Tobacco pectin lyase NtPL gene and its application

Abstract

This invention relates to the field of biotechnology, and more particularly to the tobacco pectin lyase NtPL gene and its applications. This invention is the first to experimentally demonstrate that mutants lacking pectin lyase function (as shown in SEQ ID NO:2) exhibit reduced petiole stress tolerance and increased leaf drop, while overexpressed mutants show increased petiole stress tolerance and reduced leaf drop. This provides a new means to regulate the mechanical strength of tobacco petioles and offers a new strategy for tobacco molecular breeding, which is of great significance for stabilizing tobacco yield and quality.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the tobacco pectin lyase NtPL gene and its applications. Background Technology

[0002] Cultivated tobacco (Nicotiana tabacum) is a globally important economic crop, and its yield and quality directly impact the economic benefits of related industries. Tobacco cultivation frequently faces threats from various natural disasters, such as strong winds, torrential rains, and droughts, which can damage plants, especially causing leaf breakage and drop. This not only affects tobacco yield but can also lead to a decline in leaf quality, resulting in significant economic losses for farmers. Therefore, improving the stress resistance of tobacco, especially enhancing leaf adhesion, is of great importance for stabilizing tobacco yield and quality.

[0003] Therefore, identifying genes that enhance leaf adhesion through tobacco breeding to obtain tobacco varieties with high adhesion is currently an effective method. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide the tobacco pectin lyase NtPL gene and its application.

[0005] This invention provides the application of pectin lyase as a target in regulating plant petiole stress tolerance;

[0006] The amino acid sequence of the pectin lyase is shown in SEQ ID NO:2.

[0007] The regulation includes increasing or decreasing.

[0008] The plant mentioned includes tobacco.

[0009] This invention provides plant breeding or assisted breeding products, comprising at least one of the following: A) to C:

[0010] A) Amplification primers, detection primers, and / or targeting primers using the nucleic acid encoding the pectin lyase as a template;

[0011] B) Targeting the interfering fragment of the pectin lysin;

[0012] C) gRNA targeting the pectin lysin;

[0013] D) Expression cassettes containing interfering fragments as described in B), or gRNAs as described in C);

[0014] E) A recombinant vector containing a nucleic acid encoding the pectin lyase, or an interfering fragment as described in B), or a gRNA as described in C), or an expression cassette as described in D;

[0015] F) Transformation or transfection of host cells with the recombinant vector described in E);

[0016] G), a mixture obtained by culturing host cells as described in F).

[0017] Furthermore, in the product described in this invention,

[0018] The nucleic acid encoding the pectin lyase has the nucleotide sequence shown in SEQ ID NO:1.

[0019] In this invention, the nucleic acid includes DNA or RNA, which can be a nucleotide sequence as shown in SEQ ID NO:1; or it can be a transcription product of a nucleotide sequence as shown in SEQ ID NO:1—RNA. The selection of whether the template or target used in a specific experiment is DNA or RNA depends on the experimental purpose. In this invention, the DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. Nucleic acids can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acids can be topologically linear or circular. Nucleic acids can be obtained directly from natural sources or can be prepared with the assistance of recombinant, enzymatic, or chemical techniques.

[0020] This invention provides a gRNA fragment, which is a fragment located approximately 20 bp before the recognition site (PAM site) of a CRISPR / endonuclease system (such as CRISPR / Cas9, CRISPR / Cas12a, CRISPR / Cas12b, CRISPR / Cas13a, and CRISPR / Cas14a) editing system targeting the target gene. The recognition site of the CRISPR / endonuclease system editing system varies depending on the endonuclease used; typically, the recognition site includes NGG (Ca2+). s9) and / or TTN (Cpf1), etc.; In a specific embodiment of the present invention, the CRISPR / Cas9 system is used, and its recognition site is NGG, where N represents any one of the bases A, T, C or G. The position of NGG can be any position at the 5' end, middle or 3' end of the nucleic acid encoding tobacco pectin lyase, and the present invention does not limit this; In the present invention, the nucleic acid encoding the pectin lyase is used as the target, and gene editing is performed using the CRISPR / Cas9 system. Specifically, the nucleotide sequence of the target segment of the gRNA fragment is shown in SEQ ID NO:3;

[0021] Furthermore, in this invention, a tobacco pectin lyase variant was obtained after gene editing using the CRISPR / Cas9 system. This variant has a T deletion at 461bp or 462bp of the nucleotide sequence shown in SEQ ID NO:1, resulting in a frameshift mutation that leads to the loss of pectin lyase function.

