xylem, which uses genetic engineering to over-express or inhibit the expression of the poplar CDPK6 gene.

One of the optional methods mentioned above is to transform the CDPK6 overexpression vector into poplar cells by Agrobacterium transformation to obtain CDPK6 overexpressing poplar plants, thereby promoting the development of plant xylem.

The present invention also provides a poplar CDPK6 gene, the gene sequence is shown as SEQ ID NO: 1 or the encoded protein sequence is shown as SEQ ID NO: 2.

A primer set for amplifying poplar CDPK6 gene, wherein the upstream and downstream primers of the primer set are shown in SEQ ID NO: 3-4 in the sequence listing.

A vector containing a poplar CDPK6 gene, wherein the vector is an overexpression plasmid vector.

Optionally, a method for constructing the above-mentioned vector uses pBI121 as the vector backbone, selects appropriate restriction sites to linearize the vector backbone, designs 5'-3' homologous recombination primers of the CDPK6 gene according to the restriction sites, and then uses homologous recombination to connect the CDPK6 gene to the pBI121 vector backbone to obtain a recombinant vector.

A bacterial strain containing the above vector.

Beneficial effects of the present invention:

The present invention deeply mined the transcriptome data of poplar vascular tissue, screened and successfully cloned the CDPK6 gene, and functionally verified the gene through leaf disc genetic transformation technology, explored the biological function of the CDPK6 gene, and revealed for the first time the role of this gene in regulating the development of poplar xylem, which is mainly reflected in the effects of the CDPK6 gene on the thickness of the xylem cell wall, cell number, plant height and ground stem, playing an important role in the genetic improvement of poplar wood quality.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Vector map of the pBI121 overexpression vector of the present invention.

Figure 2: Gel electrophoresis results of PCR products cloned from poplar CDPK6 gene.

Figure 3: Display of the sequencing comparison results of the cloned sequence of the poplar CDPK6 gene.

Figure 4: Identification results of CDPK6-overexpressing poplar plants.

Figure 5: Phenotypic display of wild-type plants and CDPK6-overexpressing poplar plants, *P<0.05, **P<0.01, ***P<0.001.

DETAILED DESCRIPTION

The invention identifies a Ca ²⁺ /CaM-dependent protein kinase CDPK6 involved in the formation of poplar wood based on co-expression network analysis, tissue-specific expression pattern and association analysis. The silver gland poplar (Populus alba x Populus tremulavar. glandulosa, 84K) obtained by hybridization of the female silver poplar (Populus alba) and the male glandular poplar (Populus glandulosa) is used as a material, and its RNA is extracted and reversely transcribed to obtain cDNA. The full-length CDS sequence of the gene is cloned according to the specific primers of CDPK6, and the corresponding nucleotide sequence is shown in SEQ ID NO: 1. The CDS of the CDPK6 gene is 1566bp in length, and the encoded protein consists of 522 amino acid residues, and the amino acid sequence is shown in SEQ ID NO: 2. The protein has a molecular weight of 57.42KDa and an isoelectric point of 6.14.

Based on this research, the present invention provides the following technical solutions:

Application of a poplar CDPK6 gene in forest tree breeding.

Preferably, the poplar refers to plants of the genus Populus, including Populus euphratica, Populus white, Populus cottonwood, Populus argentatus and Populus tomentosa.

One of the applications is to regulate the development of tree xylem by controlling the expression level of the gene.

Optionally, controlling gene expression includes overexpression and inhibiting expression.

Another application is to predict the development of plant xylem by detecting the expression level of the CDPK6 gene in plants.

Preferably, the biological phenotype of the plant to be tested is predicted by comparing the expression level of the CDPK6 gene in the plant with that in the standard plant.

Optionally, the method for detecting the expression level of the CDPK6 gene in the plant includes detecting the copy number of the corresponding CDPK6 gene, the transcription level of the CDPK6 gene and the translation level of the CDPK6 gene.

The present invention also provides a method for regulating the development of poplar xylem, which uses genetic engineering to over-express or inhibit the expression of the poplar CDPK6 gene.

Preferred genetic engineering methods include gene editing technology, gene silencing technology and transgenic technology.

