Poplar pagtal3 protein and application of coding gene thereof

Abstract

The application provides poplar PagTAL3 protein and an application of a coding gene thereof, relates to the technical field of genetic engineering, and the amino acid sequence of the PagTAL3 protein is shown in SEQ ID NO. 1. The single-gene expression quantity of the PagTAL3 protein is enhanced, so that the synthesis of melatonin, the chloroplast photosynthesis, the synthesis of cell wall components and the transaldolase activity of poplar are enhanced; and the gene editing mutant is reduced in the synthesis of melatonin, the chloroplast photosynthesis, the synthesis of cell wall components and the transaldolase activity. The gene has great application potential and prospect for improving the poplar variety traits.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and more particularly to a Populus PagTAL3 protein and an application of a coding gene thereof. BACKGROUND

[0002] Populus ( Populus spp. ) is an important afforestation tree species with characteristics of fast growth, high yield, strong adaptability, etc., and is widely cultivated in China, having important economic and ecological values.

[0003] In recent years, the rapid development of biotechnology has brought revolutionary changes in the field of plant breeding, continuously improving research level and promoting the innovation of plant breeding methods. The successful application of gene editing technology provides new opportunities for the research of Populus gene function and precise molecular design breeding. Through biotechnological means, Populus stress tolerance, plant height, photosynthetic performance, and wood-specific traits can be rapidly and directionally bred. The application of these technologies not only can improve the economic benefits of Populus, but also can help to save manpower and material resources, effectively saving social resources. Overall, the application of biotechnology in Populus breeding will provide new possibilities for the development of the wood industry. SUMMARY

[0004] The present application provides a Populus PagTAL3 protein and an application of a coding gene thereof.

[0005] The application of regulating the content and / or activity of PagTAL3 protein in Populus is any of the following:

[0006] A. regulating the synthesis of melatonin;

[0007] B. regulating the photosynthetic capacity of chloroplast;

[0008] C. regulating the synthesis of cell wall components;

[0009] D. regulating the activity of transaldolase;

[0010] The amino acid sequence of the PagTAL3 protein is shown in SEQ ID NO. 1.

[0011] Preferably, the content and / or activity of PagTAL3 protein in Populus is up-regulated, and the ability of the application is enhanced; the content and / or activity of PagTAL3 protein in Populus is down-regulated, and the ability of the application is weakened.

[0012] Another object of the present application is to provide an application of regulating the expression amount of a gene in Populus, which is any of the following: PagTAL3

[0013] A. regulating the synthesis of melatonin; ​

[0014] B. modulating chloroplast photosynthetic capacity;

[0015] C. modulating cell wall component synthesis;

[0016] D. modulating transaldolase activity;

[0017] The amino acid sequence encoded by the PagTAL3 gene is shown as SEQ ID NO. 1.

[0018] Preferably, the nucleotide sequence of the PagTAL3 gene is shown as SEQ ID NO. 2 or the complement sequence of SEQ ID NO. 2.

[0019] Preferably, the ability of the application is enhanced by up-regulating the expression amount of the PagTAL3 gene in poplar, and the ability of the application is weakened by down-regulating the expression amount of the PagTAL3 gene in poplar.

[0020] Another object of the present application is to provide the application of biological materials for increasing the expression amount of the PagTAL3 gene, wherein the application is:

[0021] A. increasing the synthesis of melatonin;

[0022] B. increasing the chloroplast photosynthetic capacity;

[0023] C. increasing the cell wall component synthesis;

[0024] D. increasing the transaldolase activity;

[0025] The biological material is one of the following:

[0026] a. a nucleic acid molecule capable of encoding an amino acid sequence as shown in SEQ ID NO. 1;

[0027] b. an expression cassette capable of over-expressing the nucleic acid molecule of a;

[0028] c. a recombinant vector containing the expression cassette of a;

[0029] d. a recombinant microorganism containing the expression cassette of a or the recombinant vector of b;

[0030] e. a non-reproducible plant part containing the expression cassette of a or the recombinant vector of b or the recombinant microorganism of c.