[0022] The recombinant vector described in this invention refers to a recombinant nucleic acid vector, a recombinant DNA molecule containing a desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of the operatively linked coding gene in a specific host organism or for performing the desired operation. In specific embodiments of this invention, the nucleic acid encoding the pectin lyase is linked to the pealy-Myc101 vector backbone to obtain a pectin lyase overexpression vector (pealy-Myc101-NtPL); in other embodiments of this invention, the sgRNA is integrated into a CRISPR / Cas9 gene editing vector to knock out the pectin lyase, achieving gene silencing or loss of function.

[0023] In this specification, the terms "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions and are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors, and / or simply potential genomic inserts.

[0024] The transformation methods include chemical transformation and electrotransformation; the transfection methods include calcium phosphate coprecipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, and Agrobacterium-mediated transfection. In a specific embodiment of the present invention, Agrobacterium-mediated transfection is used to obtain mutant plants for the study of the pectin lyase function described in this invention.

[0025] This invention provides a kit comprising the product and excipients described herein.

[0026] Furthermore, the excipients include at least one of the following: DNA extraction reagent, DNA reverse transcription reagent, transformation or transfection reagent, culture medium, antibiotic and / or buffer solution.

[0027] This invention provides the application of the product or kit described herein in plant breeding or assisted plant breeding.

[0028] Furthermore, the plant breeding includes regulating the adhesion of plant leaves;

[0029] The regulation includes increasing and / or decreasing;

[0030] In a specific embodiment of the present invention, the plant is tobacco, and the tobacco petiole's pressure-bearing capacity is improved by depriving or reducing the function of pectin lyase as shown in SEQ ID NO:2; and by expressing it.

[0031] This invention provides a method for plant breeding or assisted plant breeding, which includes plant breeding using the products or kits described in this invention.

[0032] This invention is the first to experimentally discover that the petiole pressure-bearing capacity of mutant strains lacking the pectin lyase function shown in SEQ ID NO:2 is reduced, while the number of fallen leaves is increased. Overexpression of the mutant strains increases the petiole pressure-bearing capacity and reduces the number of fallen leaves. This provides a new means to regulate the mechanical strength of tobacco petioles and offers a new strategy for tobacco molecular breeding, which is of great significance for stabilizing tobacco yield and quality. Attached Figure Description

[0033] Figure 1 The expression levels of the NtPL gene in different tissues are shown.

[0034] Figure 2 A schematic diagram illustrating the construction of the tobacco NtPL gene editing vector;

[0035] Figure 3 The results of NtPL sequence amplification are shown; lanes 1 and 2 are two repeated amplifications.

[0036] Figure 4 A schematic diagram illustrating the construction of a tobacco NtPL overexpression vector based on the Gateway system;

[0037] Figure 5 qPCR expression level analysis of NtPL gene overexpression lines was performed, with three replicates.

[0038] Figure 6 The leaf drop phenotype of NtPL gene knockout lines is shown.

[0039] Figure 7 The number of leaves dropped in the field was statistically analyzed for three lines: NtPL gene overexpression line, knockout line, and wild type (66 plants in each line).

[0040] Figure 8 This is a pressure gauge used to measure the pressure exerted on the petiole.

[0041] Figure 9 The critical value (N) for petiole breakage is shown in three lines: NtPL gene overexpression line, knockout line, and wild type. Detailed Implementation

[0042] This invention provides the tobacco pectin lyase NtPL gene and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0043] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0044] Example 1: Bioinformatics analysis, tissue-specific expression, and functional analysis of the NtPL gene

[0045] 1. NtPL Bioinformatics Analysis

[0046] Gene cloning analysis revealed that the NtPL gene is 1344 bp in length (the red-marked sgRNA target region), encoding a 447-amino acid protein containing the Pec_lyase (PF00544) domain, and possesses pectin lyase activity. Bioinformatics analysis showed that this gene has high homology with pectin lyases from other plants, especially exhibiting high similarity in conserved domains.