More preferably, the gene editing technology includes CRISPR/Cas gene editing technology; the gene silencing technology includes RNA interference technology; and the transgenic technology includes Agrobacterium-mediated transformation technology, electroporation technology and microinjection technology.

A method for promoting wood development of poplar plants, comprising transforming a CDPK6 overexpression vector into poplar cells by Agrobacterium transformation to obtain CDPK6 overexpression poplar plants.

The manifestations of promoting wood development of poplar plants include one or more of increased thickness of xylem cell walls, increased cell numbers, increased plant height, and thickened ground stems.

Preferably, the above method comprises at least one of the following steps:

Cloning of CDPK6 gene fragment: Using 84K silver gland poplar as material, specific primers were designed to clone the fragment according to the coding sequence of CDPK6 gene;

Construction of CDPK6 overexpression vector: The above CDPK6 gene open reading frame was cloned and transferred into a plant overexpression vector so that it was expressed under the drive of a strong promoter;

Obtaining CDPK6 overexpression vector Agrobacterium: The constructed CDPK6 gene overexpression vector is transferred into the Agrobacterium strain with strong infection ability to poplar by heat shock and other methods;

Obtaining CDPK6 overexpressing poplar plants: The overexpression vector of the CDPK6 gene was introduced into 84K silver gland poplar by Agrobacterium-mediated leaf disc transformation, and further CDPK6 overexpressing transgenic plants with antibiotic resistance were obtained;

Identification of CDPK6-overexpressing poplar plants: PCR amplification was used to detect the expression vector tag sequence and Agrobacterium sequence in the genomic DNA of transgenic plants, and real-time fluorescence quantitative PCR was used to detect the expression level of the CDPK6 gene in transgenic plants;

Phenotypic detection of poplar plants overexpressing the CDPK6 gene: The vascular tissue cells of CDPK6 overexpressing transgenic plants were observed using phloroglucinol staining and scanning electron microscopy.

A poplar CDPK6 gene, the gene sequence is shown as SEQ ID NO: 1 or the encoded protein sequence is shown as SEQ ID NO: 2.

A primer set for amplifying CDPK6 gene, wherein the upstream and downstream primers of the primer set are shown in the sequence list SEQ ID NO: 3-4. A primer set for detecting CDPK6 gene, wherein the upstream and downstream primers of the primer set are shown in the sequence list SEQ ID NO: 5-6.

A vector containing a CDPK6 gene, wherein the vector is an overexpression plasmid vector.

Optionally, overexpression vectors include pCXSN, pCAMBIA1300, and pBI121 vectors.

Preferably, the overexpression vector is a pBI121 vector.

A method for constructing the above-mentioned vector is to use pBI121 as the vector backbone (as shown in Figure 1), select appropriate restriction sites to linearize the vector backbone, design 5'-3' homologous recombination primers of the CDPK6 gene according to the restriction sites, and then use the homologous recombination method to connect the CDPK6 gene to the pBI121 vector backbone to obtain a recombinant vector.

Optionally, the endonucleases corresponding to the restriction sites in the above method are BamHI enzyme and KpnI enzyme.

Optionally, a 5'-3' homologous recombination primer set of the CDPK6 gene used in the above method has a sequence as shown in SEQ ID NO: 7-8.

A bacterial strain containing the above vector.

Preferably, the strains include Escherichia coli and Agrobacterium.

In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1

Cloning of CDPK6 gene

(1) Using 84K silver gland poplar tissue culture seedlings as materials, firstly, the 84K stem segments were fully ground in a sterile enzyme-free mortar, and the centrifugal column RNA extraction kit of Novazonic Biotech was used to extract plant total RNA;

(2) Reverse transcription was performed using the Reverse Transcription cDNA First-Strand Synthesis Kit (R312-01/02) from Nanjing Novozymes Co., Ltd. The reaction system (added in order) and conditions were as follows: cDNA was synthesized according to the instructions;

(3) Based on the CDS sequence of the CDPK6 gene, upstream and downstream primers of the CDPK6 gene were designed. The 5'-3' primers of the PCR reaction were: CDPK6-F: ATGGGAAACTGCAACAGCCTC (SEQ ID NO: 3), CDPK6-R: TTTGCGCCGTCTGGTTGG (SEQ ID NO: 4). In addition, PCR amplification and reaction conditions were carried out according to the instructions;

(4) The PCR product was separated by 2% agarose gel electrophoresis to obtain a length of about 1563 bp (excluding the terminator TGA sequence), as shown in Figure 2. After purification and recovery, it was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The sequencing results are shown in Figure 3 and SEQ ID NO.1. The nucleotide sequence length is 1566 bp, and its encoded protein sequence is shown in SEQ ID NO.2, which is a protein composed of 522 amino acids.