[0031] Another object of the present application is to provide the application of biological materials for inhibiting the expression amount of the PagTAL3 gene, wherein the application is:

[0032] A. reducing the synthesis of melatonin;

[0033] B. reducing the chloroplast photosynthetic capacity;

[0034] C. reducing cell wall component synthesis;

[0035] D. reducing transaldolase activity;

[0036] The biological material is a nucleic acid molecule in which the gene encoding the protein with the amino acid sequence shown in SEQ ID NO. 1 is deleted or inactivated by gene knockout technology.

[0037] Preferably, the biological material is a CRISPR / Cas9 system containing sgRNA, which targets the gene encoding the protein with the amino acid sequence shown in SEQ ID NO. 1.

[0038] Another object of the present application is to provide the use of substances that activate the PagTAL3 protein and / or substances that activate the nucleic acid molecule encoding the PagTAL3 protein in poplar breeding, the goal of which is to breed plants with increased melatonin synthesis, improved chloroplast photosynthesis, and increased cell wall component synthesis.

[0039] The amino acid sequence of the PagTAL3 protein is shown in SEQ ID NO. 1.

[0040] Another object of the present application is to provide the use of substances that inhibit the PagTAL3 protein and / or substances that inhibit the nucleic acid molecule encoding the PagTAL3 protein in plant breeding, the goal of which is to breed plants with reduced melatonin synthesis, reduced chloroplast photosynthesis, and reduced cell wall component synthesis.

[0041] The amino acid sequence of the PagTAL3 protein is shown in SEQ ID NO. 1.

[0042] Beneficial effects: The present application provides the use of the PagTAL3 protein and its encoding gene in poplar. PagTAL3 The enhancement of the expression of a single gene leads to the enhancement of melatonin synthesis, chloroplast photosynthesis, cell wall component synthesis, and transaldolase activity in poplar. The gene editing mutant, on the other hand, has reduced melatonin synthesis, chloroplast photosynthesis, cell wall component synthesis, and transaldolase activity. The use of this gene for improving the traits of poplar varieties has great application potential and prospects. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 The figure is a schematic diagram of the structure of recombinant plasmid BGK012 (gene editing vector) and pCAMBIA2300 (overexpression vector).

[0044] Figure 2 The figure is the sequencing results of the mutation site and its surrounding nucleotides of the mutant plant.

[0045] Figure 3Results of the determination of the melatonin content in the plants.

[0046] Figure 4 Results of the determination of the chloroplast ultrastructure.

[0047] Figure 5 Results of the determination of the cell wall components in the plants.

[0048] Figure 6 Results of the determination of the transaldolase activity level in the plants. DETAILED DESCRIPTION

[0049] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by those of ordinary skill in the art without any creative work, fall within the protection scope of the present application.

[0050] Unless otherwise defined, 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 application belongs. It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application cover modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

[0051] The term "expression cassette" generally refers to a nucleic acid construct comprising nucleic acid elements sufficient to express a gene of interest. A typical expression cassette comprises a promoter, a MCS (multiple cloning site) and a terminator. The expression cassette can also include a gene of interest, a marker gene (such as a TK gene, a DHFR gene, a CAT gene and a NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly(A) addition signal sequence and / or an mRNA splicing signal sequence, etc. The elements in the expression cassette can be directly connected or indirectly connected through a linker.

[0052] The term "vector" generally refers to a vehicle capable of carrying foreign DNA or a gene of interest into a host cell for amplification and / or expression. The vector can be a cloning vector or an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by the vector can be amplified and / or expressed in the host cell. A skilled person can select a suitable vector according to the purpose of genetic engineering and the nature of the recipient cell. The vector includes but is not limited to: plasmid, phage (such as lambda phage or M13 phage), cosmid (i.e. cos plasmid), phagemid, shuttle vector (such as yeast expression vector), Ti plasmid, artificial chromosome (such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), P1 artificial chromosome (PAC) or Ti plasmid artificial chromosome (TAC)), viral vector (such as baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, papovavirus (such as SV40), herpesvirus (such as herpes simplex virus)). A vector can contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain a replication initiation site.