[0047]

[0048] NtPL protein sequence: MAMSLLLLKKWLSFSLSSLLLLLLLLLVGVYAGVQTTDSSDVRTVEKVQLKSSENSTMAVSLEEIEEKLSKHAVDDPEEVVSMVAESIRNSTERRKLGYFSCGTGNPIDDCWRCDRNWQKNRKRLADCGIGFGRNAIGGRDGRYYVVTDSRDDDPVNPRPGTLRHAVIQEEPLWIVFKRDMVIQLKQELIMNSFKTIDARGYNVHIANGACITIQFVTNIIIHGL HIHDCKPTGNAMVRSSPSHFGWRTMADGDAVSIFGSSHIWIDHNSLSHCADGLVDAVMGSTAITISNNHFAHHNEVMLLGHSDSYTRDKQMQVTIAYNHFGEGLIQRMPRCRHYF HVVNNDYTHWEMYAIGGSANPTINSQGNRYLAPTNPFAKEVTKRVDTAAGQWKGWNWRSEGDLMLNGAYFTPSGAGASASYARASSLGAKSSSMVGAITSGAGPLACRRSRMC (SEQ ID NO: 2);

[0049] 2. Tissue-specific expression

[0050] RNA-seq sequencing analysis of NtPL gene expression in different tissues showed that NtPL gene expression was highest in stems, relatively low in leaves and flower buds, and almost non-expressed in roots. Figure 1 ).

[0051] 3. Constructing NtPL knockout vectors

[0052] Primers were designed based on the sgRNA sequence taccgggcctggggttaaca (SEQ ID NO: 3) (recognition site NGG, specifically GGG):

[0053] BsaI-sgRNA-F: 5'-tgcatgttaaccccaggcccggta (SEQ ID NO: 4);

[0054] BsaI-sgRNA-R: 5'-aaactaccgggcctggggttaaca (SEQ ID NO: 5);

[0055] The target product, double-stranded sgRNA with adapter, was obtained after annealing and purified. The linearized gene-editing vector was ligated to the target product using T4 DNA ligase. The ligated vector was then transformed with DH5α. Transformed cells were plated on LB agar plates containing antibiotics (such as ampicillin) and cultured overnight at 37°C. Positive clones were screened. Finally, single clones were selected from the positive clones for culture, plasmids were extracted, and PCR and sequencing were used to verify successful sgRNA insertion. This completed the construction of the target gene-editing vector (construction process as follows). Figure 2 ).

[0056] 4. Functional analysis of the NtPL gene

[0057] 4.1 Creation of homozygous non-transgenic knockout lines of the NtPL gene

[0058] 1 µL of the gene-editing vector plasmid was transformed into Agrobacterium competent cells LBA4404 using standard procedures. The transformed cells were then plated onto a substrate containing rifampicin-resistant (rif) and Kansas bacteria. + In resistant plates, single colonies can appear after 48 hours of growth. Single colonies are selected from those containing Kansas. + YEB liquid medium resistant to RIF was shaken to form bacteria. After 24 h, the mixture was shaken again for bacterial PCR and preservation (600 µL bacterial culture + 600 µL 50% glycerol). After the bacterial culture was verified by PCR sequencing, it was expanded and used for tobacco genetic transformation.

[0059] Tobacco leaves were infected using the leaf disc method. T0 generation transgenic positive lines were screened using kanamycin resistance screening and transgenic molecular identification primers Pos-F: 5'-ttaggtttacccgccaata-3' (SEQ ID NO: 6); Pos-R: 5'-cggtgccactttttcaagtt-3' (SEQ ID NO: 7). PCR amplification was performed using target site detection primers sgRNA-F: 5'-cagtggatgatccagaagaag-3' (SEQ ID NO: 8); sgRNA-R: 5'-gtaatggcagttgagcccat-3' (SEQ ID NO: 9). Lines with homozygous NtPL gene editing were screened to obtain T0 generation homozygous transgenic positive lines. In the T1 generation, a non-transgenic homozygous line was obtained through transgenic and target site identification. Further genome resequencing technology was used to determine that the NtPL gene of this line had a T deletion at 461 bp or 462 bp of the nucleotide sequence shown in SEQ ID NO:1.