Example 2

Construction of CDPK6 overexpression vector

(1) The expression vector pBI121 was used as the backbone, and the vector backbone was linearized using BamHI and KpnI enzymes. According to the restriction sites, the 5'-3' homologous recombination primers of the CDPK6 gene were designed as CDPK6-F: cgggggactctagaggatccATGGGAAACTGCAACAGCCTC (SEQ ID NO. 7) and CDPK6-R: tgctcaccatggtaccTTTGCGCCGTCTGGTTGG (SEQ ID NO. 8).

(2) The recombination reaction was carried out using the II One Step Cloning Kit C112 kit. The double enzyme digestion system, recombination reaction system and reaction conditions were strictly carried out according to the instructions. The positive strain was obtained by transforming the ligation product pBI121-CDPK6 into Escherichia coli, and the pBI121-CDPK6 plasmid was extracted after culturing the positive strain.

Example 3

Obtaining recombinant Agrobacterium

(1) Take the competent Agrobacterium (GV3101) stored at -80°C and melt it at room temperature or in the palm of your hand until it is in an ice-water mixture state and then insert it into ice;

(2) Add 0.01-1 μg of plasmid DNA (pBI121-CDPK6 plasmid) to 100 μL of competent cells and flick the tube to mix thoroughly;

(3) Place the samples on ice for 5 min, freeze them in liquid nitrogen for 5 min, bathe them in 37°C water for 5 min, and then bathe them in ice for 5 min.

(4) Add 700 μL of Luria-Bertani (LB) liquid medium (without any antibiotics) and shake at 28°C and 200 rpm for 3 h;

(5) Centrifuge at 6000 rpm for 1 min; discard part of the supernatant and keep about 100-120 μL of bacterial solution. Gently blow and mix to resuspend the bacterial block, spread it on a solid plate containing LB (50 mg/L kanamycin) containing the corresponding antibiotic, and let it stand for 15 min to allow the surface bacterial solution to dry;

(6) Place the incubator upside down and incubate in the dark at 28°C for 42-48 hours;

(7) Detect positive colonies using polymerase chain reaction (PCR) and store them at 4°C for subsequent transgenic infection of poplar leaves.

Example 4

Obtaining CDPK6-overexpressing poplar plants

(1) The Agrobacterium single colony or the Agrobacterium bacterial liquid preserved in glycerol that was correctly detected in Example 3 was activated and propagated, placed in a 100 mL sterile conical flask, diluted to 50 mL of LB liquid medium containing antibiotics (50 mg/L kanamycin, 17 mg/L rifampicin) at a ratio of 1:50, and cultured at 28° C. and 200 rpm for 24-36 h until the bacterial liquid color turned orange-yellow and the OD value A600 was about 1.0;

(2) Take 400-1000uL of the small shaken bacterial liquid and add it to 50mL of liquid LB medium containing antibiotics (50mg/L kanamycin and 17mg/L rifampicin). Incubate at 28°C and 200rpm with shaking until the OD600 is 0.5-0.6, which is then enough for infection.

(3) Select 84K tissue culture seedlings with good growth, place them in a clean bench, and select the 4th to 6th leaves from the top of the sterile tissue culture seedlings. Use a sterile surgical blade to make a horizontal wound on the main vein of the leaf. Place it in a differentiation medium without antibiotics for 1 to 2 days.

(4) Pour the Agrobacterium solution into a sterile 50 ml centrifuge tube in a clean bench and centrifuge at 3500 rpm for 10 min at 4°C. Pour off the supernatant and add an equal volume of sterile resuspension solution (1/2 MS, 3% sucrose, 100 uMAS). Gently shake to resuspend the cells and place on ice until use.

