CN101597609A - Monstera lectin gene and its encoded protein and application - Google Patents

Abstract

The invention discloses a monstera lectin gene, which has the nucleotide sequence shown in SEQ ID NO.1, and the monstera lectin gene encoded by SEQ ID NO.1 or having the amino acid sequence shown in SEQ ID NO.2 Bamboo Lectin Protein. The present invention also discloses the application of the above-mentioned monstera lectin gene in eukaryotic expression in tobacco and in improving insect resistance in transgenic plants, which significantly improves the insect resistance of plants and can be used for insect-resistant varieties of crops improved.

Description

Monstera lectin gene and its encoded protein and application

technical field

The invention belongs to the field of biological technology, and in particular relates to the gene and protein of the monstera lectin expressed in the monstera and the application thereof.

Background technique

Insect pests are one of the important factors causing crop yield reduction. Pests not only cause direct damage to crops, but also cause indirect damage by carrying various plant pathogens. All over the world, the annual direct loss caused by various pests accounts for more than 13% of the total harvest of crops (Gatehouse et al., 1992), and the annual loss is about hundreds of billions of dollars. Insect-resistant genes for Homopteran pests (such as aphids, brown planthoppers, and leafhoppers, which are very serious worldwide) have rarely been isolated. Therefore, the isolation and cloning of such resistance genes for molecular breeding is of great significance for reducing the losses caused by pests and diseases, ensuring the stable increase of grain production and the improvement of hygienic quality (Kai et al., 2003). In recent years, the research on the application of lectins in biology has developed rapidly. Its biggest feature is that it can recognize sugars and sugars. Each lectin has the ability to specifically bind to a special carbohydrate. And some lectin genes such as snowdrop lectin (GNA) have shown important application value in improving crop resistance to Homoptera pests.

In the analysis of the existing literature, although "European Journal of Biochemistry (European Biochemistry Journal) 1991: 202: 23-30" and "DNA sequence (nucleotide sequence), 2003: 14: 223-226" etc. It has been reported that the monocotyledonous mannose-binding lectin gene was isolated and cloned from snowdrops and leeks, but there has not been any literature report on the isolation of the monocotyledonous mannose-binding lectin gene from Monstera deliciosa. Reports of documents closely related to the subject matter of the present invention were found.

Contents of the invention

technical problem to be solved

The technical problem to be solved by the present invention is to provide a Monstera lectin gene, its encoded protein and its use, so as to fill the blank that there is no literature report on the isolation of monocotyledon mannose-binding lectin gene from Monstera, and to use The transgenic method overcomes the defect that plants have poor resistance to homopteran pests such as aphids. A monstera lectin gene and its coding protein sequence are provided. A fusion gene construct comprising the gene, a new recombinant expression vector carrying the construct, a plant cell transformed with the expression vector, and transgenic plants of the gene produced by the transformed cell and their progeny (including plant seeds and plant tissues) to obtain transgenic plants with enhanced resistance to plant diseases and insect pests.

Technical solutions

One of the technical solutions of the present invention is to provide a monstera lectin gene, which has the nucleotide sequence shown in SEQ ID NO.1, or a homologous sequence with addition, substitution, insertion or deletion of one or more nucleotides , or its alleles and their derived nucleotide sequences.

The second technical solution of the present invention is to provide a monstera lectin protein encoded by the above gene sequence. Its preferred scheme is that it has the amino acid sequence shown in SEQ ID NO.2.

The third technical solution of the present invention is to provide a plasmid containing the whole sequence or a partial fragment of the monstera lectin gene of one of the above technical solutions.

The fourth technical solution of the present invention is to provide a plant expression vector containing the whole sequence or a partial fragment of the monstera lectin gene of one of the above technical solutions.

The fifth technical solution of the present invention is to provide a host cell, which contains the gene sequence described in any one of the above-mentioned technical solutions, third or fourth. Preferably said cells are E. coli cells, or preferably Agrobacterium cells, or preferably tobacco cells.

The sixth technical solution of the present invention is to provide an application of the monstera lectin gene, including transforming tobacco cells with the plant expression vector described in the fourth technical solution, or co-cultivating the above-mentioned Agrobacterium and tobacco cells, or using The tobacco cell of claim 9 for cultivating tobacco plants. Alternatively, the sixth technical solution may also be to provide a transgenic tobacco containing the above-mentioned monstera lectin gene sequence.

