CN111004790B

CN111004790B - Alfalfa WL525 polygalacturonase MsPG1, and coding gene and application thereof

Info

Publication number: CN111004790B
Application number: CN201911395408.2A
Authority: CN
Inventors: 安渊; 李姣姣; 周鹏; 苏连泰; 吕爱敏; 张钰靖
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2022-05-17
Anticipated expiration: 2039-12-30
Also published as: CN111004790A

Abstract

The invention discloses alfalfa WL525 polygalacturonase MsPG1, a coding gene and application thereof, and relates to the technical field of plant genetic engineering, wherein the polygalacturonase MsPG1 comprises amino acid sequences shown as SEQ ID NO: 2, and active fragments and active derivatives of the protein MsPG1, and provides a coding gene of the polygalacturonase MsPG1, comprising the nucleotide sequence as set forth in SEQ ID NO: 1, the nucleotide sequence of SEQ ID NO: 1, and a variant sequence capable of hybridizing to said nucleotide sequence of SEQ ID NO: 1, and (b) 1. The invention realizes the cultivation of MsPG1 transgenic plants by using a genetic engineering technology, obviously improves the aluminum toxicity resistance of the plants, provides an important theoretical basis for the cultivation and breeding of new alfalfa aluminum toxicity-resistant varieties, and has great application value.

Description

Alfalfa WL525 polygalacturonase MsPG1, and coding gene and application thereof

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to alfalfa WL525 polygalacturonase MsPG1, and a coding gene and application thereof.

Background

Alfalfa (Medicago sativa L.) is a perennial herb of the genus Medicago of the family leguminosae and is widely cultivated worldwide. The alfalfa grows rapidly, has a long green period, strong reproductive capacity and high nutritive value, is an important feed crop and plays an important role in animal husbandry production. With the reduction of rainfall, the frequent activities of human beings, the increase of soil desertification and the rapid deterioration of ecological environment, the vegetation of the grassland is seriously degraded, and the yield of the alfalfa is greatly influenced. The continuous development of animal husbandry puts higher requirements on the yield and the quality of alfalfa. Therefore, the cultivation of new alfalfa varieties with high yield and high quality is an urgent problem to be solved.

Polygalacturonase (PG) is a hydrolase and is classified into endo-Polygalacturonase (endo-PG, ec3.2.1.15) and exo-Polygalacturonase (exo-PG, ec3.2.1.67) in terms of the mode of action. Exo-polygalacturonase hydrolyzes the non-reducing end of the pectin molecule to produce galacturonic acid. But they cannot act on rhamnose residues and esterified uronic acids in the pectin molecule. Endo-polygalacturonase hydrolytically cleaves alpha (1 → 4) galactoside linkages at different sites randomly, breaking the polygalacturonic acid chain, degrading the polygalacturonic acid in the cell wall to oligogalacturonic acid and galacturonic acid. Plant Polygalacturonases (PGs) are involved in many stages of plant development, including abscission of leaves and floral organs, fruit ripening, cell expansion, stomatal movement, and the like. Overexpression or knock-out of the PG gene in plants can increase or decrease pectin content, thereby affecting cell wall morphology and structure. Although thousands of plant PG genes are available (e.g., Arabidopsis, rice, tomato, canola, etc.), functional studies on this class of enzymes are limited to a few plants. At present, no report related to the alfalfa aluminum-resistant PG coding gene sequence exists.

Therefore, those skilled in the art are devoted to develop a novel polygalacturonase which is effective in improving the stress resistance, especially the aluminum toxicity resistance, of plants, and to realize the improvement of the stress resistance of plants, especially the aluminum toxicity resistance of alfalfa and the improvement of breeding work.

Disclosure of Invention

In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is how to provide a novel polygalacturonase, which has expression characteristics such as significantly up-regulated expression in abiotic stress, especially aluminum stress, and provides a basis and theoretical basis for improving plant resistance, especially aluminum toxicity resistance of alfalfa and breeding work.

