CN116694680A

CN116694680A - Lactoferrin plant expression vector for stably and efficiently expressing lactoferrin gene and application

Info

Publication number: CN116694680A
Application number: CN202310885128.XA
Authority: CN
Inventors: 王东芳
Original assignee: Sanya Tiedan Biotechnology Co ltd
Current assignee: Sanya Tiedan Biotechnology Co ltd
Priority date: 2023-07-19
Filing date: 2023-07-19
Publication date: 2023-09-05

Abstract

The invention discloses a lactoferrin plant expression vector for stably and efficiently expressing a lactoferrin gene and application thereof. The invention locates the target protein in plant leaf chloroplast by optimizing expression regulating element, adding proper signal peptide sequence, etc. to raise the expression amount and stability of target protein by 5-10 times, raise the expression amount of target protein from microgram level to milligram level and reach about 1 mg/g. Realizes stable and efficient expression of the lactoferrin in the leaves.

Description

Lactoferrin plant expression vector for stably and efficiently expressing lactoferrin gene and application

Field of the art

The invention relates to a lactoferrin plant expression vector and application.

(II) background art

Lactoferrin (Lactoferrin) is an iron binding protein that is widely found in mammalian milk and other body fluids. It plays an important role in protecting and regulating milk, and has various physiological activities and application potential.

Lactoferrin has several main properties:

iron binding: lactoferrin can bind and transport iron ions and play a role in regulating the iron metabolism of the body. It can bind with excessive free iron, reduce damage to cells and tissues from free iron, and release iron ions when needed.

Antibacterial action: lactoferrin has broad-spectrum antibacterial activity and can inhibit the growth of bacteria, fungi and viruses. It exerts an antibacterial effect through a variety of mechanisms including binding to anions on the surface of microorganisms, disrupting bacterial membranes, inhibiting synthesis of bacterial inner and outer membranes, and the like.

Immunomodulatory effects: lactoferrin can regulate the function of the immune system and enhance the body's resistance. It is involved in regulating inflammatory responses, stimulating activation and proliferation of immune cells, and promoting interactions between immune cells.

Antioxidant effect: lactoferrin has antioxidant activity, and is capable of scavenging free radicals and protecting cells from oxidative damage. It plays an important role in maintaining the redox balance of cells and protecting the integrity of cell membranes.

Based on the physiological activity and multiple functions of lactoferrin, the lactoferrin has wide application potential in the fields of medicines, health products, foods, cosmetics and the like. Some studies have shown that lactoferrin can be used to treat anaemia, iron deficiency diseases, infectious diseases, etc., and is also used as an ingredient of antibacterial agents, immunomodulators, antioxidants and nutraceuticals. In addition, lactoferrin is also widely used in the fields of infant formula milk powder, functional foods, oral care products, skin care products and the like.

Therefore, the research and development of lactoferrin has important scientific significance and application value, including searching for efficient production methods, exploring the bioactive mechanism thereof, optimizing the functional application thereof and the like.

Currently, plant leaf expression of lactoferrin still presents challenges and problems, mainly including the following:

the expression level is low: plant leaves are used as main tissues for expressing lactoferrin, and the natural expression level of the plant leaves is often low, so that the requirement of large-scale production cannot be met. Thus, increasing the expression level of lactoferrin in plant leaves is a critical issue.

Protein stability: lactoferrin may be degraded in plant leaves by protein degrading enzymes, resulting in a decrease in its stability. This may lead to inactivation of lactoferrin or reduce its biological activity.

Subcellular localization: the subcellular localization of lactoferrin within cells has a significant impact on its function and use. Ensuring that lactoferrin is correctly located in the plant leaf to a suitable subcellular location, such as the cytosol, endoplasmic reticulum or cell wall, has an important impact on its biological activity and application.

Purifying and extracting: purification and extraction of lactoferrin from plant leaves is a challenging step. Leaves contain a large amount of other proteins and complex components such as chlorophyll and the like, and efficient purification and extraction methods are required to obtain high purity lactoferrin.