Specifically, the definitions of the concepts involved in the technical solution of the present invention are as follows:

The DNA molecule of the monstera lectin gene in the present invention includes: a nucleotide sequence encoding a polypeptide having the activity of the monstera lectin protein, and the nucleotide sequence is the same as SEQ ID NO.1 from The nucleotide sequence at the 49th-900th nucleotide sequence has at least 70% homology; or the nucleotide sequence can be obtained from the nucleotide sequence at 40-55°C with The nucleotide sequence at position 49-900 is hybridized. Preferably, the sequence encodes a polypeptide having the amino acid sequence shown in SEQ ID NO.2. More preferably, said sequence has the nucleotide sequence from nucleotide 49-900 in SEQ ID NO.1.

The monstera lectin protein polypeptide isolated in the present invention includes: a polypeptide having the amino acid sequence of SEQ ID NO.2, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof. Preferably, the polypeptide is a polypeptide having the sequence of SEQ ID NO.2.

The DNA molecule of the present invention comprises 8-100 consecutive nucleotides in the DNA molecule.

In the present invention, "isolated" and "purified" DNA means that the DNA or fragment has been separated from the sequences on both sides of it in the natural state, and also means that the DNA or fragment has been combined with the accompanying nucleic acid in the natural state. components and have been separated from the proteins that accompany it in the cell.

In the present invention, the term "the gene encoding the monstera lectin protein (or polypeptide)" refers to: the nucleotide sequence encoding the polypeptide having the activity of the monstera lectin protein, such as No. 49- in SEQ ID NO.1 The 900 nucleotide sequence and its degenerate sequence. The degenerate sequence refers to the sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid in the nucleotides 49-900 of the coding frame of the sequence of SEQ ID NO.1 . Due to the degeneracy of codons, a degenerate sequence with as low as about 70% homology with the 49-900 nucleotide sequence in SEQ ID NO.1 can also encode the sequence described in SEQ ID NO.2.

The term also includes a nucleotide sequence capable of hybridizing to the nucleotide sequence from nucleotides 49 to 900 in SEQ ID NO.1 under moderately stringent conditions, preferably under highly stringent conditions. Also include at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% homology with the nucleotide sequence from nucleotide 49-900 in SEQ ID NO.1 % of nucleotide sequences. The term also includes variants of the open reading frame sequence of SEQ ID NO. 1 that encode a protein having the same function as the native monstera lectin protein. These variations include (but are not limited to): the deletion of several (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) nucleotides , insertion and/or substitution, and addition of several (usually within 60, preferably within 30, more preferably within 10, and most preferably within 5) at the 5' and/or 3' end ) nucleotides.

In the present invention, the term "monstera lectin protein or polypeptide" refers to the polypeptide having the sequence of SEQ ID NO.2 having the activity of monstera lectin protein. The term also includes variant forms of the sequence of SEQ ID NO. 2 that have the same function as the natural monstera lectin protein. These variations include (but are not limited to): deletions and insertions of several (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of monstera lectin protein, and derivatives that can be operably linked to signal peptides, promoters or ribosome binding site sequences.

Variations of the Monstera lectin protein polypeptide of the present invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and monstera lectin protein polypeptides that can be combined with Monstera under high or low stringent conditions The protein encoded by the DNA hybridized with the lectin protein DNA, and the polypeptide or protein obtained by using the serum of the monstera lectin protein polypeptide.

In the present invention, the term "Monstera lectin protein conservative variant polypeptide" refers to: compared with the amino acid sequence of SEQ ID NO.2, there are at most 10, preferably at most 8, more preferably at most 5 amino acids Similar or similar amino acids are replaced to form polypeptides. These conservative variant polypeptides are preferably produced by substitutions according to Table 1.

Table 1. Substituted residues in conservative variant polypeptides

initial residue representative replacement preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu

Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

The invention also includes analogs of the monstera lectin protein or polypeptide. The difference between these analogs and the natural lectin polypeptide may be the difference in the amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, Y-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their proteolytic properties or to optimize their solubility properties.

In the present invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids, cosmids and the like. When producing the monstera lectin protein of the present invention, the nucleic acid sequence of the gene encoding the monstera lectin protein can be operably linked to the expression control sequence, thereby forming a monstera lectin protein expression vector.

"Operably linked" as used herein refers to the condition that certain parts of a linear DNA sequence are capable of affecting the activity of other parts of the same linear DNA sequence. For example, a signal peptide (secretion leader) DNA is operably linked to a polypeptide DNA if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide; if a promoter controls the transcription of the sequence, it is operably linked to A coding sequence; a ribosome binding site is operably linked to a coding sequence if it is placed in a position to enable its translation. Generally, "operably linked to" means adjacent, and with respect to a secretory leader it means adjacent in reading frame.

In the present invention, the host cells are prokaryotic cells or eukaryotic cells. Commonly used prokaryotic host cells include Escherichia coli; commonly used eukaryotic host cells include yeast cells, tobacco cells and other plant cells.

The expression of the gene product encoding the monstera lectin protein can also be analyzed by Northern blot technique, that is, the presence and quantity of the RNA transcript of the monstera lectin gene in the cells can be analyzed.

