Application of LaOMT1 and its encoding gene in plant tolerance to salt stress

Abstract

The present invention discloses the application of LaOMT1 protein coding gene in improving plant tolerance to salt stress. The gene is heterologously overexpressed in wild-type Arabidopsis thaliana, and it is found that the obtained overexpressed Arabidopsis thaliana plants have increased growth under sodium chloride treatment, and the taproot length is significantly increased compared with wild-type Arabidopsis thaliana WT, indicating that the gene can be introduced into plants as a target gene to enhance the tolerance of transgenic plants to salt stress. The present invention provides new gene targets and resources for cultivating salt-tolerant plant varieties.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to application of LaOMT1 protein and its encoding gene in regulating plant salt stress tolerance. Background Art

[0002] Oxymethylation is a common reaction in different organisms. It is catalyzed by O-methyltransferase (OMT). Under the condition that S-adenosyl-L-methionine (SAM) provides a methyl group, OMT can methylate the oxygen position of the substrate to generate the corresponding methylation product, and SAM is converted into S-adenosyl-L-homocysteine ​​(SAH). Studies have shown that OMT in plants is involved in the synthesis of many compounds (pigments, floral fragrance, lignin and defensins, etc.). Moreover, different OMTs can catalyze the same substrate, the same OMT can catalyze different substrates, and when the same OMT catalyzes a substrate with multiple hydroxyl sites, its products may be multiple.

[0003] Plant OMTs are divided into Class I and Class II according to the number of amino acids they encode, the molecular weight of the protein, and the catalytic substrates. Class I OMTs have a molecular weight of about 27-30 kDa, with 231-248 amino acids, and play a key role in lignin synthesis. Except for Class I OMTs, the rest of the OMTs are classified as Class II, with a molecular weight of about 38-42 kDa, with 344-383 amino acids, and mainly play an important role in the biosynthesis of plant secondary metabolism such as flavonoids. In addition, studies have shown that plant OMTs may also play an important role in plant abiotic stress responses. At present, there is no attention on whether Class I OMTs play a role in plant tolerance to salt stress. Summary of the invention

[0004] The purpose of the present invention is to provide the application of LaOMT1 protein and its encoding gene in improving plant tolerance to salt stress.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The nucleotide sequence of the LaOMT1 protein coding gene of Hudixiao is: SEQ ID NO.1.

[0007] The amino acid sequence of the LaOMT1 protein is: SEQ ID NO.2.

[0008] A recombinant expression vector containing the LaOMT1 protein coding gene of the present invention.

[0009] When using the LaOMT1 protein coding gene to construct a plant overexpression vector, any strong promoter or inducible promoter can be added before its transcription start nucleotide. In order to facilitate the screening and identification of transgenic plants, the plant expression vector used can be processed, such as adding a selective marker gene (GUS gene, GFP gene, etc.) to the vector.

[0010] Application of LaOMT1 protein coding gene of Oleaceae in improving plant salt stress tolerance through genetic engineering; the nucleotide sequence of the LaOMT1 protein coding gene of Oleaceae is SEQ ID NO.1.

[0011] Application of a recombinant expression vector containing a LaOMT1 protein coding gene of the ophiopogon sphaerocephala in improving plant salt stress tolerance through genetic engineering; the nucleotide sequence of the LaOMT1 protein coding gene of the ophiopogon sphaerocephala is SEQ ID NO.1.

[0012] Beneficial effects of the present invention.

[0013] 1. Through research, the present invention provides the biological function of the LaOMT1 encoding gene in improving the salt stress tolerance of Arabidopsis thaliana.

[0014] 2. The plant overexpression vector pCHF1301-LaOMT1 was constructed. Figure 1 A) and heterologously expressed in wild-type Arabidopsis thaliana ( Figure 1 B). Phenotypic identification of the screened T3 positive seedlings revealed that plants overexpressing the LaOMT1 protein encoding gene had enhanced growth under salt stress ( Figure 2 ), mainly manifested in a significant increase in primary root length compared with the wild-type WT control ( Figure 3 ), indicating that this gene can be introduced into plants as a target gene to improve the salt tolerance of transgenic plants.

[0015] Description of the drawings.

[0016] Figure 1 A is a diagram of the construction of the LaOMT1 encoding gene overexpression vector.

[0017] Figure 1 B is the expression analysis of LaOMT1 protein coding gene in transgenic plants, where WT is the wild-type Arabidopsis control, and OE is different Arabidopsis transgenic lines with LaOMT1 protein coding gene.