Research is underway to solve these problems. Such as by optimizing plant gene expression systems, regulating transcription factors and signaling pathways, improving protein stability and subcellular localization, and developing efficient purification techniques, these challenges can be overcome, and the efficiency and quality of lactoferrin expressed by plant leaves can be improved, thereby promoting the use of lactoferrin in plant systems. Therefore, a method for improving the high-volume stable expression of lactoferrin in the leaves is needed.

(III) summary of the invention

The invention aims to provide a lactoferrin plant expression vector and application, which are used for improving the expression quantity of lactoferrin in plants by optimizing expression regulatory elements, applying proper signal peptide and other modes, so as to realize the expression of lactoferrin in chloroplasts, thereby improving the expression quantity of lactoferrin on one hand and the stability of target protein on the other hand.

The technical scheme adopted by the invention is as follows:

the invention provides a lactoferrin plant expression vector for stably and efficiently expressing a lactoferrin gene, wherein the lactoferrin plant expression vector comprises a lactoferrin gene sequence capable of being expressed by plants, a signal peptide coding gene sequence, a promoter and a transcription terminator.

Furthermore, the lactoferrin gene is bovine lactoferrin gene, but is optimized according to plant codon preference, the nucleotide sequence is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.

Further, the nucleotide sequence of the signal peptide coding gene is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4.

Further, the promoters include, but are not limited to, the cauliflower mosaic virus CaMV35S promoter P35S, the nucleotide sequence of which is shown in SEQ ID NO. 5; the terminator sequence includes but is not limited to cauliflower mosaic virus CaMV35S terminator T35S, and the nucleotide sequence of the terminator is shown in SEQ ID NO. 6.

The lactoferrin provided by the invention can also be derived from other animal genomes, the amino acid sequences of the proteins encoded by the lactoferrin genes are shown in table 1, and the lactoferrin genes provided by the invention comprise but are not limited to the genes and homologous genes thereof.

Table 1: protein encoded by animal lactoferrin gene

Numbering device	Animal of origin	Animal Latin name	NCBI sequence
				1	Cattle	Bos taurus	NP_851341.1
2	Yak	Bos grunniens	ALE66311.1
				3	(Cattle)	Bos indicus	ACY01187.1
4	Human body	Homo sapiens	AAB60324.1
				5	Pig	Sus scrofa	XP_020924222.1
6	Sheep	Ovis aries	NP_001020033.1
				7	Camel with top	Camelus bactrianus	XP_010965654.1

Furthermore, the expression vector of the invention also comprises a regulatory element for enhancing the expression quantity or stability of the herbicide resistant gene of a plant source, wherein the regulatory element refers to a nucleotide sequence capable of regulating the gene expression, and comprises an enhancer, a matrix attachment region sequence and the like, preferably a matrix attachment region sequence (MAR), and the nucleotide sequence is SEQ ID No: shown at 7. The regulatory element is arranged at the 5 'end of the plant source herbicide resistance gene expression frame or the 3' end of the terminator.

The basic vector used in the present invention to provide the backbone of the expression vector may be a pCambia series vector (CAMBIA, canberra, australia) or other vector, preferably a pCambia1300 vector. The selectable marker used in the present invention is the hygromycin resistance gene hyg carried by pCambia1300, alternatively the selectable marker gene may be a herbicide resistance gene or other resistance gene derived from bacteria or plants.

The invention provides application of a lactoferrin gene plant expression vector in improving the expression level of plant lactoferrin.

The invention provides an application of a lactoferrin gene plant expression vector in preparing lactoferrin, which is to transfer the lactoferrin gene plant expression vector into a plant genome to obtain a lactoferrin-enriched plant and extract the lactoferrin.

The expression vector constructed by the invention is suitable for the expression of monocotyledonous plants or dicotyledonous plants, and the monocotyledonous plants comprise: corn, rice, etc.; dicotyledonous plants include: soybean, rape, cotton, mulberry, etc.

The invention also provides a plant cell containing the lactoferrin gene plant expression vector T-DNA.

The invention also provides a plant containing the lactoferrin gene plant expression vector T-DNA.

The invention further provides a method for obtaining plant cells or plants for stably and efficiently expressing lactoferrin genes by using the expression vector, wherein the method can be an agrobacterium-mediated transformation method, a gene gun method, a protoplast infection method or other plant genetic transformation methods, and preferably the agrobacterium-mediated transformation method.