In addition, the nucleic acid molecules that can be used as probes in the present invention usually have 8-100 consecutive nucleotides of the sequence of the monstera agglutinin nucleotide coding region, preferably 15-50 consecutive nucleotides acid. The probe can be used to detect whether there is a nucleic acid molecule encoding monstera lectin protein in a sample.

The present invention relates to a method for detecting whether there is a nucleotide sequence of monstera lectin protein in a sample, which comprises using the above-mentioned probe to hybridize with the sample, and then detecting whether the probe is combined. Preferably, the sample is a product of PCR amplification, wherein the PCR amplification primers correspond to the nucleotide coding sequence of the monstera lectin protein, and can be located on both sides or in the middle of the coding sequence. Primers are generally 15-50 nucleotides in length.

In addition, according to the nucleotide sequence and amino acid sequence of the monstera lectin of the present invention, the source gene or homologous protein of the monstera lectin protein can be screened on the basis of nucleic acid homology or homology of expressed proteins.

In order to obtain a locus of Monstera cDNAs related to the Monstera lectin gene, the Monstera cDNA library can be screened with DNA probes that use ³² P for Monstera lectin under low stringency conditions. All or part of the gene sequence is radioactively labeled. The most suitable cDNA library for screening is that from Monstera. Methods for constructing cDNA libraries from cells or tissues of interest are well known in the art of molecular biology. In addition, many such cDNA libraries are commercially available, eg, from Clontech, Stratagene, Palo Alto, Cal. This screening method can identify nucleotide sequences related to the monstera lectin gene family.

The monstera lectin nucleotide full-length sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

In addition to recombinant production, fragments of the proteins of the invention can also be produced by direct peptide synthesis using solid-phase techniques (Stewart et al., (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco; Merrifield J . (1963) J. Am Chem. Soc 85:2149-2154). Protein synthesis in vitro can be performed manually or automatically. For example, peptides can be synthesized automatically using an Applied Biosystems Model 431A Peptide Synthesizer (Foster City, CA). Fragments of a protein of the invention can be chemically synthesized separately and then chemically linked to produce a full-length molecule. Utilizing the monstera lectin protein of the present invention, substances, or receptors, inhibitors or antagonists, etc. that interact with the monstera lectin protein can be screened out through various conventional screening methods.

Beneficial effect

The Monstera monocotyledon mannose-binding lectin gene provided by the present invention is cloned and prepared from Monstera for the first time, and can be used to improve the resistance of plants to Homopteran pests such as aphids through genetic engineering techniques. The results show that the transgenic Tobacco has an obvious role in the insect resistance test, and it helps to protect the healthy growth of plants. In particular, many crops have the problem of heavy insect damage and poor quality in many areas. Improving the insect resistance of crops is an effective method to solve this problem, reduce the hazards of pesticide residues, reduce agricultural investment, and improve planting benefits. The lectin of the present invention Genes have extensive application value in the improvement of crop resistance to insects, disease and antibacterial.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as molecular cloning such as Sambrook: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggestion conditions of.

Example 1

Cloning of the Gene Encoding the Monstera Lectin Protein

1. Tissue separation (isolation)

The young leaves of Monstera come from Shanghai. After harvesting the materials, they are immediately frozen in liquid nitrogen.

2. RNA isolation (RNA isolation)

Take part of the tissue, grind it with a mortar, add it into a 1.5mL EP tube filled with lysate, shake it fully, and then transfer it into a glass homogenizer. After homogenization, transfer to 1.5mL EP tube, and extract total RNA (TRIzolReagents, GIBCO BRL, USA). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

3. Full-length cloning of the gene (Cloning of Full-Iength cDNA)

According to the conserved amino acid sequence of snowdrops and other monocot mannose lectins, degenerate primers were designed, and using the principle of homologous gene cloning, the Smart-RACE method (Clonetech kit) was used for full-length cDNA cloning, which was carried out in three stages:

(1) 3′-RACE

MDAF2' (0.6kb) was obtained by PCR (UPM+F2), recovered, connected to the T-Easy vector, using SP6 or T7 as a universal primer, and using the method of terminator fluorescent labeling (Big-Dye, Perkin-Elmer, USA) , were sequenced on an ABI 377 sequencer (Perkin-Elmer, USA). The sequencing results searched the existing database (Genebank+EMBL) with BLAST and FASTA software in the GCG software package (Wisconsin group, USA), to know its nucleic acid sequence and encoded protein and known monocotyledonous mannose lectin gene (such as leek lotus It has high homology with snowdrop lectin, etc., so it is preliminarily considered to be a lectin gene.

