CN109694878B

CN109694878B - Kelp molecule breeding method based on protoplast

Info

Publication number: CN109694878B
Application number: CN201811639813.XA
Authority: CN
Inventors: 刘涛
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2021-05-14
Anticipated expiration: 2038-12-29
Also published as: CN109694878A

Abstract

The invention provides a kelp molecule breeding method based on protoplast, which relates to the technical field of transgenosis, the protoplast used in the method is loose zoospore of kelp, is natural protoplast, has good activity and high development and transformation efficiency, can be suitable for PEG fusion transformation application carrying different exogenous gene plasmids, and has wide application range. The exogenous plasmid used in the method is pBI221-Bar-GFP or pBI221-stGBSS-Bar-GFP, the conversion rate is obviously improved, and the conversion culture time is shorter. The method also optimizes the operation process of fusion transformation, overcomes the problems in the existing PEG fusion technology, is more suitable for molecular breeding of the kelp, improves the transformation rate, shortens the culture period, reduces the cost, and provides a new approach for molecular genetic breeding of the kelp.

Description

Kelp molecule breeding method based on protoplast

Technical Field

The invention relates to the technical field of transgenosis, in particular to a kelp molecule breeding method based on protoplasts.

Background

Kelp (Scacharina japonica) is a large marine brown algae growing in low-temperature seawater, and has obvious phenomena of alternation of asexual reproduction and sexual reproduction generation, namely gametophyte generation and sporophyte generation. The zoospore is released by the algal sporangium, the released spore is a single cell and is a natural protoplast, a gametophyte is formed after germination, the oocyst and the seminal vesicle of the gametophyte are combined to form a zygote, the zygote is further germinated to generate juvenile sporophyte, and the kelp is developed after long-term operation. The kelp is an important large-scale economic seaweed in China, has wide application and is mainly applied to the aspects of medicines, foods, chemical industry and the like. At present, the kelp culture area in China exceeds 24 million hectares, the culture yield is 83 million tons, the total culture yield accounts for more than 80 percent of the global total amount, and the yield and the scale are in the leading position in the world.

At present, in the aspect of cultivating main economic seaweed in China, the coverage rate of improved kelp seeds is less than 30 percent, the quantity of the improved kelp seeds and the breeding capability of excellent seedlings are seriously insufficient, and the method becomes a major bottleneck factor for restricting the sustainable, efficient and stable development of kelp industry in China. The traditional breeding method applied at present has group-level selective breeding, crossbreeding and the like, but the traditional kelp breeding has the defects of long breeding period, low strain purity, continuous strain degeneration, high cost, large risk and the like, and the molecular breeding is generated along with the rapid development of molecular biology, genomics and the like, the modern biotechnology means is integrated into the traditional breeding method, the breeding efficiency is greatly improved, and the shortening of the breeding period becomes the mainstream direction of the modern breeding. The molecular breeding comprises protoplast fusion, somatic cell crossbreeding, and target gene transfer, integration and expression, and is used for germplasm innovation, variety improvement, molecular marker-assisted breeding and the like.

The fusion between protoplasts for cell hybridization or cell reconstruction is an important means for plant cell engineering. In the existing fusion technology, polyethylene glycol (PEG) induced fusion and electrofusion are mainly used. The PEG fusion technology has the advantages of simple equipment, convenient operation and the like, is mainly applied to dicotyledonous plants and monocotyledonous plants, but is rarely applied to marine plants. At present, the method of the kelp protoplast is unstable, mainly takes the single gametophyte as a transformation object by means of the characteristic of the abnormal generation alternate life history of the kelp, and generates sporophytes through parthenogenesis or fertilization development after transformation, thereby completing the whole transgenic operation. The existing PEG fusion technology has the defects of low protoplast purity, low fusion product purity, difficult screening after fusion, few parent protoplasts for fusion and the like.

Disclosure of Invention

The invention aims to provide a kelp molecule breeding method based on protoplast. Overcomes the defects in the prior art, utilizes kelp spores as natural protoplasts, combines a PEG fusion technology and a transgenic technology, and uses PEG to induce plasmids containing target genes to be transferred into the protoplasts, optimizes the preparation and fusion transformation methods of the protoplasts, and provides a new approach for molecular genetic breeding of kelp.