[0060] 4.2 Creation of NtPL gene overexpression lines

[0061] (1) Construction of NtPL gene overexpression vector

[0062] Based on the full-length NtPL gene CDS sequence, specific primers with adapters were designed:

[0063] NtPL-attB1-F: 5'-caggctccgcggccgccaccatggcaatgagtttgttgcttttga-3' (SEQ ID NO: 10); NtPL-attB2-R: 5'-aaagctgggtcggcgcgcccgcacatgcggcttctgcg-3' (SEQ ID NO: 11);

[0064] PCR amplification was performed using tobacco K326 cDNA as a template, employing 2×Phanta Mix (Dye) high-fidelity enzyme. The PCR reaction mixture consisted of 1 µL cDNA as template, 10 µL 2×Phanta Mix (Dye), 0.5 µL each of forward and reverse primers, and water to a final volume of 20 µL. Reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ for 30 s, 56℃ for 1 min, 72℃ for 2 min, for 28 cycles. The amplified size was approximately 1.4 kJ. PCR products were detected by agarose gel electrophoresis. Figure 3 After gel extraction and purification, the target gene fragment was recombined with the linearized PUC19 cloning vector. Following the recombination reaction, the recombinant plasmid was transformed into *E. coli* DH5α competent cells. The bacterial culture was plated overnight on Carboxybenzyl chloride (CCC)-resistant plates. Positive clones were screened and sequenced. Plasmids were extracted from clones with correct sequencing results. The plasmid and the plant overexpression vector pealy-Myc101 were subjected to a logistic regression (LR) reaction. After the LR reaction, the cells were transformed into DH5α cells (CCC) resistant to K+. + In the competent state, positive results were verified, and the construction of the tobacco overexpression vector pealy-Myc101-NtPL was completed. Figure 4 ).

[0065] (2) Creation of NtPL gene overexpression lines

[0066] 1 µL of the tobacco overexpression vector pealy-Myc101-NtPL plasmid was transformed into Agrobacterium competent cells LBA4404 using standard procedures. The transformed cells were then plated onto cells containing rifampicin-resistant (rif) and Kansin-resistant strains. + In resistant plates, single colonies can appear after 48 hours of growth. Single colonies are selected from those containing Kansas. +YEB liquid medium resistant to RIF was shaken to form bacteria. After 24 h, the mixture was shaken again for bacterial PCR and preservation (600 µL bacterial culture + 600 µL 50% glycerol). After the bacterial culture was verified by PCR sequencing, it was expanded and used for tobacco genetic transformation.

[0067] Tobacco leaves were infected using the leaf disc method. Kanamycin resistance screening and transgenic molecular identification were performed using primers NtPL-Pos-F: 5'-atgacgcacaatcccactatc-3' (SEQ ID NO: 12); NtPL-Pos-R: 5'-gcacatgcggcttctgcg-3' (SEQ ID NO: 13), with an amplification length of 1569 bp. T0 generation transgenic positive lines were screened by electrophoresis. Amplification was then performed by qPCR using primers qPCR-F: tgttgcttttgaagaagtggct (SEQ ID NO: 14) and qPCR-R: tctcttccaaactcaccgcc (SEQ ID NO: 15), with an amplification length of 174 bp. One transgenic line with significantly increased expression was obtained through screening. Figure 5 ).

[0068] 4.3 Phenotypic Analysis of NtPL Gene Overexpression Lines, Gene Knockout Lines, and Wild-Type Lines

[0069] (1) Counting the number of fallen leaves in the field: Three lines, namely the NtPL gene overexpression line, the knockout line, and the wild type, were planted in the field. The leaf drop in the field was investigated during the budding stage, and the number of fallen leaves for each line was counted. The results showed that the number of fallen leaves in the NtPL gene knockout line was significantly increased ( Figure 6 The number of leaves dropped in NtPL overexpression lines was relatively reduced. Figure 7 ).

[0070] (2) Determine the pressure on the petiole: Use a pressure gauge ( Figure 8 The stress required for leaf breakage in three field-grown NtPL gene overexpression lines, knockout lines, and wild-type lines was measured, and the critical value for petiole breakage was recorded. The results showed that the NtPL gene knockout line had the lowest critical value for petiole breakage, while the critical value for petiole breakage in the NtPL gene overexpression line was higher than that in the wild type. Figure 9 ).

[0071] The above results indicate that the NtPL gene can regulate the mechanical strength of tobacco petioles. Overexpression of this gene can improve the wind resistance and natural disaster resistance of tobacco, reduce leaf drop, and thus improve economic benefits.

[0072] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of pectin lyase NtPL as a target in increasing tobacco petiole pressure tolerance; The amino acid sequence of the pectin lyase NtPL is shown in SEQ ID NO:2; The increased pressure tolerance of tobacco petioles is achieved by overexpressing tobacco pectin lyase NtPL, whose amino acid sequence is shown in SEQ ID NO:2.

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