Application of Lycoris aurea LaCOMT and its encoding gene in improving mercury stress tolerance of plants

Abstract

The present invention discloses the application of the Lycoris aurea LaCOMT protein-coding gene in improving the tolerance of plants to heavy metal mercury stress. By heterologously overexpressing this gene in the wild type of Arabidopsis thaliana, it was found that the obtained overexpressed Arabidopsis thaliana plants had improved growth and increased biomass under mercury chloride treatment. The primary root length and fresh weight were significantly increased compared to the wild type Arabidopsis thaliana WT and the Arabidopsis thaliana transformed with the GFP empty vector, indicating that this gene can be introduced into plants as a target gene to enhance the tolerance of transgenic plants to mercury stress. The present invention provides beneficial help for improving the phytoremediation of mercury pollution.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and specifically relates to the application of Lycoris aurea LaCOMT protein and its encoding gene in regulating plant tolerance to mercury stress. Background Art

[0002] Mercury and its compounds are one of the most dangerous heavy metal environmental pollutants, endangering biosafety and environmental quality globally. In the environment, mercury exists in different forms such as elemental (metallic) mercury (Hg 0 ), inorganic mercury (HgS and HgCl2), and organic mercury (mainly methylmercury, MeHg). Among them, the most common and bioavailable forms of mercury in soil are Hg(II) and MeHg.

[0003] Plants can effectively absorb heavy metals in their environment through root hairs. This ability of plants is usually used to remediate heavy metal-polluted soils and is called phytoremediation. A large number of studies have shown that transferring genes related to heavy metal detoxification from different biological sources (animals, microorganisms, and plants) into high-biomass plants and heterologously overexpressing target genes can mediate the tolerance and high accumulation of transgenic plants to heavy metals and their analogs. Therefore, transgenic technology has important application value and broad prospects in phytoremediation of environmental pollution.

[0004] Caffeic acid O-methyltransferase (COMT) belongs to type II O-methyltransferase (OMT) and plays an important role mainly in the biosynthesis of plant secondary metabolites such as lignin and flavonoids. Although the substrate preference of COMT is caffeic acid, it can also catalyze the conversion of N-acetylserotonin to melatonin, etc. It has been demonstrated that in addition to participating in the biosynthesis of plant secondary metabolites, COMT may also play a role in abiotic stress. For example, COMT and its catalytic product melatonin play a role in salt stress tolerance. However, there is no report on whether COMT is involved in the function of heavy metal mercury stress tolerance.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide the application of Lycoris aurea LaCOMT protein and its encoding gene in improving plant tolerance to mercury stress.

[0007] The purpose of the present invention can be achieved by the following technical solutions.

[0008] The encoding gene of Lycoris aurea LaCOMT protein, whose nucleotide sequence is: SEQ ID NO.1.

[0009] Lycoris aurea LaCOMT protein, whose amino acid sequence is: SEQ ID NO.2.

[0010] A recombinant expression vector containing the Lycoris aurea LaCOMT protein-encoding gene of the present invention.

[0011] When constructing a plant overexpression vector using the LaCOMT protein-encoding gene, any strong promoter or inducible promoter can be added before the transcriptional start nucleotide. For facilitating the screening and identification of transgenic plants, the used plant expression vector can be processed, such as adding a selectable marker gene (GUS gene, GFP gene, etc.) to the vector.

[0012] Application of the Lycoris aurea LaCOMT protein-encoding gene in improving plant tolerance to mercury stress by genetic engineering; the nucleotide sequence of the Lycoris aurea LaCOMT protein-encoding gene is SEQ ID NO.1.

[0013] Application of a recombinant expression vector containing the Lycoris aurea LaCOMT protein-encoding gene in improving plant tolerance to mercury stress by genetic engineering; the nucleotide sequence of the Lycoris aurea LaCOMT protein-encoding gene is SEQ ID NO.1.

[0014] Advantages of the present invention.