To achieve the above object, the present invention provides alfalfa WL525 polygalacturonase MsPG1, wherein polygalacturonase MsPG1 is a polypeptide having the sequence as SEQ ID NO: 2, a variant polypeptide of said polypeptide, and an active fragment or an active derivative of said polygalacturonase MsPG 1.

Further, the variant polypeptide has the same function as the polygalacturonase MsPG1, comprising: a homologous amino acid sequence polypeptide, a conservative variant, an allelic variant, a natural mutant, an induced mutant, a protein encoded by a DNA capable of hybridizing to a DNA related to the polygalacturonase MsPG1, a polypeptide or protein obtained using antisera to the polygalacturonase MsPG 1.

Further, the homologous amino acid sequence polypeptide comprises a sequence identical to the sequence SEQ ID NO: 2, a polypeptide having 1 to 50 amino acid deletions, insertions, and/or substitutions, and 1 to 20 amino acids added to the C-terminus and/or the N-terminus.

Further, the conservative variant is specifically a variant of the sequence SEQ ID NO: 2 is a polypeptide formed by 1-10 amino acid substitutions compared with the polypeptide.

The invention also provides a coding gene for the alfalfa WL525 polygalacturonase MsPG1 as described above, the coding gene comprising the nucleotide sequence as set forth in SEQ ID NO: 1, the nucleotide sequence of SEQ ID NO: 1, and a variant sequence capable of hybridizing to said nucleotide sequence SEQ ID NO: 1, and (b) 1.

Further, the degenerate sequence has a nucleotide sequence identical to the nucleotide sequence of SEQ ID NO: 1 has at least 70% homology; preferably, said degenerate sequence is identical to said nucleotide sequence SEQ ID NO: 1 has at least 90% homology.

Further, the variant sequence comprises a nucleotide sequence identical to the nucleotide sequence of SEQ ID NO: 1 is deleted, inserted and/or substituted by 1 to 90 nucleotides compared with the other 1, and a sequence formed by adding 1 to 60 nucleotides to the 5 'and/or 3' end.

The invention also provides application of the encoding gene of the alfalfa WL525 polygalacturonase MsPG1 in aluminum toxicity resistance.

Further, the application specifically includes: constructing an expression vector containing the coding gene of the polygalacturonase MsPG1, transforming a plant host, and culturing and screening to obtain a transgenic plant.

Further, the application also comprises the preparation of inhibitors and antagonists related to the polygalacturonase MsPG 1.

Compared with the prior art, the invention at least has the following beneficial technical effects:

(1) the polygalacturonase MsPG1 and the coding gene thereof have obvious up-regulation expression performance in an aluminum stress environment, and can provide a basis for culturing transgenic plants with excellent characteristics of aluminum toxicity resistance and the like;

(2) the invention successfully constructs an expression vector containing the MsPG1 gene, cultivates a tobacco and arabidopsis transgenic plant body capable of overexpressing the MsPG1 gene, obviously reduces the aluminum content in the plant body, improves the aluminum toxicity resistance of the original plant, provides an important basis for cultivating the aluminum toxicity resistance alfalfa and improving the breeding work of the aluminum toxicity resistance alfalfa, and has great application value.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a diagram illustrating the result of the evolutionary tree analysis of the alfalfa WL525MspG1 gene according to a preferred embodiment of the present invention;

FIG. 2 is a diagram showing the results of homology comparison (DNAMAN) between the nucleotide sequences of the mRNA of alfalfa WL525MspG1 gene and Medicago truncatula PG gene according to a preferred embodiment of the present invention;

FIG. 3 is a diagram illustrating the results of homology comparison (DNMAN) of the amino acid sequences of alfalfa WL525MspG1 gene and the PG genes of 4 species in accordance with a preferred embodiment of the present invention;

FIG. 4 is a statistical result of the expression level change of the alfalfa WL525MspG1 gene during Al stress according to a preferred embodiment of the present invention;