Compared with the prior art, the invention has the beneficial effects that: the invention locates the target protein in plant leaf chloroplast by optimizing expression regulating element, adding proper signal peptide sequence, etc. to raise the expression amount and stability of target protein by 5-10 times, raise the expression amount of target protein from microgram level to milligram level and reach about 1 mg/g. Realizes stable and efficient expression of the lactoferrin in the leaves.

(IV) description of the drawings

Fig. 1: schematic of T-DNA structure of BLF plant expression vector. P35S is CaMV35S promoter, T35S is CaMV35S terminator, BLF is codon optimized BLF gene, hyg is hygromycin resistance gene, CTP is chloroplast signal peptide, MAR is matrix attachment region sequence (MAR).

(fifth) detailed description of the invention

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

modifications and substitutions of steps, methods, or conditions of the present invention without departing from the spirit of the invention are intended to be within the scope of the present invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.

Example 1, vector construction

To construct the vectors required in the present invention, the following sequences were synthesized artificially: 1) The nucleotide sequence of the codon optimized bovine lactoferrin gene BLF is shown as SEQ ID No:1, the amino acid sequence is shown as SEQ ID No:2 is shown in the figure; 2) The nucleotide sequence of the chloroplast signal peptide CTP is shown as SEQ ID No:3, the amino acid sequence is shown as SEQ ID No:4 is shown in the figure; 3) The cauliflower mosaic virus CaMV35S promoter P35S has a nucleotide sequence shown in SEQ ID NO. 5; 4) The nucleotide sequence of the cauliflower mosaic virus CaMV35S terminator T35S is shown in SEQ ID NO. 6; 5) A matrix attachment region sequence (MAR) having a nucleotide sequence set forth in SEQ ID No: shown at 7.

1. Lactoferrin control expression vector 1300-p35S-BLF-T35S construction

(1) The vector 1300 from which the hygromycin gene was deleted was recovered by digestion of pCambia1300 with restriction enzyme XhoI using pCambia1300 vector (NCBI Accession Number: AF 234296) as the starting vector;

(2) The BLF gene (primer BLF-F, BLF-R) was obtained by PCR cloning using the artificially synthesized BLF genome as a template.

(3) And then, connecting the vector recovered in the step (1) and the BLF gene fragment in the step (2) by utilizing recombinase, and cloning to obtain a transitional vector p35S-BLF-T35S.

(4) Then, the pCambia1300 vector was subjected to double cleavage with restriction enzymes BamHI and EcoRI, and the cleaved vector was recovered.

(5) The plasmid DNA of the transition vector p35S-BLF-T35S was used as a template, and the BLF gene expression cassette (primer SBLF-F, SBLF-R) was cloned by PCR to recover a p35S-CTP-BLF-T35S fragment having a fragment size of about 3.0 kb.

(6) And then cloning after connecting the vector recovered in the step (4) and the p35S-CTP-BLF-T35S fragment in the step (5) by utilizing recombinase to obtain a final vector 1300-p35S-BLF-T35S.

BLF-F:5’-CTCTCTACAAATCTATCTCTAAAGAACAATGGCCCCACGCAAGAA CGTG；

BLF-R:5’-CACACATTATTATGGAGAAATCATTAGCGGGTGAGGAAGGCGCAG GCC；

SBLF-F:5’-CTGCAGGTCGACTCTAGAGGATCCTAGAGCAGCTTGCCAACATGSBLF-R:5’-CAGCTATGACCATGATTACGTAATTCGGGGGATCTGGATTTTAG

2. Construction of optimized lactoferrin expression vector 1300-p35S-CTP-BLF-T35S

(1) The vector 1300 from which the hygromycin gene had been deleted was recovered by digestion of pCambia1300 with the restriction enzyme XhoI using the pCambia1300 vector (NCBI Accession Number: AF 234296) as the starting vector.

(2) The BLF gene (primer BLF-F, BLF-R) was obtained by PCR cloning using the artificially synthesized DNA of the nucleotide sequence of the BLF gene as a template, and a BLF fragment having a fragment size of about 2.1kb was recovered.