(5) Place the cut leaves in the resuspended Agrobacterium solution and immerse them for 10-15 minutes, shaking them slowly during the process to allow the injured parts of the leaves to fully contact the solution.

(6) Use sterile tweezers to remove the leaf and place it on sterile filter paper to absorb excess bacterial solution. Finally, place the leaf after absorbing the bacterial solution in symbiotic culture medium without antibiotics, with the front side of the leaf facing down, and culture it in the dark at 25°C for 60-70 h until bacterial colonies grow near the leaf.

(7) In a clean bench, place the dark-treated co-cultured leaves on pre-sterilized filter paper to absorb excess bacteria, and then transfer them to a selective medium with screening pressure (30 mg/L kanamycin) and sterilization (200 mg/mL timentin) for screening and culture. The light cycle is 16 h of light/8 h of darkness, and the temperature is 25°C. During this period, the culture medium is replaced every 8-10 days until resistant buds grow. After the adventitious buds grow to about half a centimeter, use sterile tweezers or a scalpel to cut off the leaf disc with adventitious buds and place new selective medium to allow them to continue growing.

(8) When the adventitious buds grow to 1-2 cm, use sterile tweezers or scalpels in a clean bench to cut off the adventitious buds individually and place them on a rooting medium containing a screening pressure (30 mg/L kanamycin) and a sterilized medium (200 mg/mL timentin) for rooting culture. After about two weeks, the adventitious buds will take root and then continue to subculture. The first subculture uses a rooting medium containing a screening pressure (30 mg/L kanamycin) and a sterilized medium (200 mg/mL timentin), while the subsequent subcultures use a rooting medium containing only a sterilized medium (200 mg/mL timentin). Molecular testing of leaves can be performed on the first rooted transgenic seedlings.

Example 5

(1) The leaf genomic DNA of the CDPK6 overexpressing poplar clones grown for one month was extracted using the FastPure PlantDNA Isolation Mini Kit of Nanjing Novezan Biotechnology Co., Ltd., and the vector sequences and Agrobacterium sequences at both ends of the cloning site were detected by PCR technology. The presence of the vector sequences at both ends of the cloning site in the PCR results, the correct band length, and the absence of the Agrobacterium sequence band indicated that the seedlings were positive. The 5'-3' primer sequences were: pBI121-F: acagttgcgcagcctgaatg (SEQ ID NO.9), pBI121-R: gatgcatcaggccgacagtc (SEQ ID NO.10); GV3101-VirD2-F: TTCGTCACGGCATAGTCCTG (SEQ ID NO.11), GV3101-VirD2-R: TTGCTCGGTACGGATGGAAG (SEQ ID NO.12);

The results are shown in FIG4 . For the CDPK6 overexpressing poplar #1, #2, #5, #6, #7, #8 and #9 strains obtained by the present invention, the vector sequences at both ends of the PCR cloning site are present ( FIG4A ), the band length is correct, and the Agrobacterium sequence band is absent ( FIG4B );

(2) The total RNA of the plant was extracted using the centrifugal column RNA extraction kit of Novazome Biotech Co., Ltd., and then reverse transcribed and synthesized into cDNA using the reverse transcription cDNA first-strand synthesis kit (R312-01/02). The expression level of the CDPK6 gene in the overexpressed poplar strain was then detected by real-time fluorescence quantitative PCR. Compared with the control plants, the expression level of the CDPK6 gene in the CDPK6 overexpressed poplar plants was significantly increased, and the overexpressed poplar plants were positive plants. The 5'-3' primer sequence of the CDPK6 gene was: CDPK6-qPCR-F:

GGACACGGACAACAATGGAACAATC (SEQ ID NO.5), CDPK6-qPCR-R: CTGCCTCACTTCAGACTCAGAAAGC (SEQ ID NO.6); 18S was selected as the internal reference in the experiment, and the 5'-3' primer sequences of the internal reference were 18S-F: GGCATGGAAGGTGATGCAGATC; 18S-R: CTGTGTCAAACAAGAACTTGTCC, and the PCR reaction system was configured, gently mixed thoroughly, and quickly centrifuged to avoid wall adhesion, followed by PCR reaction. The specific PCR program was set as follows: ① pre-denaturation: 95℃/30 seconds; ② PCR reaction (denaturation, annealing and extension): 95℃/5 seconds, 60℃/35 seconds (adjustable according to the copy number), 40 cycles; ③ melting curve: 95℃/15s, 60℃/1min, 95℃/15s.