FIG. 5 is a fluorescent image of the alfalfa WL525MspG1 gene in the positioning of tobacco leaf epidermal cells, wherein A is the positioning of p35S-YFP, B is the positioning of p35S-YFP after plasmolysis, C is the positioning of p35S-MspG1-YFP, D-E is the positioning of p35S-MspG1-YFP and pm-rk co-transgenic tobacco plasmolysis, respectively, and F is the combination of D and E;

FIG. 6 is a table type observation of wild type and Arabidopsis thaliana overexpressing MsPG1 gene under Al stress according to a preferred embodiment of the present invention, wherein A is a photograph of root growth, B is statistics of root length, and C is the relative expression level of MsPG1 gene;

FIG. 7 is a table showing the phenotype observation of wild type and Arabidopsis thaliana overexpressing the MsPG1 gene under Al stress according to a preferred embodiment of the present invention, wherein A is the root relative dry weight increase and B is the leaf relative dry weight increase;

FIG. 8 is a diagram illustrating Al content determination under Al stress of wild type and Arabidopsis thaliana overexpressing the MsPG1 gene according to a preferred embodiment of the present invention, wherein A is root Al content statistics and B is leaf Al content statistics.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or as suggested by the instructions for use of the reagents.

Example 1 cloning of alfalfa MspG1 Gene

1. Obtaining plant material

Taking the leaf tissue of the alfalfa WL525 subjected to the Al treatment for extracting RNA;

extraction of RNA

Extracting total RNA by using a TransZol Up plant total RNA extraction kit, identifying the integrity of the RNA by gel electrophoresis, and determining the purity and concentration of the RNA by using a spectrophotometer (Thermo Scientific Nanodrop 1000);

3. full Length cloning of Gene

According to the nucleotide sequence of the medicago truncatula 'A17' A _27_ P130781 and the protein function annotation result, the full length of the medicago sativa WL525MspG1 gene is obtained.

The result of the A _27_ P130781 nucleic acid sequence is combined with the prediction of ORF filing (http:// www.ncbi.nlm.nih.gov/gorf) of NCBI, and the ORF reading frame of alfalfa WL525MspG1 gene is found. According to the obtained sequence, specific primers ORF-F (5'-ATGTTTCATACAATGGAGTGTTTTTTCAC-3') and ORF-R (5'-CTTGAGCAAGCAGTTTAGAATTG-3') are designed from the start codon and the stop codon respectively, PCR is carried out by taking cDNA of alfalfa WL525 as a template, and the full-length coding sequence (SEQ ID NO.1) of 1215bp alfalfa WL525MsPG1 protein is obtained through amplification. Sequencing results show that a database (GenBank) is subjected to BLAST (http:// blast.ncbi.nlm.nih.gov /) comparison at NCBI website, the homology of a nucleic acid sequence and a coding protein of the database and the known PG gene of medicago truncatula, soybean (Glycine max L.) and chickpea (Cicer arietinum L.) is very high, and the database is preliminarily considered to be the PG gene.

Example 2 sequence information and homology analysis of the alfalfa WL525MspG1 Gene

The full-length open reading frame sequence of the alfalfa WL525MspG1 is 1215bp, and the detailed sequence is shown in a sequence shown in SEQ ID NO. 1. The amino acid sequence of alfalfa WL525MspG1 protein is deduced according to the open reading frame sequence, and has 404 amino acid residues in total, the molecular weight of 43.83kDa, the isoelectric point (pI) of 9.52, and the detailed sequence shown in SEQ ID NO. 2.

The amino acid sequences of 51 PG from different plant species were aligned using the MUSCLE program in MEGA6.0, with the full amino acid sequence of the PG gene. The 51 PG amino acid sequences were from the NCBI website (http:// www.ncbi.nlm.nih.gov/genbank /). And (4) constructing the evolutionary tree by using a neighbor joint method (NJ). The main parameters are set as a distance model, Poisson model; detecting the robustness of the gene tree by adopting a bootstrap method, and repeating for 1000 times; and processing vacancy missing data, and deleting between every two data. The constructed evolutionary tree is classified according to the classification criteria of Park et al (Park et al.2010). As shown in FIG. 1, the PG gene evolutionary tree is divided into branches A to G7, and the alfalfa WL525MspG1 gene (marked by red stars) is classified as the branch F, so that it can be seen that the alfalfa WL525MspG1 gene may be a PG gene.