(2) 5′-RACE

According to the result of 3' RACE, the reverse specific primer R2 was designed, and MDAR2' (0.75kb) was obtained by PCR (UPM+R2) (the process was the same as (1)). Recovery, connected to the T-Easy vector, using SP6 or T7 as a universal primer, using the method of terminator fluorescent labeling (Big-Dye, Perkin-Elmer, USA), on the ABI 377 sequencer (Perkin-Elmer, USA) Perform sequencing. The sequencing results were compared with the 3'RACE results and spliced to obtain the full-length fragment sequence.

(3) Sequence and assemble the 5'RACE sequencing results with the 3'RACE sequencing results to obtain the full-length fragment sequence information, and design a pair of specific primers for PCR amplification of the MDA coding region (MDAKF1+MDAKR1) to obtain the MDA coding region (0.852kb) (the process is the same as (1)).

The results of BLAST and molecular modeling proved that the newly obtained gene from Monstera is indeed a plant lectin gene. Since the known homologous agglutinin (GNA) gene has a strong resistance to Homoptera insects, including aphids and planthoppers (Hilder et al., 1995; Powell et al., 1993, 1995;), it is speculated that this gene has the same Function.

By using the above three methods in combination, the nucleic acid sequence of the full-length coding gene of the candidate monstera t-lectin protein was obtained. On the basis of splicing to obtain the full length (including at least the complete open reading frame), the primer MDAF1: 5'-TAGCAATAGTATTGTACACTAAC-3' (SEQ ID NO.3) was further designed as the forward primer, and the oligonucleotide MDAR1: 5' -GGGGAAATGTTTCTAACGTTC-3'(SEQID NO.4) was used as reverse primer, and total RNA was used as template to carry out RT-PCR amplification. 35 cycles of 1 minute at 72°C and 2 minutes at 72°C, with a final extension at 72°C for 10 minutes. The PCR amplification product was detected by electrophoresis, and the length of the amplified fragment obtained was 1186bp. Then, the PCR amplification product was cloned and sequenced according to conventional methods to obtain the sequence shown in SEQ ID NO.1.

Example 2

Sequence Information and Homology Analysis of Monstera Lectin Gene

The length of the novel monstera lectin full-length cDNA of the present invention is 1186 bp, and the detailed sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at nucleotides 49-900. The amino acid sequence of monstera lectin was deduced according to the full-length cDNA, which has a total of 283 amino acid residues, a molecular weight of 31.656KD, and a pl of 8.34. See SEQ ID NO.2 for the detailed sequence.

The full-length cDNA sequence of monstera lectin and its encoded protein were compared with the nucleotides in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR databases using BLAST program The protein homology search showed that it had a certain homology with the gene of galactanus agglutinin (GNA). At the amino acid level, it has 36% identity and 52% similarity with the 14th-142nd amino acid residues of snowdrop lectin (GenBank Accession No. L07475) (see Table 2). It can be seen from the above that there is a high homology between the monstera lectin gene and the snowdrop lectin gene at the protein level, so it can be considered that the lectin also has a similar effect on improving the insect resistance of plants.

Table 2. Homologous comparison (FASTA) table of the amino acid sequences of the monstera lectin protein of the present invention and the snowdrop lectin

Query 14 LVLLHAVLLALLTPARAGEQVLIANRTLYAGQSLYSMNRNYRFIMQWDCSLALYEFQKPL 73

L++L + L ++TP+ E +L + TL G SL S ++ FIMQ DC+L LY KP+

Sbjct 6 LLILTTIFLGVITPSCLSENILYSGETLPTGGSLTS--GSFVFIMQQDCNLVLYNVDKPI 63

Query 74 WISTTDRQPGTFCDATLDAGSARLAVTQRNTNKQVWSSTTSFYAFKYVLVLQTDLNVVIY 133

W + T G D ++ + V +NK +W+S T YV +LQ D NVVIY

Sbjct 64 WATNTG---GLSSDCSISLQTDGDLVVYTPSNKPIWASNTDGQNGNYVCILQKDRNVVIY 120

Query 134 SRPVWSTNT 142

W+T T

Sbjct 121 GTNRWATGT 129

Among them, Query represents the amino acid sequence of Monstera m-lectin protein; Sbict represents the amino acid sequence of Snowdrop g-Lectin protein (GenBank Accession No.AAL07475); the same amino acid is marked with a single character of amino acid between the two sequences.