[0053] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes, algae, etc.

[0054] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by connecting an exogenous gene of interest with a vector in vitro. It can be constructed in any suitable way, as long as the constructed recombinant vector can carry the exogenous gene of interest into the recipient cell, and provide the exogenous gene of interest with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0055] The term "recombinant microorganism" generally refers to a recombinant microorganism obtained by manipulating and modifying the genes of a microorganism of interest, so that the function of the recombinant microorganism is changed. For example, an exogenous gene of interest or a recombinant vector is introduced into the microorganism of interest, or the endogenous genes of the microorganism of interest are directly genetically edited.

[0056] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0057] Unless otherwise specified, the quantitative tests in the following examples are all set up with three repeated experiments, and the results are averaged.

[0058] Example 1

[0059] 1. PagTAL3 Creation of transgenic poplar trees with overexpressed genes

[0060] Following the method of Sun Tiantian et al. (Analysis of drought stress resistance function of Populus aquaporin gene PagPIP1;3 [J]. Journal of Northwest Forestry University, 2023, 38(04):11-17.), genomic DNA was extracted from Populus (84K) and analyzed. PagTAL3 Gene (SEQ ID NO.2) was amplified, and the amplification product was used as a template to select... Kpn I and Bam HI restriction site, poplar PagTAL3 The gene was cloned into the expression vector pCAMBIA2300 (see attached diagram for its skeletal structure). Figure 1 The recombinant expression vector pCAMBIA2300- was obtained from the above. PagTAL3 Under the premise of ensuring correct reading frame, the recombinant expression vector was transferred into Agrobacterium GV3101 to obtain recombinant Agrobacterium. Using the Agrobacterium infection method, the recombinant Agrobacterium was genetically transformed into sterile leaves of 84K poplar, then resistant poplar materials were screened, followed by differentiation and regeneration culture, and then rooting culture to obtain regenerated plants.

[0061] PagTAL3 protein sequence:

[0062] MATISKLSNPSPAASLSAPSRSSSLPKVFLGFSTKTSLSKVRLSLKNTQPSPRTSLVVRCSQSSGNGSPIKRTTLHDLYEREGQSPWYDNLCRPVTDLIPLIESGVRGVT SNPAIFQKAISSSNAYNDQFRELVQSGKDIETAYWELVVKDIQDACKLFESIYDQTDAGDGYVSVEVSPRLADDTQGTVEAAKWLHKVVDRPNVYIKIPATAPCIPSIKE VISLGISVNVTLIFSLTRYDAVIDAYLDGLEASGLSDLSRVTSVASFFVSRVDTLIDKMLEKIGTPEALDLRGKAAVAQAALAYKLYQKKFSGPRWEALVKKGAKKQRLL WASTSVKNPAYADTLYVAPLIGPDTVSTMPDQALQAFVDHGSVARTIDSNVSEAEGIYNALEKLGIDWGYVGNQLEVEGVDSFKKSFDSLLDTLQEKANSLKLVSL, SEQ ID NO.1.

[0063] PagTAL3 CDs nucleotide sequence:

[0064]

[0065] 2、 PagTAL3 Creation of gene editing mutant poplar

[0066] Reference Tianma bamboo (application CRISPR / Cas9 technology directional mutation tobacco CYP71D16 Gene research[D / OL]. Guizhou: Guizhou University, 2019.) The method of editing the target gene and transferring it into the BGK012 gene editing vector plasmid, using CRISPR / Cas9 online design tool ( http: / / skl.scau.edu.cn / ), based on PagTAL3 sequence design target, according to the construction requirements of BGK012 vector, synthesize sgRNA sequence primer, obtain AtU6-sgRNA1-AtU6-sgRNA2 fragment and transfer it into BGK012 vector plasmid to obtain PagTAL3 gene editing vector.