The invention provides a kelp molecule breeding method based on protoplast, which comprises the following steps:

(1) preparation of natural kelp protoplast

Drying seed sea in the shade at 8 deg.C overnight, and spraying herba Zosterae Marinae with cold sea water to obtain spore solution, i.e. herba Zosterae Marinae natural protoplast;

(2) protoplast transformation

Adding exogenous plasmid pBI221-stGBSS-Bar-GFP or pBI221-Bar-GFP into the spore liquid obtained in the step (1), adding PEG4000 solution, uniformly mixing, and standing and culturing in a dark place to obtain a spore culture solution;

adding a W5 solution into the spore culture solution, standing, centrifuging, removing supernatant, collecting precipitate, adding sterilized seawater into the precipitate, and performing conversion culture in a dark place to obtain a spore solution after the conversion culture;

the gene sequence of the exogenous plasmid pBI221-stGBSS-Bar-GFP is shown as SEQ ID NO: 1 is shown in the specification;

the gene sequence of the exogenous plasmid pBI221-Bar-GFP is shown as SEQ ID NO: 2 is shown in the specification;

(3) culturing seedlings

Culturing the seedling curtain: and (3) adding the culture solution obtained after the transformation culture in the step (2) into a large tray, putting a seedling curtain and a glass slide into the large tray, taking out the seedling curtain after the adhesion culture, putting the seedling curtain into a culture water tank, adding sterilized seawater into the culture water tank, and continuously culturing to obtain the sporophyte seedlings.

Specifically, the method comprises the following steps:

(1) preparation of natural kelp protoplast

Selecting seed herba Zosterae Marinae with well developed sporangia, washing with filtered seawater to remove surface impurities, drying in shade at 8 deg.C overnight, cooling with boiled filtered sterilized cold seawater to stimulate the seed herba Zosterae Marinae to release free spores with the release amount of 0.5-2.0 × 10⁶Obtaining spore liquid, namely the kelp natural protoplast;

(2) protoplast transformation

Adding exogenous plasmid pBI221-stGBSS-Bar-GFP or pBI221-Bar-GFP into the spore liquid obtained in the step (1), adding PEG4000 solution, uniformly mixing, and standing and culturing at 8 ℃ in a dark place for 30min to obtain a spore culture solution;

adding W5 solution into spore culture solution, standing at 8 deg.C, centrifuging at 2500rpm for 15min, and settling cells at the bottom of the tube; removing supernatant, adding sterilized seawater into the precipitate, performing transformation culture at 8 deg.C in dark place for 8-168 hr, and performing fluorescence observation under fluorescence microscope Blue to obtain culture solution after transformation culture;

(3) culturing seedlings

Culturing the seedling curtain: and (3) adding the culture solution obtained after the transformation culture in the step (2) into a large tray, putting a seedling curtain and a slide, performing adhesion culture for 2 hours, taking out the seedling curtain, putting the seedling curtain into a culture water tank, adding sterilized seawater, and continuing to culture for 30 days after the number of cells adhered to the slide reaches 28-70 under the field of view of 10 multiplied by 10, thereby obtaining the sporophyte seedlings.

In some specific embodiments, the method wherein the protoplasts of step (2) are converted into:

taking a 5000mL triangular flask, firstly adding 400mL of the spore solution obtained in the step (1), then adding 40mg of exogenous plasmid, uniformly mixing, adding 400mL of PEG4000 solution, uniformly mixing, and standing and culturing for 30min at 8 ℃ in a dark place;

adding a W5 solution into the mixed solution to make the system reach 4000mL, and standing for 5min at 8 ℃ to obtain a spore solution; centrifuging at 2500rpm for 15min to deposit the cells on the bottom of the tube; discarding the supernatant, adding 4000ml of sterilized seawater, keeping out of the sun at 8 ℃, and performing transformation culture;

the amount of the free spores released from the kelp seeds in the spore liquid in the step (1) is 0.5-2.0 multiplied by 10⁶Per mL; preferably 0.8X 10⁶one/mL.

Specifically, the PEG4000 solution in step (2) is an aqueous PEG4000 solution, has a pH of 5.7, and is prepared from filtered and sterilized seawater, and the volume percentage content of the PEG4000 is 20 to 50%, preferably 30 to 40%, and more preferably 35%.

Preferably, the aqueous solution of PEG4000 further comprises 0.4M mannitol, pH 5.7, prepared from filtered sterilized seawater, and the volume percentage of PEG4000 is 20-50%, preferably 30-40%, and more preferably 35%.