Example 3

Structural and functional studies of Monstera md-lectin protein:

1. Precursor protein and mature protein analysis of the amino acid sequence of Monstera md-lectin protein. Through the comparative analysis of the nucleotide sequence and amino acid sequence of Monstera md-lectin protein and other plant lectins, it is found that the Monstera md-lectin protein gene encodes a precursor protein: containing signal peptide sequence, mature protein sequence, According to the rules of van Heijne (1986) et al. to predict the signal peptide of lectin protein and compare the md-lectin protein of Monstera with other plant lectins such as GNA protein, the signal peptide cleavage position of md-lectin protein of Monstera was identified The point is between the 30th amino acid (A) and the 31st amino acid (G), which is also consistent with most known lectins that specifically bind mannose, such as snowdrop lectin (GNA, Van Damme et al., 1991) , Clivia lectin (Van Damme et al., 1994) and other signal peptide cleavage sites of lectin proteins.

2. Specific binding glycosylation analysis of Monstera md-lectin protein. Through the specific binding glycosylation analysis of Monstera md-lectin protein and other plant lectin proteins such as GNA (GNA), Clivia lectin, Narcissus lectin, etc., it was found that Monstera md-lectin protein is the same as Most mannose-specific binding lectins, such as galanthus agglutinin (GNA), have two typical mannose-specific binding site cassettes (QDNY), as shown in Table 3. Therefore, it is proved that the monstera md-lectin protein is also similar to galanthus agglutinin (GNA), which is a lectin protein specifically binding to mannose.

Table 3. Monstera md-lectin protein mannose-specific binding site box

(The mannose-specific binding site and QDNY are boxed in the table)

MAATSLLFSA APLLVLLHAV LLALLTPARA GEQVLIANRT LYAGQSLYSM NRNYRFIMQW 60

DCSLALYEFQ KPLWISTTDR QPGTFCDATL DAGSARLAVT QRNTNKQVWS STTSFYAFKY 120

L

V VI

SRPVWST NTGVWDSVEF QNGDQILLSS GTLKVGQYLS NNELNFRFIM180VLVL T

L

V VI

SRPVWST NTGVWDSVEF QNGDQILLSS GTLKVGQYLS NNELNFRFIM180

QDDCDLVLYD DDRRIWSSET SGKGTGCNAK LNGLTGQLIV RDNNGRIVWS SKNRAVPIKK 240

P

RVVV

TGRVWA SDTSIPENQG GLQDAGRIEM VTN                   283HILLL

P

R VVV

TGRVWA SDTSIPENQG GLQDAGRIEM VTN 283

Example 4

Prokaryotic Expression and Purification of Monstera Lectin Protein or Polypeptide in Escherichia coli

In this example, the full-length Monstera md-lectin coding sequence or fragment was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

The monstera md-lectin polypeptide was prokaryotically expressed in Escherichia coli in the form of fusion protein.

Construction of prokaryotic expression vector and transformation of Escherichia coli

According to the amino acid sequence of Monstera md-lectin, design primers to amplify the protein coding region after excising the signal peptide, and introduce restriction endonuclease sites on the forward and reverse primers (this is based on the selected pET32a(+) vectors) in order to construct expression vectors. Using the amplification product obtained in Example 1 as a template, after PCR amplification, the md-lectin gene of Monstera bamboo was cloned into the pET32a(+) vector (Novagen) under the premise of ensuring the correct reading frame. The identified expression vector was transformed into Escherichia coli BL21 by the CaCl ₂ method, and the engineering strain BL21-pET32a(+)-md-lectin containing the pET32a(+)-md-lectin expression vector was screened and identified.

Isolation and Identification of Engineering Bacteria Expressing Trx-md-lectin Recombinant Protein

Pick a single colony of BL21-pET32a(+)-md-lectin engineering bacteria and shake it overnight in 3ml LB medium containing 100μg/ml ampicillin, then pipette the culture solution into a new LB medium at a concentration of 1:100 (containing 100 μg/ml ampicillin) and cultured for about 3 hours until the OD ₆₀₀ reached 0.5, then added IPTG to a final concentration of 1 mmol/L and continued to culture at 37°C for 0, 1, 2 and 3 hours respectively. Centrifuge 1ml of bacterial solution with different culture time, add lysate (50μl of 2×SDS loading buffer, 45μl of distilled water, 5μl of dimercaptoethanol) to the bacterial sediment, suspend the bacterial sediment, boil in boiling water bath for 5 minutes, 10000rpm Centrifuge for 1 minute, add the supernatant to 12% SDS-PAGE gel electrophoresis. After staining, it was observed that the amount of protein with expected molecular weight increased with the increase of IPTG induction time, which was the engineering bacteria expressing Trx-md-lectin fusion protein.