(3) The CTP polypeptide coding sequence DNA synthesized manually is used as a template, CTP fragments (primer CTP-F, CTP-R) are obtained through PCR cloning, and CTP fragments with the fragment size of about 0.25kb are recovered;

(4) And then cloning the vector recovered in the step (1), the BLF fragment in the step (2) and the CTP fragment in the step (3) after three-section connection by utilizing recombinase to obtain a transitional vector p35S-CTP-BLF-T35S.

(5) Then, the pCambia1300 vector was subjected to double cleavage with restriction enzymes BamHI and EcoRI, and the cleaved vector was recovered.

(6) The plasmid DNA of the transition vector p35S-CTP-BLF-T35S was used as a template, and the BLF gene expression cassette (primer SCBLF-F, SCBLF-R) was cloned by PCR to recover a p35S-CTP-BLF-T35S fragment having a fragment size of about 3.3 kb.

(7) And (3) connecting the vector recovered in the step (5) and the fragment in the step (6) by utilizing recombinase, and cloning to obtain the final vector 1300-p35S-CTP-BLF-T35S.

BLF-F:5’-CAGCTGCTAAGGCTGAGGCCCCACGCAAGAACGTGC；

BLF-R:5’-CACACATTATTATGGAGAAATCATTAGCGGGTGAGGAAGGCGCAG GCC；

CTP-F:5’-CTCTCTACAAATCTATCTCTAAAGAACAATGGCTGCTACTATGGCT TC；

CTP-R:5’-GCCTCAGCCTTAGCAGCTGGAGCAGCAACAGAAGAAGAAG；

SCBLF-F:5’-CTGCAGGTCGACTCTAGAGGATCCTAGAGCAGCTTGCCAACAT G；

SCBLF-R:5’-CAGCTATGACCATGATTACGTAATTCGGGGGATCTGGATTTTAG

3. Construction of lactoferrin expression vector 1300-MAR-p35S-CTP-BLF-T35S optimized by adding expression regulatory element

(1) Double-enzyme cutting is carried out on the 1300-p35S-CTP-BLF-T35S vector by using restriction enzymes BamHI and HindIII, and the carrier after enzyme cutting is recovered;

(2) MAR fragments (primer MAR-F, MAR-R) were obtained by PCR cloning using the artificially synthesized MAR nucleotide sequence DNA as a template, and MAR fragments having a fragment size of about 1.2kb were recovered.

(3) And then cloning after connecting the vector recovered in the step (1) and the MAR fragment in the step (2) by utilizing recombinase to obtain a final vector 1300-MAR-p35S-CTP-BLF-T35S.

MAR-F:5’-GTAAAACGACGGCCAGTGCCAAGCTTGATTAAAAATCCCAATTAT ATTTGG；

MAR-R:5’-CATGTTGGCAAGCTGCTCTAGGATCCACTATTTTCAGAAGAAGTT CC。

4. Obtaining Agrobacterium containing T-DNA vector

The obtained T-DNA vectors (1300-p 35S-BLF-T35S, 1300-p35S-CTP-BLF-T35S and 1300-MAR-p 35S-CTP-BLF-T35S) are subjected to electric shock transformation with agrobacterium EHA105, strains containing plant transformation vectors are obtained through kan screening, and the strains are stored to a-80 ℃ refrigerator after glycerol is added for transgenic crop infection.

Example 2 tobacco conversion

(1) The Agrobacterium of example 1 (1300-p 35S-BLF-T35S, 1300-p35S-CTP-BLF-T35S and 1300-MAR-p 35S-CTP-BLF-T35S) was streaked onto YEP plates containing 1mg/L kanamycin and 1mg/L rifampicin, incubated at 28℃for 2 days, single colonies were picked from the plates, inoculated into 20mL of YEP liquid medium containing 50mg/L kanamycin (kan) and 50mg/L rifampicin (Rif), and incubated on a thermostated shaking bed at 180r/min to an OD of 0.8 (approximately 17 h) at 28 ℃.