Preferably, the W5 solution described in step (2) contains 154mM NaCl and 5mM KCl, has a pH of 5.7, and is prepared from autoclaved seawater.

Preferably, the exogenous plasmid in the step (2) is pBI221-Bar-GFP, and is transformed and cultured for 24-48h at 8 ℃ in a dark place.

Preferably, the exogenous plasmid in the step (2) is pBI221-stGBSS-Bar-GFP, and is protected from light at 8 ℃ and transformed and cultured for 8 h.

Compared with the prior art, the invention has the advantages that:

1. the protoplast used by the method is a natural protoplast of the kelp, the protoplast has good activity and high development and transformation efficiency, and the PEG fusion efficiency can be improved; meanwhile, the protoplast can be used for large-batch PEG fusion experiments, and more genetically transformed plants can be obtained at one time; in addition, the protoplast can be applied to PEG fusion transformation of plasmids carrying different exogenous genes, and has wide application range. Solves the problems of low purity of protoplast, small number of parent protoplast for fusion and the like in the PEG fusion technology.

2. The exogenous plasmid used by the method provided by the invention is pBI221-stGBSS-Bar-GFP or pBI221-Bar-GFP, the conversion rate is obviously improved, and the conversion culture time is shorter. When the exogenous plasmid is pBI221-Bar-GFP, the transformation culture is carried out for 24-48h, and the average transformation rate can reach 1.83% -1.92%; when the exogenous plasmid is pBI221-stGBSS-Bar-GFP, the transformation culture is carried out for 8 hours, and the average transformation rate reaches 2.54 percent. The method provided by the invention also optimizes the operation process of fusion transformation, shortens the period, obviously reduces the cell death rate and lowers the economic cost, and provides a new approach for molecular genetic breeding of the kelp.

Drawings

FIG. 1 is a map of plasmid pBI221-GFP of example 2;

FIG. 2 is a map of plasmid pBI221-stGBSS-Bar-GFP of example 2;

FIG. 3 is a map of plasmid pBI221-Bar-GFP of example 2;

FIG. 4 is a view showing observation under a fluorescence microscope and a normal microscope at the time of transformation culture for 8 hours in example 2;

FIG. 5 is a view showing observation under a fluorescence microscope and a normal microscope at the time of transformation culture for 24 hours in example 2;

FIG. 6 is a view showing observation under a fluorescence microscope and a normal microscope at the time of transformation culture for 48 hours in example 2;

FIG. 7 is a view showing observation under a fluorescence microscope and a normal microscope at the time of transformation culture for 72 hours in example 2;

FIG. 8 is a view showing observation under a fluorescence microscope and a normal microscope at 168 hours of the transformation culture in example 2;

Detailed Description

The following description of the embodiments is only intended to aid in the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The following description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions.

Example 1

Preparation of natural kelp protoplast

Selecting seed kelp with well developed sporangia, slightly washing the seed kelp to remove surface impurities (not touching, slightly washing) with filtered seawater, placing in a foam box, adding an ice bottle at 8 ℃ (namely 3 storehouses), drying in the shade overnight, using a burnt filter sterilization cold seawater and ice bottle to stimulate the kelp to diffuse free spores in a big box to obtain spore liquid (namely kelp natural protoplast), and counting the cell concentration of the spore liquid before and after enrichment under a blood counting cell plate.

The amount of zoospore is 0.8 × 10⁶one/mL was used for subsequent experiments.

Example 2

PEG induced transformation

(1) Plasmid pBI221-stGBSS-Bar-GFP

The plasmid pBI221-GFP (map: FIG. 1, available from Shanghai enzyme research Biotechnology Co., Ltd., product No.: MJ1389) was transformed into pBI221-stGBSS-Bar-GFP plasmid, the map of which is shown in FIG. 2, and the gene sequence is shown in SEQ ID NO: 1 is shown.