The open reading frame sequence of Medicago sativa WL525MsPG1 and the amino acid sequence of the protein encoded by it were subjected to nucleotide and protein homology searches at NCBI using the BLAST program, and as a result, they were found to have 97% identity at the nucleotide level to the Medicago truncatula PG1 gene (accession number: XP-003597886.1), as shown in FIG. 2, to have very high similarity at the amino acid level to the Medicago truncatula PG (accession number: XP-003597886.1), Glycine max (accession number: XP-028203424.1), chick pea (accession number: XP-004486675) and Arabidopsis thaliana (accession numbers: NM123851 and CAA20037.1) PG genes, and to have four conserved domains typical of plant PG genes, SPNTN (motif I), GDDC (motif II), CGPGHGISIGSLG (motif III) and RIK (motif IV), as shown in FIG. 3. Therefore, the alfalfa WL525MspG1 gene has high homology with PG genes of other known species in terms of nucleic acid or protein level.

Example 3 expression differences of alfalfa WL525MspG1 gene under abiotic stress

1. Obtaining of materials: and performing Al treatment on the alfalfa WL525 for 0h,1h,3h,6h,12h and 24h respectively. Wrapping the samples with aluminum-platinum paper respectively, putting the wrapped samples into liquid nitrogen, and then transferring the samples into an ultra-low temperature refrigerator at minus 80 ℃ for storage for later use;

RNA extraction, RNA integrity, purity, concentration determination and cDNA acquisition reference example 1;

3. designing specific primers to carry out Real-time fluorescent quantitative PCR analysis on the expression quantity of genes in each tissue, designing specific primers for quantitative analysis of MsPG1 genes in Real-time PCR according to the obtained alfalfa WL525MsPG1 gene sequence, wherein the specific primers are qPG1-F (5'-TCCAGGGCATGGTATCAGCA-3'), qPG1-R (5'-CAAATCCACCTCCACCCTGC-3'), an internal reference gene is an elongation factor EF-alpha gene, and the primers are EF-F (5'-GCACCAGTGCTCGATTGC-3') and EF-R (5'-TCGCCTGTCAATCTTGGTAACAA-3');

4. of the target gene and reference geneStandard curve: by ddH₂Performing gradient dilution on the standard cDNA solution, performing Real-time PCR amplification by using the diluted cDNA solution as a template and specific primers of a target gene and an internal reference gene respectively, and drawing a dissolution curve and a standard curve after the reaction is finished; analyzing the dissolution curve, and judging whether the dissolution curves of the target gene and the internal reference gene obtain a single peak or not so as to judge whether a single PCR amplification product can be obtained by using the primer or not; determining the proper dilution multiple of the template cDNA by a standard curve;

5. real-time fluorescent quantitative analysis of target genes in a sample to be detected: using the first strand of the synthesized cDNA as a template, respectively amplifying by using specific primers of a target gene and an internal reference gene for fluorescent quantitative analysis, carrying out Real-time PCR reaction on a BIO-RAD CFX Real-time quantitative apparatus with a reaction system of 20 mu L, carrying out three-step method by adopting the reaction, carrying out denaturation at 94 ℃ for 20s, and then carrying out 40 cycles: 15s at 94 ℃; 15s at 55 ℃; 25s at 72 ℃; after each amplification is finished, a dissolution curve is made to check the specificity of the amplified product;

6. by using 2^-△△CtRelative quantitative analysis is carried out by the method, and the result shows that the expression level of the alfalfa WL525MsPG1 in roots and leaves is obviously increased under various abiotic stresses (figure 4).