(2) Transferring the bacterial liquid with the OD600 of 0.8 in the step (1) into a newly prepared YEP liquid culture medium containing 50mg/L of Rif according to the proportion of 1.5% of volume concentration, and simultaneously adding 400 mu mol/L of acetosyringone. The culture was continued under the same conditions for another 6 hours, and transformation was started when the OD600 was 0.5.

(3) And (3) 50mL of the bacterial liquid with the OD600 of 0.5 in the step (2) is subjected to centrifugal precipitation at 4 ℃ and 2500r/min, the bacterial liquid is resuspended in 40mL of MS liquid culture medium on an ultra-clean workbench, the resuspended bacterial liquid is poured into a glass plate, tobacco leaves (tobacco germinated for about one month) cut into 5X 5mm size are put into the bacterial liquid, soaked for 10min, and the leaves are taken out and placed on sterile filter paper to absorb the attached bacterial liquid to be used as explants.

(4) The explant from step (3) was placed on RMOP medium and co-cultured for 3 days at 28℃under dark culture conditions.

(5) The explant subjected to the co-culture in the step (4) is transferred to an RMOP medium (500 mg/L of carbenicillin is added to inhibit the growth of the agrobacteria) to which an antibiotic (20 mg/L hyg (hygromycin)) is added, and the selection culture is performed under the conditions of 25 mu mol.m.s and 25 ℃.

(6) After 2-3 weeks of selective culture, the transformed cells of the explants will differentiate resistant adventitious buds or produce resistant callus, and these resistant material will be transferred to corresponding selective medium (RMOP+20 mg/L hyg+500mg/L Cb (carbicillin)) for subculture at 26 ℃.

(7) When the adventitious bud length of the step (6) is more than 1cm, cutting and inserting the adventitious bud length into a tobacco rooting culture medium (MS+0.1 mg/L NAA+100mg/L kan+500mg/L Cb) containing selective pressure (10 mg/L hygromycin), carrying out rooting culture at 26 ℃ for about two weeks to grow adventitious roots, and carrying out molecular identification after the adventitious roots grow slightly longer.

Example 3 detection of lactoferrin expression level in tobacco leaves

To detect the expression level of bovine lactoferrin in tobacco leaves in example 2, we purchased a bovine lactoferrin ELISA detection kit (abcam, bovine Lactoferrin ELISA Kit (ab 274406)).

Method for checking the expression level of BLF gene in tobacco: cutting 0.1g leaf into pieces, adding 1-1.5mL PBS, grinding in a homogenizer, and strictly operating according to the specification of BLF protein detection kit (ab 274406). Specifically, the sample extract was diluted 500-fold with 1×pbs. The same tissue extract of non-transgenic tobacco plants containing the same dilution fold was used as a control to detect background signals due to the test tissue. Each elisa plate contained BLF standard of known concentration for generating a standard curve (curve equation y= 1.2769 ×)

(1.0151－e ^-0.0329x) ). Each ELISA plate contains a blank buffer for detecting the background signal of the extraction buffer. Abcam corporation kit (cat No. ab 274406) was used to detect BLF content. Samples, controls and standards were added to the elisa plate and incubated for 30 minutes at room temperature. Then, the elisa plates were washed, an antibody conjugate solution was added to each of the elisa plates, and incubated at room temperature for 30 minutes. After antibody conjugation incubation, the elisa plate was washed. The substrate solution was added to the elisa plate and incubated at room temperature for 30 minutes. After incubation, the reaction is reversedThe stop solution was added to the ELISA plate, and the OD value was read at 450 and nm, and converted to the expression level according to the standard curve equation (Table 2).

TABLE 2 average BLF Gene content in different tobacco leaves (. Mu.g/g.+ -. SD)

Note that: fresh weight mean, standard deviation and measurement ranges are based on all readings for each tissue type (n=3 different leaves). Student-t test analysis was performed on the mean values of each group, with differences in letters indicating the presence of a significant difference (p < 0.05) between the two groups of data.