[0067] The constructed gene editing vector is transferred into Agrobacterium GV3101 to obtain recombinant Agrobacterium. Using Agrobacterium infection method, the sterile leaves of 84K poplar are genetically transformed by recombinant Agrobacterium, and then the resistant poplar materials are screened, then differentiation and regeneration culture is carried out, then rooting culture is carried out, and then the regenerated plants are obtained.

[0068] The sgRNA sequence is as follows:

[0069] GAACCTCTGCAGGCCTGTGA (5'-3'), SEQ ID NO. 3,

[0070] GTACTGGGAACTTGTGGTGA (5'-3'), SEQ ID NO. 4.

[0071] 3. Identification of overexpression transgenic and mutant plants

[0072] Reference Sun T (Analysis of drought stress tolerance function of poplar aquaporin gene PagPIP1; 3 [J]. Journal of Northwest Forestry University, 2023, 38(04): 11-17.) and Liu TT (Application of CRISPR / Cas9 technology in efficient knockout of multiple target genes in poplar [J]. Genetics, 2015, 37(10): 1044-1052.) The verification method of poplar overexpression transgenic material and gene editing mutant material, respectively collect the leaves of overexpression transgenic plants and mutant plants, extract genomic DNA, and then perform PCR amplification using appropriate primers, and then sequence the PCR amplification products.

[0073] The results show that the sequence of the overexpression transgenic plant is correct, and the expression amount is 9.63 times that of the wild type, indicating that the overexpression plant is successfully constructed, which is named OE#11.

[0074] The sequencing results of the homozygous mutant plant (i.e. the mutation of both chromosomes is consistent) show that, compared with the genomic DNA of the wild type poplar (denoted as WT), the difference lies in the following mutations in the gene encoding the PagTAL3 protein: “CTCTGCAGGCCTGTGTCTCTGG” (SEQ ID NO. 5) is mutated to “CTCTGCAGGCCTGTGAATCTCTGG” (SEQ ID NO. 6), and “GTCAGGTGCTCTCAATCCAGTGG” (SEQ ID NO. 7) is mutated to “GTCAGGTGCTCTCAATCAGTGG” (SEQ ID NO. 8), causing a frameshift mutation of the subsequent nucleotides. The sequencing results of the mutation site and the surrounding nucleotides are shown in the following table: Figure 2 The mutant plant is named as OE#11. tal3#3 ko .

[0075] Example 2

[0076] PagTAL3 Comparison of the production traits of the gene overexpression transgenic poplar and the mutant

[0077] Tested plants: wild type poplar 84K plant, OE#11 plant and tal3#3 ko Plant

[0078] The content of melatonin, photosynthetic performance (chloroplast ultrastructure) and cell wall components of the tested plants were determined. At least 3 plants of each plant material were measured.

[0079] The aseptic seedlings with consistent growth vigor were cultured in an artificial sterile room, the same leaf sequence leaves were taken, and then the content of melatonin was detected by using the plant melatonin (MT) ELISA kit (Jiangsu Enzyme Immune Industry Co., Ltd., Plant Melatonin (MT) ELISA Kit 96T, Item No. MM-08480I) according to the instructions; the same leaf sequence leaves were taken, and the starch granule accumulation in the chloroplasts of the leaves was observed according to Yang Jiajun (Effect of High Temperature and Drought Stress on Growth and Ultrastructure of ‘Nanlin 895 Poplar’ Cuttings [J]. Forestry Science, 2020, 56(05): 176-183.) to determine the change of photosynthetic capacity; the same internode stems were taken, and the cell wall components in the stems were determined according to Xue Xiaoming (Comparative Study on Wood Anatomical Structure Characteristics and Infrared Spectra of Cinnamomum camphora and Phoebe sheareri [J]. Journal of Sichuan Agricultural University, 2016, 34(02): 178-184.).