[0018] Figure 2 The phenotypes of each plant under different concentrations of sodium chloride treatment, where WT is wild-type Arabidopsis, and OE is different Arabidopsis transgenic lines with LaCOMT protein encoding gene.

[0019] Figure 3Figure 1 shows the main root length statistics of each plant under different concentrations of sodium chloride treatment. WT is wild-type Arabidopsis, and OE is different Arabidopsis transgenic lines with LaOMT1 protein coding gene. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, instruments, etc. used in the following examples are all commercially available unless otherwise specified. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

[0021] Example 1: Genetic transformation of LaOMT1 protein encoding gene in Arabidopsis thaliana.

[0022] 1. Construction of recombinant vector

[0023] Total RNA was extracted from the seedlings of P. hudixiao and the RNA was synthesized into cDNA using reverse transcriptase.

[0024] The overexpression vector of the LaOMT1 protein coding gene was constructed by double restriction enzyme digestion method, and primers containing the complete open reading frame (ORF) of the LaOMT1 protein coding gene and two restriction enzyme cutting sites of Kpn I and Xba I (excluding the stop codon) were designed. The primer sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4. PCR amplification obtained the PCR product containing the LaOMT1 protein coding gene.

[0025] The above PCR amplification product and pCAMBIA1301-GFP vector plasmid were double-digested with Kpn I and Xba I, respectively. The PCR product after digestion was connected with the backbone vector using T4 ligase at 25°C for 2 h to obtain a recombinant vector. The recombinant vector with the correct sequence was named pCHF1301-LaOMT1. The partial structure of pCHF1301-LaOMT1 is shown in the figure. Figure 1 As shown in A, pCHF1301-LaOMT1 can express the protein shown in SEQ ID NO.2.

[0026] The recombinant expression vector pCHF1301-LaOMT1 obtained above was transformed into Agrobacterium tumefaciens EHA105 to obtain Agrobacterium containing the recombinant expression vector.

[0027] 2. Genetic transformation and screening of Arabidopsis

[0028] The obtained Agrobacterium tumefaciens EHA105 containing pCHF1301-LaOMT1 was transformed into Arabidopsis Columbia-0 ecotype by floral infection, and T1 generation seeds were harvested. T1 generation Arabidopsis seeds were sown on 1 / 2 MS solid medium containing 20 mg / L for hygromycin resistance screening. The transgenic seedlings resistant to hygromycin were transplanted into pots filled with vermiculite, irrigated with 1 / 2 MS liquid medium in a timely manner, and grown under long-day conditions at 22 ºC. The growth and development process and phenotypic traits of T2 and T3 generation transgenic Arabidopsis were observed and recorded.

[0029] The total RNA of the T3 homozygous transgenic Arabidopsis obtained by screening was extracted and reverse transcribed to obtain cDNA. The expression level was detected using the designed primers for amplification of the LaOMT1 protein coding gene (primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6). At the same time, Arabidopsis Actin2 was used as an internal reference to design primers (primer sequences are shown in SEQ ID NO.7 and SEQ ID NO.8). The results are shown in Figure 1 As shown in B, the LaOMT1 gene could not be detected in the wild-type Arabidopsis WT plants, while the expression of the LaOMT1 gene could be detected in the three Arabidopsis lines that overexpressed the LaOMT1 protein encoding gene.

[0030] Example 2: Comparison of tolerance of transgenic Arabidopsis thaliana expressing the LaOMT1 protein encoding gene with wild-type plants under sodium chloride stress.

