Tobacco pectin lyase NtPL gene and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a tobacco pectin lyase NtPL gene and application thereof. It is found for the first time through tests that the petiole pressure bearing capacity of a pectin lyase function deletion mutant strain shown in the amino acid sequence such as SEQ ID NO: 2 is reduced, the number of fallen leaves is increased, and the petiole pressure bearing capacity of an overexpressed mutant strain is increased, and the number of fallen leaves is reduced; a new means is provided for regulating and controlling the mechanical strength of the tobacco petiole, a new strategy is provided for tobacco molecular breeding, and the method has important significance for stabilizing the yield and quality of tobacco.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a tobacco pectin lyase NtPL gene and an application thereof. Background Art

[0002] Cultivated tobacco (Nicotiana tabacum) is an important economic crop in the world. Its yield and quality directly affect the economic benefits of related industries. During the cultivation process, tobacco is often threatened by various natural disasters, such as extreme climatic conditions such as strong winds, heavy rains and droughts, which may cause plant damage, especially leaf breakage and shedding. This not only affects the yield of tobacco leaves, but may also cause the quality of tobacco leaves to decline, causing significant economic losses to farmers. Therefore, improving the stress resistance of tobacco, especially improving the adhesion of leaves, is of great significance for stabilizing tobacco yield and quality.

[0003] Therefore, it is currently an effective means to discover genes that improve leaf adhesion through tobacco breeding and obtain tobacco varieties with high adhesion. Summary of the invention

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

[0005] The present invention provides the use of pectin lyase as a target in regulating the stress tolerance of plant petioles;

[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 plants include tobacco.

[0009] The present invention provides a product for plant breeding or assisted breeding, which includes 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), an interfering fragment targeting the pectin lyase;

[0012] C), gRNA targeting the pectin lyase;

[0013] D), an expression cassette containing the interference fragment as described in B), or the gRNA as described in C);

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

[0015] F), transforming or transfecting the host cell with the recombinant vector as described in E);

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

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

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

[0019] In the present invention, the nucleic acid includes DNA or RNA, which can be a nucleotide sequence as shown in SEQ ID NO: 1; it can also be a transcription product of the nucleotide sequence as shown in SEQ ID NO: 1 - RNA; it is selected whether the template or target used in a specific experiment is DNA or RNA according to different experimental purposes; in the present 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 linear or circular in topology. Nucleic acids can be obtained directly from natural sources, or can be prepared with the assistance of recombination, enzymatic methods or chemical techniques.

[0020] The present invention provides a gRNA fragment, which is a fragment targeting a target gene and located about 20 bp before the recognition site (PAM site) of the CRISPR / endonuclease system (such as CRISPR / Cas9, CRISPR / Cas12a, CRISPR / Cas12b, CRISPR / Cas13a and CRISPR / Cas14a) editing system; the recognition site of the CRISPR / endonuclease system editing system is different according to the different endonucleases. Generally, the recognition site includes NGG (Ca 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, wherein N represents any one of A, T, C or G base species, and the position of the NGG can be at any position of the 5' end, the middle or the 3' end of the nucleic acid encoding tobacco pectin lyase, and the present invention is not limited to this; In the present invention, the nucleic acid encoding the pectin lyase is used as a target, and the CRISPR / Cas9 system is used for gene editing. Specifically, the nucleotide sequence of the target segment of the gRNA fragment is shown in SEQ ID NO: 3;

[0021] Furthermore, in the present invention, a tobacco pectin lyase variant was obtained after gene editing by the CRISPR / Cas9 system, in which the base T at 461bp or 462bp of the nucleotide sequence shown in SEQ ID NO:1 was deleted, resulting in a frameshift mutation, leading to the loss of pectin lyase function.

[0022] The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule, which contains the desired coding sequence and the appropriate nucleic acid sequence or element that is essential for the expression of the operably linked coding gene in a specific host organism or the completion of the target operation. In a specific embodiment of the present invention, the nucleic acid encoding the pectin lyase is connected to the pealy-Myc101 vector backbone to obtain a pectin lyase overexpression vector (pealy-Myc101-NtPL); in other embodiments of the present invention, the sgRNA is integrated into the CRISPR / Cas9 gene editing vector to knock out the pectin lyase, so as to achieve the purpose of silencing or loss of gene function.

[0023] In this specification, "plasmid" and "vector" are sometimes used interchangeably, as plasmid is the most commonly used vector form. However, the present invention is intended to include such other forms of expression vectors that play an equivalent role, which are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors and / or only potential genomic insertions.

[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, Agrobacterium-mediated transfection, etc. 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 of the present invention.