[0015] 1. Through research, the present invention for the first time provides the biological function of the Lycoris aurea LaCOMT encoding gene in improving Arabidopsis thaliana tolerance to mercury stress.

[0016] 2. After treatment with exogenous mercuric chloride, the gene expression level of the LaCOMT protein-encoding gene is induced, indicating that this gene can respond to mercury stress ( Figure 1 ).

[0017] 3. A plant overexpression vector pCAMBIA1301-LaCOMT-GFP ( Figure 2 ) was constructed and heterologously expressed in the wild type of Arabidopsis thaliana ( Figure 3 ). Phenotypic identification was carried out on the screened T3 generation positive seedlings, and it was found that the plants overexpressing the LaCOMT protein-encoding gene had enhanced growth under mercury treatment, mainly manifested as a significant increase in the primary root length and fresh weight compared with the wild type and the GFP empty vector control ( Figure 4 ), indicating that this gene can be introduced into plants as a target gene to improve the mercury tolerance of transgenic plants.

[0018] Description of the drawings.

[0019] Figure 1 Induced expression of the LaCOMT protein-encoding gene under 200 μM mercuric chloride stress.

[0020] Figure 2 Diagram for constructing the overexpression vector.

[0021] Figure 3Analysis of the expression level of the LaCOMT protein-encoding gene in transgenic plants. Among them, WT is the wild-type Arabidopsis thaliana control, GFP is the control of the empty vector pCAMBIA1301-GFP, and OE is different Arabidopsis thaliana transgenic lines transfected with the LaCOMT protein-encoding gene.

[0022] Figure 4 Shows the phenotypes of each plant under 10 μM mercuric chloride treatment. Among them, WT is the wild-type Arabidopsis thaliana, GFP is the control of the empty vector pCAMBIA1301-GFP, and OE is different Arabidopsis thaliana transgenic lines transfected with the LaCOMT protein-encoding gene.

[0023] Figure 5 Shows the statistical results of the fresh weight and main root length of each plant under 10 μM mercuric chloride treatment. Among them, WT is the wild-type Arabidopsis thaliana, GFP is the control of the empty vector pCAMBIA1301-GFP, and OE is different Arabidopsis thaliana transgenic lines transfected with the LaCOMT protein-encoding gene. Specific implementation mode

[0024] The following further describes the present invention in detail in combination with specific implementation modes. The provided examples are only for clarifying the present invention, rather than limiting the scope of the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial channels unless otherwise specified. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence list is the 5'-terminal nucleotide of the corresponding DNA, and the last position is the 3'-terminal nucleotide of the corresponding DNA.

[0025] Example 1. Gene expression pattern of the LaCOMT protein-encoding gene in Lycoris aurea under mercuric chloride stress

[0026] The present invention provides a Lycoris aurea LaCOMT protein, the amino acid sequence of which is SEQ ID NO.2, and the nucleotide sequence of the LaCOMT protein-encoding gene in Lycoris aurea is SEQ ID NO.1.

[0027] Under normal and mercuric chloride stress treatment conditions, the expression characteristics of the LaCOMT protein-coding gene in the roots and leaves of Lycoris aurea were detected: The Lycoris aurea seedlings that had grown in pots for 1 year were removed from the soil, washed, cultured in sterile water for 1 day, and after carefully sucking out the water, they were respectively placed in sterile water (control) and 200 μM mercuric chloride solution for treatment. Samples of the roots and leaves of the plants were taken at 0, 3, 6, 10, 24, and 48 hours after treatment. The total RNA of the samples was extracted, reverse transcribed to obtain cDNA, and primers were designed for real-time fluorescence quantitative pCR (qRT-PCR) amplification of the LaCOMT protein-coding gene. The primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4. At the same time, primers were designed using the Lycoris aurea LaTIP41 gene as an internal reference, and the primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0028] The results are as Figure 1 shown. Under the control (sterile water) treatment, with the extension of the treatment time, the gene expression of the LaCOMT protein-coding gene showed small fluctuations; while under the 200 μM mercuric chloride stress treatment, its gene expression showed a significant induced expression trend. Among them, the expression level of the LaCOMT protein-coding gene in the roots of Lycoris aurea reached the peak at 3 h after mercuric chloride treatment, and the expression level of the LaCOMT protein-coding gene in the leaves of Lycoris aurea roots reached the peak at 10 h after mercuric chloride treatment.