[0031] Wild-type Arabidopsis WT and different strains of Arabidopsis overexpressing the LaOMT1 protein gene (OE-4, OE-15 and OE-22) were sown simultaneously in 1 / 2 MS solid culture medium containing 0 mM (control), 50 mM, 100 mM and 150 mM sodium chloride, and the changes in the root growth state of Arabidopsis were observed after 7 days of treatment. The results showed that on the normal 1 / 2 MS culture medium, there were slight differences in the morphology of Arabidopsis overexpressing the LaOMT1 protein gene and the wild-type Arabidopsis WT Arabidopsis plants. On the 1 / 2 MS solid culture medium containing different concentrations of sodium chloride, the transgenic plants were larger and grew better ( Figure 2 ). Specifically, the underground root system is more developed, and the main root length of the transgenic plants is significantly longer than that of the wild-type Arabidopsis WT ( Figure 3 ). This indicates that the gene can be introduced into plants as a target gene to improve the ability of transgenic plants to tolerate salt stress. Sequence Listing <110> Institute of Botany, Chinese Academy of Sciences, Jiangsu Province <120> Application of LaOMT1 and its encoding gene in plant tolerance to salt stress <160> 8 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 717 <212> DNA <213> Lycoris aurea (L'Hér.) Herb <400> 1 atgggtgcta gccaagatga ttatccacta atccacaaga atattttgca tagtgaagat 60 cttcttaagt acatattgga gactagtgtt tacccaagag agcatgaaca gctcaagggg 120 ttgagggagg tgactgagaa acatgaatgg agtacggcgc ttgtcgcagc cgatgaagga 180 ttatttcttt ctatgttgtt aaagctcatg aatgccaaga gaaccattga gattggtgta 240 tacaccggtt attctctgct cacaaccgct ttggctttac cagaagatgg aaagataacg 300 gcaattgacg tcaacaagtc ctactttgag ataggactgc catttattca gaaagcagga 360 gttgagcata agatcaattt cattgaatca gaagcacttc ctgttcttga tcatatgctt 420 caagagatga aggaagaaga cctctacgac tttgcatttg tcgatgcaga caaaccaaac 480 tatgctaatt accacgagcg attagtgaag cttgtcaggg ttggaggagc aatcgtctac 540 gandacacgc tctggttcgg aactgtagca ttccaggaat atccaggcct tcatccggaa 600 gaggaggagt gtagggtctc ttcagaac ctgaataagc tctggcagc tgatccccgt 660 gtcgagatat cccaagtctc agtcggcgat ggactgacta ttgtcgacg tctttat 717 <210> 2 <211> 239 <212> PRT <213> Lycoris aurea (L'Hér.) Herb) <400> 2 Met Gly Ala Ser Gln Asp Asp Tyr Pro Leu Ile His Lys Asn Ile Leu 1 5 10 15 His Ser Glu Asp Leu Leu Lys Tyr Ile Leu Glu Thr Ser Val Tyr Pro 20 25 30 Arg Glu His Glu Gln Leu Lys Gly Leu Arg Glu Val Thr Glu Lys His 35 40 45 Glu Trp Ser Thr Ala Leu Val Ala Ala Asp Glu Gly Leu Phe Leu Ser 50 55 60 Met Leu Leu Lys Leu Met Asn Ala Lys Arg Thr Ile Glu Ile Gly Val 65 70 75 80 Tyr Thr Gly Tyr Ser Leu Thr Thr Ala Leu Ala Leu Pro Glu Asp 85 90 95 Gly Lys Ile Thr Ala Ile Asp Val Asn Lys Ser Tyr Phe Glu Ile Gly 100 105 110 Leu Pro Phe Ile Gln Lys Ala Gly Val Glu His Lys Ile Asn Phe Ile 115 120 125 Glu Ser Glu Ala Leu Pro Val Leu Asp His Met Leu Gln Glu Met Lys 130 135 140 Glu Glu Asp Leu Tyr Asp Phe Ala Phe Val Asp Ala Asp Lys Pro Asn 145 150 155 160 Tyr Ala Asn Tyr His Glu Arg Leu Val Lys Leu Val Arg Val Gly Gly 165 170 175 Ala Ile Val Tyr Asp Asn Thr Leu Trp Phe Gly Thr Val Ala Phe Pro 180 185 190 Glu Tyr Pro Gly Leu His Pro Glu Glu Glu Glu Cys Arg Val Ser Phe 195 200 205 Arg Asn Leu Asn Lys Leu Leu Ala Ala Asp Pro Arg Val Glu Ile Ser 210 215 220 Gln Val Ser Val Gly Asp Gly Leu Thr Ile Cys Arg Arg Leu Tyr 225 230 235 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <400> 3 cgcaggtacc atgggtgcta gccaagatga ttatg 35 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence <400> 4 cagctctaga tcaataaaga cgtcgacaaa tagtcagtc 39 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 gatatcccaa gtctcagtcg 20 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <400> 6 ctcgcaagtt gatagcagac ata 23 <210> 7 <211> twenty four <212> DNA <213> Artificial Sequence <400> 7 gacaatggta ccggtatggt gaag 24 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <400> 8 catatcatcc cagttgctaa caaca 25

Claims

1. Application of the LaOMT1 protein coding gene in improving plant tolerance to salt stress; characterized by: The transgenic plants obtained by introducing the LaOMT1 protein encoding gene into plants for overexpression have enhanced resistance to salt stress. The amino acid sequence of the protein encoded by the LaOMT1 protein encoding gene is shown in SEQ ID NO.2, and the plant is Arabidopsis thaliana.

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