Extraction and Purification of Trx-md-lectin Fusion Protein

The engineering bacteria BL21-pET32a(+)-md-lectin expressing Trx-md-lectin fusion expression protein was induced by the above method, and the bacterial cells were collected by centrifugation, and were treated with BugBuster reagent and Benzonase nuclease according to the instructions of the manufacturer (Novagen). Purification of inclusion bodies. Inclusion bodies can be dissolved with lysis buffer (50mM CAPS, pH 11.0, 0.3% N-lauroylsarcosine), and then dialyzed with dialysis buffer (200mM Tris-HCl, pH 8.5). Then affinity chromatography was carried out with histidine binding (His·Bind) resin, and the Trx-md-lectin fusion protein was collected by elution with elution buffer (1M imidazole, 500mM NaCl, 20mM Tris-HCl pH 7.9). The expression protein of md-lectin can be isolated after the fusion protein is digested with enterokinase at 20°C for 16 hours.

Example 5

Eukaryotic expression of monstera lectin protein or polypeptide in tobacco and identification of insect resistance of transgenic plants

The construction of the expression vector containing the target gene (monstera lectin gene), according to the full-length gene sequence (SEQ ID NO.1) of the monstera lectin protein, design primers to amplify the complete coding reading frame, and Restriction endonuclease sites were introduced into the upstream and downstream primers (this can depend on the vector selected), so as to construct the expression vector. Using the amplification product obtained in Example 1 as a template, after PCR amplification, the monstera lectin gene cDNA was cloned into an intermediate vector (such as pBluescript), and further cloned into a binary expression vector (such as pBI121 and improved pCAMBIA2300 ), under the premise of ensuring the correct reading frame, the identified expression vector was transformed into Agrobacterium, and the model plant Nicotiana tabacum was transformed using the leaf disc method.

Transformation of Tobacco Using the Leaf Disk Method

1. Use a sterile toothpick to pick up positive colonies on the YEB selection plate, inoculate them in 2ml of YEB liquid (Sm+, Kan+), culture at 28 degrees, 200rpm for 24-36 hours with shaking;

2. Centrifuge at 4,000g for 10min at room temperature;

3. Discard the supernatant, suspend the bacteria with 1/2MS liquid medium, and dilute to 5-20 times the original volume, so that the OD600 of the bacteria solution is about 0.5;

4. Take the aseptic leaves of tobacco that have grown for about two weeks, remove the main veins, and cut them into small leaves of about 1 square centimeter;

5. Put the leaves into the prepared bacterial solution, soak for 2-5 minutes, and blot the bacterial solution on sterile filter paper;

6. Put the infected leaves on MS medium and culture in dark at 28°C for 48 hours;

7. Transfer the leaves to the callus medium (MS+6-BA 1.0mg/L+NAA 0.1mg/L+Kan50mg/L+cb 250mg/L), culture under light at 25-28°C for 7-15 days Visible callus formation;

8. Differentiated buds can be seen to grow after about 20 days. After the buds grow up, cut them off and place them on the rooting medium (1/2MS+NAA 0.5mg/L+Kan 25mg/L) for rooting culture, 2-7 days take root left and right;

9. After the root system develops, take out the plant, wash the attached solid medium with sterile water, move it into the soil, cover it with a glass cover for a few days at the beginning, remove the glass cover after the plant is strong, and cultivate it in the greenhouse.

Using Western blot to detect the expression of MDA protein in transgenic tobacco plants

1. Extraction of protein: a) Take 50mg leaves, add 100ul 1*PBS (KH ₂ PO ₄ 0.2g/l, Na ₂ HPO ₄ 1.15g/l, KCl 0.2g/l, NaCl 8g/l) in 1.5ml Grinding in an Eppendorf tube; b) Centrifuge at 13,000 rpm at 4°C for 10 minutes; c) Take the supernatant and set aside. Note: The above process was carried out on ice.

2. Protein quantification: refer to Bradford method (Bradford, 1976). Take 2ul protein sample, add 1ml Bradford reagent, mix well, measure OD595 by spectrophotometer; protein content calculation: _1OD595 =28.57μg.

3. SDS-PAGE protein separation: The preparation of SDS-PAGE refers to "Molecular Cloning" (Sambrook et al., 1989); Buffer: glycerol 2.4g, 1M Tris-HCl pH6.81ml; bromophenol blue 0.01%, H ₂ O to 20ml), boiled for 10 minutes; b) polyacrylamide gel electrophoresis at room temperature at 100V until the indicator (bromophenol blue) leading edge reaches the bottom of the gel.

4. Protein transfer to nitrocellulose membrane: a) before transfer, equilibrate gel and nitrocellulose membrane with transfer buffer (39mmol glycine, 48mmol Tris Base, 0.037% SDS, 20% methanol) for 30 minutes; b) use at room temperature Transfer to a semi-dry electroporation apparatus for 1 hour, and place 3 layers of Whatman filter paper on both sides of the gel;

5. Detection of protein on the membrane: a) Soak the nitrocellulose membrane in blocking solution, shake slowly at 37°C, and block for 60 minutes (blocking solution: take 5g skimmed milk powder and dissolve it in 100ml 1*PBS (containing 0.5g sodium azide) ; b) Soak the filter membrane in washing buffer, wash twice at 37°C for 15 minutes each time; c) add primary antibody (antibody against Monstera md-Lectin), incubate at 37°C for 30 minutes; d) Same as step b, wash three times; e) add secondary antibody (avidin-alkaline phosphatase complex), incubate at 37°C for 30 minutes; same step b), wash twice; f) add substrate to display Color observation of protein bands.