[0029] Example 2: Arabidopsis thaliana transformed with the Lycoris aurea LaCOMT protein-coding gene showed improved mercury stress tolerance.

[0030] I. Construction of the recombinant vector.

[0031] The total RNA of Lycoris aurea seedlings was extracted, and the RNA was reverse transcribed into cDNA using reverse transcriptase.

[0032] An overexpression vector of the LaCOMT protein-coding gene was constructed by double digestion. Primers containing the complete open reading frame (ORF) of the LaCOMT protein-coding gene and two restriction enzyme sites, Spe I and Xba I (excluding the stop codon), were designed, and the primer sequences are shown in SEQ ID NO.7 and SEQ ID NO.8. A PCR product containing the LaCOMT protein-coding gene was obtained by PCR amplification.

[0033] The above PCR amplification products and the plasmid of pCAMBIA1301-GFP vector were double digested with Spe I and Xba I respectively. The digested PCR products and the backbone vector were ligated with T4 ligase at 25 ºC for 2 h to obtain the recombinant vector. The recombinant vector with the correct sequence was named pCAMBIA1301-LaCOMT-GFP. Part of the structure of pCAMBIA1301-LaCOMT-GFP is as Figure 2 shown, and pCAMBIA1301-LaCOMT-GFP can express the protein shown in SEQ ID NO.2.

[0034] The obtained recombinant expression vector pCAMBIA1301-LaCOMT-GFP was transformed into Agrobacterium tumefaciens GV3101 to obtain Agrobacterium containing the recombinant expression vector.

[0035] II. Genetic transformation and screening of Arabidopsis thaliana.

[0036] The obtained Agrobacterium tumefaciens GV3101 containing pCAMBIA1301-LaCOMT-GFP was transformed into Arabidopsis thaliana Columbia-0 ecotype by the floral dip method, and the T1 generation seeds were harvested. The T1 generation Arabidopsis thaliana seeds were sown on 1 / 2 MS solid medium containing 20 mg / L hygromycin for hygromycin resistance screening. The screened transgenic plant lines resistant to hygromycin were transplanted into pots filled with vermiculite and irrigated with 1 / 2 MS liquid medium in a timely manner, and grown under long-day conditions at 22 ºC. Observe and record the growth and development process and phenotypic traits of T2 and T3 generation transgenic Arabidopsis thaliana.

[0037] Total RNA was extracted from the screened T3 generation homozygous transgenic Arabidopsis thaliana, reverse transcribed into cDNA, and the expression level was detected using the designed qRT-PCR amplification primers for the LaCOMT protein-coding gene (the primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4). At the same time, Arabidopsis thaliana Actin2 was used as an internal reference, and primers were designed (the primer sequences are shown in SEQ ID NO.9 and SEQ ID NO.10). The results are as Figure 3 shown. In wild-type Arabidopsis thaliana WT and Arabidopsis thaliana plants transformed with the empty vector pCAMBIA301-GFP, the LaCOMT gene could not be detected, while the LaCOMT gene expression could be detected in 7 screened Arabidopsis thaliana lines overexpressing the LaCOMT protein-coding gene. Among them, the expression levels of the LaCOMT protein-coding gene in OE#4, OE#6, and OE#7 lines were relatively high.