Example 4 alfalfa WL525MspG1 Gene transformation of the model plant Arabidopsis thaliana

1. Construction of plant expression vectors

Specific primers PG1-F (5'-GCTCTAGAATGTTTCATACAATGGAGTGTTTTTTCAC-3') and PG1-R (5'-CGAGCTC CTTGAGCAAGCAGTTTAGAATTG-3') are designed from the start codon and the stop codon respectively, Xba I and Sac I enzyme cutting sites are introduced to two sides of the full-length sequence of the gene respectively, and the alfalfa WL525cDNA is used as a template for PCR amplification. PCR products were recovered and ligated into pMD18-T Simple vector, and monoclonal plaques were picked for PCR validation. Extracting positive clone bacterial liquid plasmid. Carrying out Xba I and Sac I double enzyme digestion on the target fragment plasmid and the PHB binary transformation vector, recovering the enzyme digested PHB vector and the MspG1 fragment, connecting the fragments at 16 ℃ overnight by using T4 ligase, and transforming the vector into agrobacterium GV 3101.

2. Transformation of Arabidopsis thaliana

(1) Pre-shaking agrobacterium: selecting positive monoclonal antibody to 5ml YEP liquid culture medium containing 50mg/L Kan, 50mg/L gentamicin and 25mg/L Rif, shaking bacteria at 28 deg.C and 200rpm for 24 h;

(2) expanding and culturing agrobacterium: expanding and culturing the pre-shaken agrobacterium liquid into a YEP culture medium containing the same resistance at a ratio of 1:100, culturing for 13-16h at 28 ℃ and 200rpm until the absorbance OD is obtained₆₀₀Collecting bacteria at 18 deg.C, 3500rpm, 15min to 0.6-1.5;

(3) transformation of Arabidopsis thaliana: (all the siliques and full-bloomed and white flowers on the plants are cut off before transformation) 300mL of 1/2MS solution containing 2% of sucrose is prepared, a small amount of MS solution is used for resuspending the collected agrobacterium, 0.01% (v/v) of Tween-20 is added into the residual sucrose solution, the stems and inflorescences of the plants are soaked in bacterial liquid for 5mins, the bacterial liquid is taken out and drained, and the bacterial liquid is placed into a disposable plastic bag for sealing and moisture preservation. After all the plants are transformed, covering a black box, and culturing for 24 hours in a dark place. Then taking out the plants, vertically placing the plants, watering to ensure that the plants have sufficient water.

3. Screening of transgenic Positive lines

After the transformed Arabidopsis thaliana is completely matured, the seeds are harvested, dried, filtered by a 50-mesh stainless steel sieve, the siliques are removed, and the transgenic T is collected₀Seeding in a plug tray, and performing seedling resistance screening with 0.05% (v/v) Basta to obtain T₁Transgenic plants are generated and continuously screened until T is obtained₃The generation of homozygous transgenic plants.

Example 5 alfalfa MspG1 tobacco leaf subcellular localization analysis

The identified GV3101 strain was inoculated into 5mL YEP (containing Kan 50mg/L) and shake-cultured at 28 ℃ to OD₆₀₀About 0.5; adding 1mL of the bacterial liquid into 50mL of YEP liquid culture medium, and performing shake culture at 28 ℃ until OD₆₀₀About 0.5; taking 5mL of bacterial liquid, and centrifuging at 4000rpm/min for 10 min; suspending the cells to OD with MS liquid Medium₆₀₀About 0.6, adding AS (final concentration of 0.2mM) and MES (final concentration of 10mM), and standing at room temperature for 3 hrs; uniformly mixing the P19 with the MS suspended bacterial liquid in a volume of 1: 1; injecting tobacco leaf, and standing in shade for 48 hrs; infecting with AgrobacteriumThe tobacco leaves are placed under a laser confocal microscope for microscopic observation. And (3) setting p35S-YFP transformed tobacco as a negative control, setting a membrane-positioned pm-rk vector as a positive control, and treating with 1M mannitol for 5 minutes to perform plasmolysis. FIG. 5 is a schematic illustration of the p35S-YFP alignment of FIG. 5A; FIG. 5B shows the p35S-YFP plasmolysis positioning; FIG. 5C shows the positioning of p35S-MspG 1-YFP; FIGS. 5D-F show the p35S-MspG1-YFP (FIG. 5D) and pm-rk (FIG. 5E) co-transformed tobacco plasmodesmata after separation and FIG. 5F shows the combination of FIGS. 5D and 5E. The results show that the MspG1 gene of alfalfa WL525 is localized on the cell membrane.