Identification of Insect Resistance of Transgenic Plants Containing Monstera Lectin Gene (MDA)

In view of the fact that the gene encoding mannose-specifically binding plant lectins such as snowdrop agglutinin (GNA) has been proved to be effective against pests such as piercing-sucking mouthparts such as aphids (Hilder et al., Transgenic Res, 1995, 4: 18-25 ), planthoppers (Tang et al., Plant Breeding, 2002, 121:93～95) and insects with chewing mouthparts such as the tomato moth Lahanohia olerucea (Gatehouse et al., Molecular Breeding, 1997, 3:49～63) are all resistant , and the monstera agglutinin (MDA) is also a mannose-specific binding lectin and has a high homology with the snowdrop agglutinin (GNA), so the gene (MDA) encoding the monstera agglutinin is the same The snowdrop lectin gene (gna) is similar and should also have the function of insect resistance. We further identified the insect resistance of the transgenic plants expressing the monstera agglutinin gene (MDA). According to the method of Hilder et al. (Transgenic Res, 1995, 4: 18-25), the transgenic tobacco plants (20-30 cm high) expressing the Monstera agglutinin gene (MDA) were carried out for aphid (Peach aphid) resistance identification. Its effect on aphid survival and developmental progress. In the study on the survival rate of aphids, after inoculating 10 first-instar nymphs of aphids on each plant, the survival number of aphids was measured every 2 days (14 days in total). In the study on the progress of aphid development, five first-instar nymphs of aphids were inoculated on each plant, and the number of surviving aphid adults and nymphs was measured 13 days later. The results showed that the number of aphids surviving on the transgenic tobacco plants expressing the monstera agglutinin gene (MDA) was significantly reduced (P<0.05) compared with the non-transgenic control. Compared with the non-transgenic control, the developmental progress of aphids surviving on the transgenic tobacco plants of MDA was also significantly slowed down (P<0.05), and the number of adults developed after 13 days was also significantly reduced (P<0.05). Therefore, the results of insect resistance prove that the monstera agglutinin gene (MDA) is indeed resistant to insects (such as aphids), and the monstera agglutinin gene (MDA) can be used in the research and industrialization of plant pest control using transgenic technology .

Nucleotide Sequence Listing

SEQUENCE LISTING

<110> Shanghai Normal University

<120>Monstera lectin gene and its encoded protein and application

<160>4

<170>PatentIn version 3.3

<210>1

<211>1186

<212>DNA

<213>Monstera deliciosa

<220>

<221> CDS

<222>(49)..(900)