[0038] III. Phenotypic identification of transgenic Arabidopsis thaliana plants under mercury chloride stress

[0039] Wild-type Arabidopsis thaliana WT, Arabidopsis thaliana transformed with the empty vector pCAMBIA301-GFP, and different lines of Arabidopsis thaliana overexpressing the LaCOMT-encoding protein gene (OE#4, OE#6, and OE#7) were simultaneously sown on 1 / 2 MS solid medium containing 0 μM (control) and 10 μM mercuric chloride. After 10 days of treatment, the changes in the growth status of Arabidopsis roots and the fresh weight of Arabidopsis plants were observed. The results showed that on normal 1 / 2 MS medium, there were no obvious morphological differences between Arabidopsis thaliana overexpressing the LaCOMT-encoding protein gene and wild-type Arabidopsis thaliana WT and Arabidopsis thaliana transformed with the empty vector pCAMBIA301-GFP. However, on 1 / 2 MS solid medium containing 10 μM mercuric chloride, the transgenic plants had a larger morphology and better growth Figure 4 ). Specifically, the leaves in the above-ground part were larger and the roots in the underground part were more developed. Among them, the fresh weight of the above-ground part of the transgenic plants was significantly greater than that of the wild-type plants, and the main root length was significantly greater than that of wild-type Arabidopsis thaliana WT and Arabidopsis thaliana transformed with the empty vector pCAMBIA301-GFP Figure 5 ). This indicates that this gene can be introduced into plants as a target gene to improve the mercury stress tolerance of transgenic plants. Sequence Listing <110> Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences <120> Application of Lycoris aurea LaCOMT Protein and Its Encoding Gene in Improving Plant Mercury Stress Tolerance <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1098 <212> DNA <213> Lycoris aurea (L'Hér.) Herb <400> 1 atgggttcag cccaaaactt catcacacca gcaccccaaa caacagatga agaagcatgc 60 atatttgcca tgcagctcac aagcttctca atattaccca tgactctgaa agcagccatc 120 gagctcgatc tcttcgagat catcgcaaag gctgggccta atgcttatct ttcgccgagc 180 gaggtcgcga gccaactgcc taataacaag aacccacagg ctgcaatcat gttggatagg 240 atcatgaggt tgttatccag ttacaatatc ttgacttgca gtgttgagac aagtaatgtt 300 aaagatggga gagttgagag gaaatatgga gcagcacctg ttgtcaagta cttgaccaag 360 aacgaggacg gggtttccat ggctgctctt ggcctcatga accaggacaa gatcctcatg 420 gagagctggt actatttgaa ggatgcggtc ttggacgggg gcatcccatt caacaaggct 480 tacggaatga gtgcattcga ataccatggc actgacccga ggttcaacaa ggtgttcaat 540 gaagggatga ggaatcactc caccatcatc accaagaaga tccttgaaac ctatacgggt 600 tttgacgatc tcaaagttct cgtcgatgtt ggaggaggag ttggagggac cattaatatg 660 atcgtcaaga aacatcctca cttaaaaggg atcaacttcg atttgcccca tgttatttca 720 gaagcagaac ctattcctgg agtggagcat gttggtggag atatgtttga gagcgtgcct 780 agtggagatg ccattttcat gaagtggatt ttgcatgact ggagcgacga gcactgccta 840 aagctactga agaactgctg gaaggctctg ccagacaatg gcaaggtgat cctgatggaa 900 agcatccttc cggaataccc tgaacctacg cccgcatcgc aaggcatgat ccacgtagac 960 atgatcatgt tggctcacaa ccccggaggg aaagagcgaa ccgcgatgga gtttgatttg 1020 