Example 6 Al stress analysis of Arabidopsis plants overexpressing MspG1

1. Analysis of expression difference of over-expressed MsPG1 in Arabidopsis plants

1.1. Obtaining of materials: wild-type and transgenic MspG1 Arabidopsis lines (OX1 and OX2) T₃Seed disinfection was performed by dibbling on MS medium (pH5.8, 1% sucrose) and culturing for about 10 days to obtain the material. Wrapping the samples with aluminum-platinum paper respectively, putting the wrapped samples into liquid nitrogen, and then transferring the samples into an ultra-low temperature refrigerator at minus 80 ℃ for storage for later use;

RNA extraction, determination of RNA integrity, purity and concentration and cDNA acquisition reference example 1;

1.3. designing a standard curve of a specific primer, a target gene and an internal reference gene, and referring to example 3 for real-time fluorescent quantitative analysis of the target gene in a sample to be detected;

1.4. by using 2^-△△CtThe results of relative quantitative analysis by the method are shown in FIG. 6C, which shows that the expression level of MsPG1 gene in transgenic Arabidopsis strains (OX1 and OX2) is increased, and MsPG1 gene is successfully transferred into Arabidopsis.

2. Transgenic Arabidopsis Al stress phenotype analysis

Wild type and transgenic Arabidopsis lines (OX1 and OX2) T₃Seed Disinfection was separately spotted on MS medium (pH5.8, 1% sucrose) and when cultured at approximately 2cm root length, transferred to CaCl containing 0, 30 and 50. mu.M Al₂The medium was treated for 24h, photographed (as shown in FIG. 6A), and the root length statistics were measured with a ruler (as shown in FIG. 6B). The result shows that the Arabidopsis plant over-expressing MspG1 is stressed by AlThe root length is significantly higher than the wild type.

3. Analysis of relative Dry weight growth of roots and leaves after Al stress in transgenic Arabidopsis

Mixing wild type and T₃Seed Disinfection was individually spotted on MS medium (pH5.8, 1% sucrose) and when cultured at about 2cm root length, transferred to CaCl containing 50. mu.M Al₂Treating in culture medium for 24h, cutting off root and leaf in control group and experimental group, deactivating enzyme at 80 deg.C for 2h, drying at 60 deg.C until the mass is constant, and measuring weight by balance. Relative dry weight gain (dry weight after treatment-dry weight before treatment)/dry weight before treatment, as shown in fig. 7, the root relative dry weight gain of two lines (OX1 and OX2) of transgenic arabidopsis thaliana was higher after aluminum treatment than that of the wild type (fig. 7A), the leaf relative dry weight gain was not significantly different between the aluminum treated and the wild type (fig. 7B), and the results combined with the above fig. 6 show that the over-expressed MsPG1 can improve the aluminum toxicity resistance of plants in arabidopsis thaliana plants.

Example 7 analysis of Al content in roots and leaves of Arabidopsis plants overexpressing MspG1 after Al stress

Mixing wild type and T₃Seed Disinfection was individually spotted on MS medium (pH5.8, 1% sucrose) and when cultured at about 2cm root length, transferred to CaCl containing 50. mu.M Al₂Treating in culture medium for 24h, cutting root and leaf of control group and experimental group, deactivating enzyme at 80 deg.C for 2h, oven drying at 60 deg.C until the mass is constant, weighing dry weight, placing into digestion tube, adding 2ml of 4:1 HNO₃:HClO₄Digesting the mixed solution at 60 ℃ for 2h, digesting at 120 ℃ until the digested solution is colorless, clear and transparent, diluting with double distilled water to a constant volume of 10ml, and measuring the aluminum content in the solution by ICP. The results show (as shown in fig. 8) that the content of Al in roots (fig. 8A) and leaves (fig. 8B) of an overexpression MsPG1 arabidopsis plant is significantly lower than that of the wild type, which indicates that the overexpression of the MsPG1 gene in arabidopsis plants can reduce the content of aluminum in the plant body and improve the aluminum toxicity resistance of the plants.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Sequence listing