The results are shown in FIG4C . In the CDPK6 overexpressing poplar lines #1, #7 and #9 obtained by the present invention, compared with the control plants, the expression levels of the CDPK6 gene in the above CDPK6 overexpressing poplar lines increased by 9.33 times, 13.02 times and 16.18 times, respectively.

(3) Combining the results of PCR cloning and real-time fluorescence quantitative PCR, the CDPK6 overexpressing poplar lines obtained in this experiment were #1, #7 and #9, as shown in FIG5A .

Example 6

Phenotypic detection of poplar plants overexpressing CDPK6 gene

(1) First, the growth and physiological traits of CDPK6-overexpressing poplars were measured, mainly including plant height, ground diameter, length of the 15th internode and width of the 15th internode. As shown in Figure 5B-C, compared with the control plants, the plant height of CDPK6-overexpressing poplars increased significantly by 26.56%, the ground diameter of CDPK6-overexpressing poplars increased significantly by 12.09%, the length of the 15th internode of CDPK6-overexpressing poplars increased significantly by 23.56%, and the width of the 15th internode of CDPK6-overexpressing poplars increased significantly by 18.12%.

(2) The xylem cell morphology and cell wall thickness of CDPK6-overexpressing poplars were observed and analyzed using pyrogallol staining and biological scanning electron microscopy. As shown in Figure 5D-F, compared with the control plants, the number of xylem layers and cell wall thickness in the CDPK6-overexpressing poplar lines increased significantly by 42.10% and 18.02%, respectively.

(3) In addition, the study also found that the transcription level of the CDPK6 gene in the overexpression plants was positively correlated with the growth phenotype (plant height, ground diameter, number of xylem layers, and cell wall thickness) of the overexpression plants, and the correlation coefficients (r) were 0.98, 0.96, 0.94, and 0.97, respectively.

In summary, the present invention reveals that the CDPK6 gene has a positive regulatory function on the growth phenotype of poplar plants (plant height, ground diameter, number of xylem layers, cell wall thickness). The present invention provides an application of obtaining poplar materials with excellent wood quality traits by obtaining CDPK6 overexpressing poplar plants, and predicting the development of plants by detecting the expression of the CDPK6 gene in plant leaves, thereby providing technical support for the screening of plants with excellent traits.

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (10)

1. An application of poplar CDPK6 gene in tree breeding.

2. The use according to claim 1, wherein the development of the xylem of the tree is regulated by controlling the expression level of said gene.

3. The use of claim 1, wherein the development of the xylem of the tree is predicted by detecting the expression level of the CDPK6 gene in the tree.

4. A method for regulating and controlling the development of poplar xylem, which is characterized in that the overexpression or the expression of poplar CDPK6 genes is inhibited by means of genetic engineering.

5. The method of claim 4, wherein the CDPK6 overexpression vector is transformed into poplar cells by agrobacterium transformation to obtain a poplar plant with CDPK6 overexpression, promoting xylem development in the plant.

6. A poplar CDPK6 gene, wherein the gene sequence is as set forth in SEQ ID NO:1 or the coded protein sequence is shown as SEQ ID NO: 2.

7. A primer set for amplifying the gene in claim 6, wherein the upstream primer and the downstream primer of the primer set are shown in a sequence table SEQ ID NO: 3-4.

8. A vector comprising the gene of claim 6, wherein said vector is an over-expression plasmid vector.

9. The method for constructing a vector according to claim 8, wherein pBI121 is used as a vector backbone, a proper cleavage site is selected to linearize the vector backbone, a 5'-3' homologous recombination primer of the CDPK6 gene is designed according to the cleavage site, and the CDPK6 gene is connected with the pBI121 vector backbone by homologous recombination to obtain the recombinant vector.

10. A strain comprising the vector constructed in claim 8 or 9.