As can be seen from Table 2, the average expression level of the BLF gene in the leaf of vector 1300-p35S-CTP-BLF-T35S is significantly higher than that in the leaf of vector 1300-p35S-BLF-T35S, indicating that the chloroplast signal peptide CTP significantly increases the expression level of BLF in tobacco leaf. At the same time, the carrier

The average expression level of the BLF gene in the leaf of 1300-MAR-p35S-CTP-BLF-T35S is obviously higher than that of the vector 1300-p35S-CTP-BLF-T35S, which indicates that the application of the expression control element MAR further obviously improves the expression level of the BLF in tobacco leaves.

Therefore, compared with the common expression vector (1300-p 35S-BLF-T35S), the expression level of BLF in tobacco leaves can be obviously improved by utilizing the chloroplast signal peptide CTP in the invention, and meanwhile, on the basis, the expression level of BLF in tobacco leaves can be further improved by utilizing regulatory elements such as MAR and the like.

Example 4 lettuce transformation and measurement of Lactoferrin expression level in lettuce leaves

Agrobacterium EHA105 constructed in example 2 containing the expression vectors 1300-p35S-BLF-T35S, 1300-p35S-CTP-BLF-T35S and 1300-MAR-p35S-CTP-BLF-T35S, respectively, was streaked onto YEP plates containing 1mg/L kanamycin and 1mg/L rifampicin frozen at-80℃and cultured overnight at 28 ℃. Picking single colony, inoculating in YEP liquid culture medium, shake culturing at 28deg.C and 220r/min until bacterial liquid concentration is OD600 about 1.0; and (3) centrifuging at 4000r/min for 5min, collecting thalli, washing twice by using a 1/2MS liquid culture medium, and re-suspending in the 1/2MS liquid culture medium to dilute the thalli until the OD600 is 0.6, thus obtaining the dip dyeing liquid.

Cutting the edge of sterile lettuce seedling She Qiequ into 3-6 mm ² Leaf discs of (2) are subjected to dip dyeing for 20 minutes at room temperature in a dip dyeing liquid; sterilizing filter paper, sucking bacterial liquid, inoculating to co-culture Medium (MS), culturing at 28deg.C for 3d, inoculating to bud induction screening medium (MS+1.5 mg/L6-BA+0.2mg/L IAA+20mg/L Hyg+300mg/L Cb), culturing at 25deg.C under 2000lx illumination intensity, and culturing under illumination for 14 h/d. The differentiated shoots were excised and inserted into 1/2MS medium (30 mg/L, 20mg/L Hyg and 300mg/L Cb for sucrose addition) to induce rooting at 26 ℃.

The bovine lactoferrin ELISA test kit (abcam, bovine LactoferrinELISAKit (ab 274406)) of example 3 was used to test the expression level of bovine lactoferrin in lettuce leaves, and the results are shown in Table 3.

TABLE 3 average BLF Gene content in different raw vegetable leaves (. Mu.g/g.+ -.SD)

As can be seen from Table 3, the average expression level of the BLF gene in the leaf of vector 1300-p35S-CTP-BLF-T35S is significantly higher than that in the leaf of vector 1300-p35S-BLF-T35S, indicating that the chloroplast signal peptide CTP significantly increases the expression level of BLF in the leaf of Shengmai. At the same time, the carrier

The average expression level of the BLF gene in the 1300-MAR-p35S-CTP-BLF-T35S leaf is obviously higher than that in the vector 1300-p35S-CTP-BLF-T35S leaf, which indicates that the application of the expression control element MAR further obviously improves the expression level of the BLF in the raw vegetable leaf.

Therefore, compared with the common expression vector (1300-p 35S-BLF-T35S), the expression level of BLF in the raw vegetable leaves can be obviously improved by utilizing the chloroplast signal peptide CTP in the invention, and meanwhile, on the basis, the expression level of BLF in the raw vegetable leaves can be further improved by utilizing regulatory elements such as MAR and the like.