<110> Shanghai university of transportation

<120> alfalfa WL525 polygalacturonase MsPG1, and coding gene and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1215

<212> DNA

<213> alfalfa (Medicago sativa)

<400> 1

atgtttcata caatggagtg ttttttcaca ttgttaatga ttctaagtat gtttaaactc 60

ggattgagta gcacaacaac tttcaatgta ttgcaatatg gagcagttgg agatggaaaa 120

accaatgatt ctcctgcttt tctgaaagca tggaaagatg tgtgtcaaag caaatcaggc 180

acatctcgtc tcatcatacc agcagccaag acattcttac tgaggccaat agctttccgt 240

ggtccatgca aatctaatta tatttacatt gagctttcag ggaatattgc tgcacctaaa 300

acaaaatcgg aatattcagg atcccaaata aacacatgga ttggtttctc atttgtgaat 360

ggtttgatta taagtggaaa agggactatt gatggtcgag gctctacgtg gtggaaacaa 420

ccttgcatag gaaatccacc acccgggaca agttgccgtc caccaacagc aataacattg 480

aatagatgct atcgatttca aataaaagga tatacaagtg ttaatccagc aagaagccat 540

ataactctaa caagctgcaa aaagggcatt atctctaaca tccgtttaat tgctcctgga 600

gagagcccta atactgatgg cattgatatt tctgcctcaa gagacattca agtgcttaac 660

tctttcattg caactggtga tgattgcatt gctattagtg ctggatcatc agtaatcaaa 720

ataactgcta taacatgtgg tccagggcat ggtatcagca ttggatcatt gggggcacgt 780

ggagacaccg atattgtaga ggatgtgcat gtgaagaatt gtacgttgac tgaaacatta 840

actggagtaa gaatcaagac aaagcagggt ggaggtggat ttgctaggag gatcacgttt 900

gaaaatatta gatttgttcg agcccataac cccattatga ttgaccaatt ttattgtgtt 960

aatcaaatgg tctgcaaaaa catgactaaa gcaatcaaag tgagtgatgt aacatataga 1020

gggattagtg gaacatcatt aacagacaag gcaataaatt tgaactgcga ccaaaatgtg 1080

gggtgctcta accttgtatt tgatcgtgtt tatgtaagat ctgctgttcc taagatgaag 1140

gttttctcct tctgccataa tgctcatgga agagcctcac acaccaaacc aattctaaac 1200

tgcttgctca agtag 1215

<210> 2

<211> 404

<212> PRT

<213> alfalfa (Medicago sativa)