[0025] The present invention provides a kit, which comprises the product of the present invention and auxiliary materials.

[0026] Furthermore, the auxiliary materials include: at least one of a DNA extraction reagent, a DNA reverse transcription reagent, a transformation or transfection reagent, a culture medium, an antibiotic and / or a buffer.

[0027] The present invention provides application of the product or the kit in plant breeding or assisted plant breeding.

[0028] Further, 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 stress tolerance of tobacco petioles is improved by loss of function of pectate lyase as shown in SEQ ID NO: 2 or reduction of the stress tolerance of tobacco petioles; and by expression.

[0031] The present invention provides a method for plant breeding or assisted plant breeding, which comprises using the product of the present invention or the kit of the present invention to carry out plant breeding.

[0032] The present invention discovers for the first time through experiments that the petiole pressure tolerance of the pectate lyase function-deficient mutant strain with an amino acid sequence as shown in SEQ ID NO: 2 is reduced, the number of fallen leaves is increased, and the petiole pressure tolerance of the overexpression mutant strain is increased, and the number of fallen leaves is reduced; this provides a new means for regulating the mechanical strength of tobacco petioles and a new strategy for tobacco molecular breeding, which is of great significance for stabilizing tobacco yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Shows the expression level of NtPL gene in different tissues;

[0034] Figure 2 Schematic diagram showing the construction of tobacco NtPL gene editing vector;

[0035] Figure 3 Shows the results of NtPL sequence amplification, lanes 1 and 2 are two repeated amplifications;

[0036] Figure 4 Schematic diagram showing the construction of tobacco NtPL overexpression vector based on Gateway system;

[0037] Figure 5 Shown is the qPCR expression analysis of NtPL gene overexpression lines, repeated 3 times;

[0038] Figure 6 Shows the leaf-drop phenotype of the NtPL gene knockout line;

[0039] Figure 7 The number of fallen leaves in the field of three strains, namely, NtPL gene overexpression strain, knockout strain and wild type, is shown (66 strains in each strain);

[0040] Figure 8 Shows the pressure gauge used to measure the pressure on the petiole;

[0041] Fig. 9 The critical value (N) of petiole breakage of three strains, namely, NtPL gene overexpression strain, knockout strain and wild type, is shown. DETAILED DESCRIPTION

[0042] The present invention provides tobacco pectin lyase NtPL gene and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0043] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

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

[0045] 1. NtPL bioinformatics analysis

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

[0047]

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

[0049] 2. Tissue-specific expression

[0050] Based on RNAseq sequencing, the expression of NtPL gene in different tissues was analyzed. The results showed that NtPL gene was expressed at the highest level in stems, relatively low in leaves and flower buds, and almost not in roots ( Figure 1 ).

[0051] 3. Construction of NtPL knockout vector

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

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

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

[0055] After annealing, a double-stranded sgRNA target product with a linker is obtained, and the target product is purified; the linearized gene editing vector and the target product are connected using T4 DNA ligase, and the connected product vector is transformed into DH5α. The transformed cells should be spread on LB agar plates containing antibiotics (such as ampicillin) and cultured overnight at 37°C to screen positive clones. Finally, a single clone is selected from the positive clones for culture, plasmids are extracted, and the vector in which the sgRNA is successfully inserted is verified by PCR and sequencing, thus completing the construction of the target gene editing vector (the construction process is as follows Figure 2 ).

[0056] 4. Analysis of NtPL gene function

[0057] 4.1. Creation of homozygous non-transgenic knockout strains of NtPL gene

[0058] 1 µL of the gene editing vector plasmid was transferred into the competent Agrobacterium LBA4404. After transformation, the plasmid was spread onto the plasmid containing rifampicin resistance (rif) and Kan + After 48 hours of growth, a single colony may appear on the plate. + The cells were shaken in YEB liquid medium with rif resistance, and after 24 h, the culture solution was shaken for PCR and preservation (600 µL of culture solution + 600 µL of 50% glycerol). The correct culture solution was verified by PCR sequencing and then expanded for tobacco genetic transformation.