Example 5 Mulberry transformation

Agrobacterium EHA105 constructed in example 2 containing the expression vectors 1300-p35S-BLF-T35S, 1300-p35S-CTP-BLF-T35S and 1300-MAR-p35S-CTP-BLF-T35S, respectively, was streaked onto YEP plates containing 1mg/L kanamycin and 1mg/L rifampicin frozen at-80℃and cultured overnight at 28 ℃. Picking single colony, inoculating in YEP liquid culture medium, shake culturing at 28deg.C and 220r/min until bacterial liquid concentration is OD600 about 1.0; and (3) centrifuging at 4000r/m for 5min, collecting the thalli, washing twice by using a 1/2MS liquid culture medium, and re-suspending in the 1/2MS liquid culture medium to dilute the thalli until the OD600 is 0.3, thus obtaining the dip dyeing liquid.

Immersing a cut leaf disc in a dye solution under sterile conditions, slightly oscillating, immersing for 1h at room temperature, taking out the leaf disc, sucking redundant bacterial liquid on sterile filter paper, inoculating to a co-culture medium (MS+TDZ1.0 mg/L+NAA0.2 mg/L+fructose 20 g/L+agar powder 5g/L; pH5.8), co-culturing for 2d at 26 ℃, washing the leaf disc with a liquid co-culture medium (MS+TDZ1.0 mg/L+NAA0.2 mg/L+fructose 20g/L; pH5.8) for 3 times to remove excessive agrobacterium, transferring to a primary screening medium (MS+TDZ1.0 mg/L+NAA0.2 mg/L+hyg+Cef 400 mg/L+fructose 20 g/L+agar powder 5g/L; pH5.8), culturing for 15-30 d at 26 ℃, replacing the medium once every 7 times, and transferring the leaf disc to a secondary screening medium (MS+TDZ1.0.0 mg/L+NAA0.2 mg/L+NAA0+2 mg/L+fruit powder 5.2 mg/L+fruit powder 5 g/L) when the leaf disc starts growing and culturing for a callus or transferring to the secondary screening medium (NAA0.0+0.0+0.2 mg/L+NAA0+0.2+0+5.2 mg/L). Culturing at 26 ℃ and carrying out 25d secondary culture.

When the hygromycin buds screened for the second time grow to 2cm high, transferring the hygromycin buds into a rooting culture medium (1/2MS+NAA0.5 mg/L+BA 2.0mg/L+hyg10 mg/L+Cef50mg/L+fructose 20 g/L+agar powder 5g/L; pH 5.8), inducing rooting at 26 ℃, and performing seedling hardening and transplanting to culture into complete plants.

It should also be noted that the above-recited list only illustrates a few embodiments of the present invention. The invention is not limited to the above embodiments, but may be extended and expanded in many ways. All extensions that one of ordinary skill in the art could directly derive from or envision from the disclosure of the present invention should be considered as the scope of the present invention.

Claims

1. A lactoferrin plant expression vector for stably and efficiently expressing a lactoferrin gene, wherein the lactoferrin plant expression vector comprises a plant-expressible lactoferrin gene sequence, a signal peptide-encoding gene sequence, a promoter, and a transcription terminator.

2. The lactoferrin plant expression vector of claim 1, wherein the lactoferrin gene sequence is set forth in SEQ ID No. 1.

3. The lactoferrin plant expression vector of claim 1, wherein the signal peptide encoding gene sequence is shown in SEQ ID No. 3.

4. The lactoferrin plant expression vector of claim 1, wherein said promoter comprises a cauliflower mosaic virus CaMV35S promoter P35S and said terminator sequence comprises, but is not limited to, a cauliflower mosaic virus CaMV35S terminator T35S.

5. The lactoferrin plant expression vector of claim 4, wherein said promoter P35S nucleotide sequence is set forth in SEQ ID No. 5; the nucleotide sequence of the terminator T35S is shown in SEQ ID NO. 6.

6. The lactoferrin plant expression vector of claim 1, wherein said expression vector comprises a regulatory element comprising an enhancer, matrix attachment region sequence.

7. The lactoferrin plant expression vector of claim 6, wherein said regulatory element is a matrix attachment region sequence and the nucleotide sequence is SEQ ID No: shown at 7.

8. Use of the lactoferrin gene plant expression vector of claim 1 to increase the expression level of lactoferrin in plants.

9. Use of a lactoferrin gene plant expression vector of claim 1 in the preparation of lactoferrin.

10. A plant cell comprising the lactoferrin gene plant expression vector T-DNA of claim 1.