<400> 2

Met Phe His Thr Met Glu Cys Phe Phe Thr Leu Leu Met Ile Leu Ser

1 5 10 15

Met Phe Lys Leu Gly Leu Ser Ser Thr Thr Thr Phe Asn Val Leu Gln

20 25 30

Tyr Gly Ala Val Gly Asp Gly Lys Thr Asn Asp Ser Pro Ala Phe Leu

35 40 45

Lys Ala Trp Lys Asp Val Cys Gln Ser Lys Ser Gly Thr Ser Arg Leu

50 55 60

Ile Ile Pro Ala Ala Lys Thr Phe Leu Leu Arg Pro Ile Ala Phe Arg

65 70 75 80

Gly Pro Cys Lys Ser Asn Tyr Ile Tyr Ile Glu Leu Ser Gly Asn Ile

85 90 95

Ala Ala Pro Lys Thr Lys Ser Glu Tyr Ser Gly Ser Gln Ile Asn Thr

100 105 110

Trp Ile Gly Phe Ser Phe Val Asn Gly Leu Ile Ile Ser Gly Lys Gly

115 120 125

Thr Ile Asp Gly Arg Gly Ser Thr Trp Trp Lys Gln Pro Cys Ile Gly

130 135 140

Asn Pro Pro Pro Gly Thr Ser Cys Arg Pro Pro Thr Ala Ile Thr Leu

145 150 155 160

Asn Arg Cys Tyr Arg Phe Gln Ile Lys Gly Tyr Thr Ser Val Asn Pro

165 170 175

Ala Arg Ser His Ile Thr Leu Thr Ser Cys Lys Lys Gly Ile Ile Ser

180 185 190

Asn Ile Arg Leu Ile Ala Pro Gly Glu Ser Pro Asn Thr Asp Gly Ile

195 200 205

Asp Ile Ser Ala Ser Arg Asp Ile Gln Val Leu Asn Ser Phe Ile Ala

210 215 220

Thr Gly Asp Asp Cys Ile Ala Ile Ser Ala Gly Ser Ser Val Ile Lys

225 230 235 240

Ile Thr Ala Ile Thr Cys Gly Pro Gly His Gly Ile Ser Ile Gly Ser

245 250 255

Leu Gly Ala Arg Gly Asp Thr Asp Ile Val Glu Asp Val His Val Lys

260 265 270

Asn Cys Thr Leu Thr Glu Thr Leu Thr Gly Val Arg Ile Lys Thr Lys

275 280 285

Gln Gly Gly Gly Gly Phe Ala Arg Arg Ile Thr Phe Glu Asn Ile Arg

290 295 300

Phe Val Arg Ala His Asn Pro Ile Met Ile Asp Gln Phe Tyr Cys Val

305 310 315 320

Asn Gln Met Val Cys Lys Asn Met Thr Lys Ala Ile Lys Val Ser Asp

325 330 335

Val Thr Tyr Arg Gly Ile Ser Gly Thr Ser Leu Thr Asp Lys Ala Ile

340 345 350

Asn Leu Asn Cys Asp Gln Asn Val Gly Cys Ser Asn Leu Val Phe Asp

355 360 365

Arg Val Tyr Val Arg Ser Ala Val Pro Lys Met Lys Val Phe Ser Phe

370 375 380

Cys His Asn Ala His Gly Arg Ala Ser His Thr Lys Pro Ile Leu Asn

385 390 395 400

Cys Leu Leu Lys

<210> 3

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

atgtttcata caatggagtg ttttttcac 29

<210> 4

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

cttgagcaag cagtttagaa ttg 23

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

tccagggcat ggtatcagca 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

caaatccacc tccaccctgc 20

<210> 7

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

gcaccagtgc tcgattgc 18

<210> 8

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

tcgcctgtca atcttggtaa caa 23

<210> 9

<211> 37

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

gctctagaat gtttcataca atggagtgtt ttttcac 37

<210> 10

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

cgagctcctt gagcaagcag tttagaattg 30

Claims

1. Alfalfa WL525 polygalacturonase MsPG1, wherein polygalacturonase MsPG1 is a polypeptide having the sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

2. A gene encoding alfalfa WL525 polygalacturonase MsPG1 according to claim 1, characterized by the amino acid sequence of SEQ ID NO: 1, which is identical to the degenerate sequence of SEQ ID NO: 1 has at least 70% homology.

3. The gene of alfalfa WL525 polygalacturonase MsPG1 of claim 2, having the nucleotide sequence of SEQ ID NO: 1.

4. use of the alfalfa WL525 polygalacturonase MsPG1 of claim 1 for resisting aluminum toxicity.

5. The use of the gene encoding alfalfa WL525 polygalacturonase MsPG1 according to claim 4 for aluminum toxicity resistance, in particular comprising: constructing an expression vector containing the coding gene of the polygalacturonase MsPG1, transforming a plant host, and culturing and screening to obtain a transgenic plant.

6. The use of the alfalfa WL525 polygalacturonase MsPG1 gene encoding an aluminum toxin resistance according to claim 5, further comprising the preparation of an inhibitor or antagonist related to the polygalacturonase MsPG 1.