<223>

<400>1

tagcaatagt attgtacact aacacaagtt cagatttggc attccccaat ggcagcaact 60

tcccttcttt tttcagcagc tcctctcctc gtcctcctcc atgcggtcct cctcgccctc 120

ttaacacccg ccagggcggg ggagcaggtc ctcatcgcca accgcacgtt gtatgccgga 180

cagtccctgt acagcatgaa ccgcaactac aggttcatca tgcagtggga ctgctccttg 240

gccctgtacg agttccagaa gcccctctgg atctcgacca ccgatcggca gccgggcact 300

ttctgcgacg ccactctcga tgccggctcc gcccgcctcg cggtcacgca gcggaacacc 360

aacaagcaag tctggtcgag caccacgagt ttctacgcat tcaagtacgt cctcgtcctg 420

cagaccgacc tgaacgtggt catctacagc agacctgtct ggtccaccaa cacgggcgtc 480

tgggactccg tggagttcca gaatggggac cagatcttgt tgtcctccgg cacgctcaag 540

gttgggcagt acctgtccaa caacgagctc aacttcaggt ttattatgca ggacgactgc 600

gacctggtcc tctacgacga cgaccggcgc atctggagct cggagacctc cggcaagggc 660

accggctgca acgcaaagtt gaacggtctc accggccagc tcatcgtccg ggacaacaac 720

ggcaggatcg tatggagcag caagaaccgc gccgtcccaa ttaagaagca catcctgctg 780

ctgcagcccg atcggaacgt tgtcgtctac accggacgcg tctgggcctc cgacacgagc 840

atcccggaga accaaggagg gctgcaggat gctgggcgga tcgagatggt caccaactaa 900

attagcgtgc ctcgtgtact gctcatcgtg cggccatttg cgttatttac tataaacaag 960

acttaatact aaaataagtg gtcggggctg gtgcgtcgtc acgtctgtat ctcgttttcg 1020

cggccgtcat gataaactat cctttgtgcg gtaagtctat atgtttctat ggcgtgagct 1080

tcacttggcg tgtaccttgt ccataaagaa aagtttgcat ctgttcattt aattataata 1140

acgtacgaac gttagaaaca tttcccccaa aaaaaaaaaa aaaaaa 1186

<210>2

<211>283

<212>PRT

<213>Monstera deliciosa

<400>2

Met Ala Ala Thr Ser Leu Leu Phe Ser Ala Ala Pro Leu Leu Val Leu

1 5 10 15

Leu His Ala Val Leu Leu Ala Leu Leu Thr Pro Ala Arg Ala Gly Glu

20 25 30

Gln Val Leu Ile Ala Asn Arg Thr Leu Tyr Ala Gly Gln Ser Leu Tyr

35 40 45

Ser Met Asn Arg Asn Tyr Arg Phe Ile Met Gln Trp Asp Cys Ser Leu

50 55 60

Ala Leu Tyr Glu Phe Gln Lys Pro Leu Trp Ile Ser Thr Thr Asp Arg

65 70 75 80

Gln Pro Gly Thr Phe Cys Asp Ala Thr Leu Asp Ala Gly Ser Ala Arg

85 90 95

Leu Ala Val Thr Gln Arg Asn Thr Asn Lys Gln Val Trp Ser Ser Thr

100 105 110

Thr Ser Phe Tyr Ala Phe Lys Tyr Val Leu Val Leu Gln Thr Asp Leu

115 120 125

Asn Val Val Ile Tyr Ser Arg Pro Val Trp Ser Thr Asn Thr Gly Val

130 135 140

Trp Asp Ser Val Glu Phe Gln Asn Gly Asp Gln Ile Leu Leu Ser Ser

145 150 155 160

Gly Thr Leu Lys Val Gly Gln Tyr Leu Ser Asn Asn Glu Leu Asn Phe

165 170 175

Arg Phe Ile Met Gln Asp Asp Cys Asp Leu Val Leu Tyr Asp Asp Asp

180 185 190

Arg Arg Ile Trp Ser Ser Glu Thr Ser Gly Lys Gly Thr Gly Cys Asn

195 200 205

Ala Lys Leu Asn Gly Leu Thr Gly Gln Leu Ile Val Arg Asp Asn Asn

210 215 220

Gly Arg Ile Val Trp Ser Ser Lys Asn Arg Ala Val Pro Ile Lys Lys

225 230 235 240

His Ile Leu Leu Leu Gln Pro Asp Arg Asn Val Val Val Tyr Thr Gly

245 250 255

Arg Val Trp Ala Ser Asp Thr Ser Ile Pro Glu Asn Gln Gly Gly Leu

260 265 270

Gln Asp Ala Gly Arg Ile Glu Met Val Thr Asn

275 280

<210>3

<211>23

<212> DNA

<213>Monstera deliciosa

<400>3

tagcaatagt attgtacact aac 23

<210>4

<211>21

<212>DNA

<213>Monstera deliciosa

<400>4

ggggaaatgt ttctaacgtt c 21

Claims (10)

1. a Monstera deliciosa agglutinin gene has the nucleotide sequence shown in the SEQ ID NO.1, perhaps add, replace, insert or delete the homologous sequence of one or more Nucleotide, or its allelotrope and deutero-nucleotide sequence thereof.

2. a Monstera deliciosa agglutinant protein matter is coded by the described gene order of claim 1.

3. Monstera deliciosa agglutinant protein matter according to claim 2 is characterized in that having the aminoacid sequence shown in the SEQ IDNO.2, perhaps add, replace, insert or delete one or more amino acid whose homologous sequences.

4. one kind contains claim 1 described Monstera deliciosa agglutinin gene complete sequence or the segmental plasmid of part.

5. one kind contains claim 1 described Monstera deliciosa agglutinin gene complete sequence or the segmental plant expression vector of part.

6. host cell, this cell contains each described gene order in claim 1 or 4 or 5.

7. host cell according to claim 6 is characterized in that, said cell is a Bacillus coli cells.

8. host cell according to claim 6 is characterized in that, said cell is an agrobatcerium cell.

9. host cell according to claim 6 is characterized in that, said cell is a tobacco cell.

10. the application of a Monstera deliciosa agglutinin gene comprises with claim 5 described expression vector transformation of tobacco cell or with the described Agrobacterium of claim 8 and tobacco cell is cultivated altogether or cultivate tobacco plant with the described tobacco cell of claim 9.