ttagcgaagg aggccggatt ctcgagctcc aaggtcattt gtggatatgc tgcttcttgg 1080 gtgcttgagt tctacaaa 1098 <210> 2 <211> 366 <212> PRT <213> Lycoris aurea (L'Hér.) Herb <400> 2 Met Gly Ser Ala Gln Asn Phe Ile Thr Pro Ala Pro Gln Thr Thr Asp 1 5 10 15 Glu Glu Ala Cys Ile Phe Ala Met Gln Leu Thr Ser Phe Ser Ile Leu 20 25 30 Pro Met Thr Leu Lys Ala Ala Ile Glu Leu Asp Leu Phe Glu Ile Ile 35 40 45 Ala Lys Ala Gly Pro Asn Ala Tyr Leu Ser Pro Ser Glu Val Ala Ser 50 55 60 Gln Leu Pro Asn Asn Lys Asn Pro Gln Ala Ala Ile Met Leu Asp Arg 65 70 75 80 Ile Met Arg Leu Leu Ser Ser Tyr Asn Ile Leu Thr Cys Ser Val Glu 85 90 95 Thr Ser Asn Val Lys Asp Gly Arg Val Glu Arg Lys Tyr Gly Ala Ala 100 105 110 Pro Val Val Lys Tyr Leu Thr Lys Asn Glu Asp Gly Val Ser Met Ala 115 120 125 Ala Leu Gly Leu Met Asn Gln Asp Lys Ile Leu Met Glu Ser Trp Tyr 130 135 140 Tyr Leu Lys Asp Ala Val Leu Asp Gly Gly Ile Pro Phe Asn Lys Ala 145 150 155 160 Tyr Gly Met Ser Ala Phe Glu Tyr His Gly Thr Asp Pro Arg Phe Asn 165 170 175 Lys Val Phe Asn Glu Gly Met Arg Asn His Ser Thr Ile Ile Thr Lys 180 185 190 Lys Ile Leu Glu Thr Tyr Thr Gly Phe Asp Asp Leu Lys Val Leu Val 195 200 205 Asp Val Gly Gly Gly Val Gly Gly Thr Ile Asn Met Ile Val Lys Lys 210 215 220 His Pro His Leu Lys Gly Ile Asn Phe Asp Leu Pro His Val Ile Ser 225 230 235 240 Glu Ala Glu Pro Ile Pro Gly Val Glu His Val Gly Gly Asp Met Phe 245 250 255 Glu Ser Val Pro Ser Gly Asp Ala Ile Phe Met Lys Trp Ile Leu His 260 265 270 Asp Trp Ser Asp Glu His Cys Leu Lys Leu Leu Lys Asn Cys Trp Lys 275 280 285 Ala Leu Pro Asp Asn Gly Lys Val Ile Leu Met Glu Ser Ile Leu Pro 290 295 300 Glu Tyr Pro Glu Pro Thr Pro Ala Ser Gln Gly Met Ile His Val Asp 305 310 315 320 Met Ile Met Leu Ala His Asn Pro Gly Gly Lys Glu Arg Thr Ala Met 325 330 335 Glu Phe Asp Leu Leu Ala Lys Glu Ala Gly Phe Ser Ser Ser Lys Val 340 345 350 Ile Cys Gly Tyr Ala Ala Ser Trp Val Leu Glu Phe Tyr Lys 355 360 365 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <400> 3 catgactctg aaagcagcca tc 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <400> 4 caacaggtgc tgctccatat t 21 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <400> 5 gcaaccatcc aaagtttaac tgct 24 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <400> 6 aatgtgcaag cagggctagt aa 22 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <400> 7 cagcactagt atgggttcag cccaaaactt catca 35 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <400> 8 cgcatctaga tttgtagaac tcgataaccc aagaa 35 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <400> 9 gacaatggta ccggtatggt gaag 24 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <400> 10 catatcatcc cagttgctaa caaca 25

Claims

1. Application of Lycoris aurea LaCOMT protein-coding gene in improving plant tolerance to mercury stress, including: Overexpress the LaCOMT protein-encoding gene in plants to obtain transgenic plants; The obtained transgenic plants have enhanced resistance to mercury stress; the amino acid sequence of the LaCOMT protein is as shown in SEQ ID NO.2, and the plant is Arabidopsis thaliana.

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