[0059] Tobacco leaves were infected by the leaf disc method, and T0 generation transgenic positive lines were screened by kanamycin resistance screening and transgenic molecular identification primers Pos-F: 5'-ttaggtttacccgccaata-3' (SEQ ID NO: 6); Pos-R: 5'-cggtgccacttttcaagtt-3' (SEQID 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' (SEQID NO: 9) to screen for lines with homozygous editing of the NtPL gene, and obtain T0 generation homozygous transgenic positive lines. In the T1 generation, a non-transgenic homozygous strain was screened through transgenic and target site identification, and genome resequencing technology was further used to determine that the NtPL gene of the strain had a base 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 strains

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

[0062] According to the CDS sequence of the full-length NtPL gene, 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 the cDNA of tobacco K326 as a template, and 2×Phanta Mix (Dye) high-fidelity enzyme was used for amplification. The PCR reaction system was: 1µL cDNA as a template, 10µL 2×Phanta Mix (Dye), 0.5µL each of forward and reverse primers, and water was added to 20µL. Reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ 30s, 56℃ 1min, 72℃ 2min, 28 cycles. The amplified size was about 1.4 k, and the PCR product was detected by agarose gel electrophoresis ( Figure 3 ) and then gel-cut and purified, the target gene fragment was recombined with the linearized PUC19 cloning vector, and the recombinant plasmid was transformed into the competent E. coli DH5α, the bacterial solution was coated on the plate (with resistance of Car) and cultured overnight, the positive clones were screened for sequencing, and the plasmids were extracted from the clones with correct sequencing. The plasmid and the plant overexpression vector pealy-Myc101 were subjected to LR reaction, and then transformed into DH5α (with resistance of K) after the LR reaction. + ) in competent state, verify the positive clones, and complete the construction of tobacco overexpression vector pealy-Myc101-NtPL ( Figure 4 ).

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

[0066] 1 µL of the tobacco overexpression vector pealy-Myc101-NtPL plasmid was transferred into the competent Agrobacterium LBA4404. After transformation, the plasmid was spread onto the plasmid containing rifampicin resistance (rif) and Kan + After 48 hours of growth, a single colony may appear on the plate. +The cells were shaken in rif-resistant YEB liquid medium, and after 24 hours, the culture solution was shaken for PCR and preservation (600µL culture solution + 600µL 50% glycerol). The correct culture solution was verified by PCR sequencing and then expanded for tobacco genetic transformation.

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

[0068] 4.3 Phenotypic analysis of NtPL gene overexpression strains, gene knockout strains and wild type

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

[0070] (2) Determine the pressure on the petiole: Use a pressure gauge ( Figure 8 ) The pressure required to break the leaves of three strains, namely, NtPL gene overexpression strain, knockout strain and wild type, was measured in the field, and the critical value of petiole breakage was recorded. The results showed that the critical value of petiole breakage in NtPL gene knockout strain was the lowest, and the critical value of petiole breakage in NtPL gene overexpression strain was higher than that in wild type ( Fig. 9 ).

[0071] The above results show that the NtPL gene can regulate the mechanical strength of tobacco petioles. Overexpression of this gene can improve tobacco's wind resistance and resistance to natural disasters, reduce leaf drop, and thus achieve improved economic benefits.

[0072] The above are only preferred embodiments 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 principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of pectin lyase as a target in regulating plant petiole stress tolerance; The amino acid sequence of the pectin lyase is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that: The regulation includes increasing or decreasing.

3. The use according to any one of claims 1 or 2, characterized in that: The plants include tobacco.

4. A plant breeding or assisted breeding product, characterized in that: Including at least one of the following A) to C: A), amplification primers, detection primers and / or targeting primers using the nucleic acid encoding the pectin lyase as a template; B), an interfering fragment targeting the pectin lyase; C), gRNA targeting the pectin lyase; D), an expression cassette containing the interference fragment as described in B), or the gRNA as described in C); E), a recombinant vector containing a nucleic acid encoding the pectin lyase, or an interference fragment as described in B), or a gRNA as described in C), or an expression cassette as described in D); F), transforming or transfecting the host cell with the recombinant vector as described in E); G), culturing the mixture obtained by the host cells described in F).

5. The product according to claim 4, characterized in that The nucleic acid encoding the pectin lyase has a nucleotide sequence as shown in SEQ ID NO:

1.

6. The product according to claim 4, characterized in that The nucleotide sequence of the gRNA is shown in SEQ ID NO:

3.

7. A kit, characterized in that The invention comprises the product and auxiliary materials described in any one of claims 4 to 6.

8. The kit according to claim 7, characterized in that The auxiliary materials include: at least one of a DNA extraction reagent, a DNA reverse transcription reagent, a transformation or transfection reagent, a culture medium, an antibiotic and / or a buffer.

9. Use of the product according to any one of claims 4 to 6 or the kit according to claim 7 or 8 in plant breeding or assisted plant breeding.

10. A method for plant breeding or assisted plant breeding, characterized in that The method comprises using the product according to any one of claims 4 to 6 or the kit according to claim 7 or 8 to carry out plant breeding.

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