[0080] The results show that, compared with the wild type strain, the melatonin content of the OE#11 strain is increased, the photosynthetic capacity is enhanced, the cell wall component (lignin) accumulation is increased, and the tal3#3 koThe melatonin content of the strain is reduced, the photosynthetic capacity is reduced, and the accumulation of cell wall components (lignin) is reduced (see the following Figure 3 for the melatonin content, the photosynthetic performance in the following Figure 4 , and the cell wall components in the following Figure 5 ).

[0081] Example 3

[0082] PagTAL3 Measurement of transaldolase activity level of the gene overexpression transgenic poplar and mutant

[0083] Test plants: 84K wild type plants, OE#11 plants and tal3#3 ko plants.

[0084] The aseptic seedlings with the same growth vigor were cultured in an artificial aseptic room, the leaves of the same leaf sequence were taken, and total protein was extracted. Then, the plant transaldolase activity enzyme-linked immunoassay kit (Jiangsu Enzyme Immune Industry Co., Ltd., Plant Transaldolase (TAL) ELISA Kit 96T, Item No. MM-107001) was used to detect the transaldolase activity level according to the instructions.

[0085] The results show that, compared with the wild type, the transaldolase activity level of the OE#11 plant is improved, which can promote the primary metabolism of the plant, tal3#3 ko and the transaldolase activity level of the mutant plant is reduced.

[0086] Each of the embodiments in the specification is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other.

[0087] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of regulating the content of PagTAL3 protein in poplar, characterized in that, The application is as follows: Regulates the synthesis of melatonin in poplar trees; The amino acid sequence of the PagTAL3 protein is shown in SEQ ID NO.

1. Upregulating the content of PagTAL3 protein in poplar increases the content of melatonin in poplar; downregulating the content of PagTAL3 protein in poplar decreases the content of melatonin in poplar.

2. Regulating poplar trees PagTAL3 The application of gene expression levels is characterized by, The application is as follows: Regulates the synthesis of melatonin in poplar trees; The PagTAL3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.1, and it upregulates the amino acid profile of poplar. PagTAL3 Gene expression levels were increased in poplar trees, while melatonin levels were downregulated in poplar trees. PagTAL3 Gene expression levels were reduced, and melatonin content was decreased in poplar trees.

3. The method for regulating poplar in accordance with claim 2 PagTAL3 The application of gene expression levels is characterized by, The PagTAL3 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. Improve PagTAL3 The application of biomaterials for gene expression level is characterized by, The application is as follows: Increase the synthesis of melatonin in poplar trees; The biomaterial is one of the following: a. Nucleic acid molecules encoding amino acid sequences such as SEQ ID NO. 1; b. An expression cassette capable of overexpressing the nucleic acid molecules described in a; c. A recombinant vector containing the expression cassette described in b; d. Recombinant microorganisms containing the expression cassette described in b or the recombinant vector described in c.

5. Inhibition PagTAL3 The application of biomaterials for gene expression level is characterized by, The application is as follows: Reduces melatonin synthesis in poplar trees; The biological material is a CRISPR / Cas9 system containing sgRNA, wherein the sgRNA targets the gene encoding the protein whose amino acid sequence is shown in SEQ ID NO.1, and the sequence of the sgRNA is shown in SEQ ID NO.3 and SEQ ID NO.

4.

6. The application of substances that inhibit PagTAL3 protein and / or substances that inhibit nucleic acid molecules encoding PagTAL3 protein in plant breeding, wherein the breeding objective is to cultivate poplar trees with reduced melatonin synthesis; The substance that inhibits the PagTAL3 protein and / or the substance that inhibits the nucleic acid molecule encoding the PagTAL3 protein is a CRISPR / Cas9 system containing sgRNA, the sequence of which is shown in SEQ ID NO.3 and SEQ ID NO.4, and the amino acid sequence of which is shown in SEQ ID NO.1.

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