Firstly, taking a 5000mL triangular flask, adding 400mL enriched spore liquid, then adding 40mg exogenous plasmid pBI221-stGBSS-Bar-GFP, and uniformly mixing. Adding 400mL of 35% PEG4000 solution, mixing uniformly, standing and culturing for 30min at 8 ℃ in the dark to obtain a mixed solution;

adding 3200mL of W5 into the mixed solution to make the system reach 4000mL, standing at 8 ℃ for 5min, centrifuging at 2500rpm for 15min, and depositing cells at the bottom of the tube;

③ removing the supernatant, adding 4000mL of sterilized seawater, keeping out of the sun at 8 ℃, and carrying out transformation culture to obtain the spore liquid. Fluorescence observation is carried out under a fluorescence microscope Blue grade at 8h, 24h, 48h, 72h and 168h (7d) (the transformed spores show red-yellow overlapped fluorescence and the untransformed spores show red-overlapped fluorescence), and the observation results are respectively shown in FIG. 4, FIG. 5, FIG. 6, FIG. 7 and FIG. 8. More than 20 visual fields are randomly selected for each observation (the total number of cells to be observed is ensured to be more than or equal to 200 cells), the total number of the cells successfully transformed is respectively counted, the transformation efficiency is calculated, the number of dead cells is counted, the death rate is calculated, and the result is shown in table 1.

Cell attachment culture:

glass slide: dripping 70ul of transformation culture solution at the groove of each slide to obtain spore solution, culturing 15 slides in each group of the experimental group with 7 groups, 105 slides and 210 grooves in total in each group, putting the slides in culture dishes for culturing for 24 hours, adding 50mL of seawater culture solution into each culture dish, and culturing in a dark place;

seedling curtain: adding 10L of water into each large tray, simultaneously placing 10 seedling curtains (for double-layer culture), 4 glass slides (for detecting the attachment density), totally 6 culture boxes with 60 seedling curtains, attaching for 2 hours, taking out the seedling curtains, placing the seedling curtains into a culture water tank, and adding sterilized seawater for culture after the number of cells attached to the glass slides reaches 70, 29, 60, 36, 45 and 43 cells per visual field under the visual field of 10 multiplied by 10.

(2) Plasmid pBI221-Bar-GFP

The plasmid pBI221-GFP is transformed into a pBI221-Bar-GFP plasmid, the map of which is shown in figure 3, and the gene sequence is shown in SEQ ID NO: 2, respectively.

Firstly, taking a 2000mL triangular flask, adding 100mL enriched spore liquid, then adding 10mg exogenous plasmid pBI221-Bar-GFP, and uniformly mixing. Adding 100mL of 35% PEG4000 solution, mixing, and standing and culturing for 30min in the dark at 8 deg.C (3 days to obtain a mixed solution);

adding 800mLW5 to the mixed solution to make the system reach 1000mL, standing at 8 ℃ for 5min, centrifuging at 2500rpm for 15min, and depositing the cells at the bottom of the tube;

③ removing the supernatant, adding 1000mL of sterilized seawater, keeping out of the sun at 8 ℃, and carrying out transformation culture to obtain the spore liquid. Fluorescence observation is carried out under a fluorescence microscope Blue grade at 8h, 24h, 48h, 72h and 168h (7d) (the transformed spores show red-yellow overlapped fluorescence and the untransformed spores show red-overlapped fluorescence), and the observation results are respectively shown in FIG. 4, FIG. 5, FIG. 6, FIG. 7 and FIG. 8. More than 20 visual fields are randomly selected for each observation (the total number of cells to be observed is ensured to be more than or equal to 200 cells), the total number of the cells successfully transformed is respectively counted, the transformation efficiency is calculated, the number of dead cells is counted, the death rate is calculated, and the result is shown in table 2.

Cell attachment culture:

seedling curtain: adding 10L of water into each large tray, simultaneously placing 10 seedling curtains (for double-layer culture), 4 glass slides (for detecting the attachment density), totally 1 culture box with 10 seedling curtains, attaching for 2 hours, taking out the seedling curtains, placing the seedling curtains into a culture water tank, and adding sterilized seawater for culture after the number of cells attached to the glass slides reaches 28 cells per visual field under the visual field of 10 multiplied by 10.

(3) Control

Firstly, taking a 1000mL triangular flask, adding 100mL enriched spore liquid, and uniformly mixing. Adding 100mL of 35% PEG4000 solution, mixing, and standing and culturing for 30min in the dark at 8 deg.C (3 days to obtain a mixed solution);

adding about 800mLW5 into the mixed solution to make the system reach 1000mL, standing for 5min at 8 ℃ (only in 3 banks), centrifuging for 15min at 2500rpm, and depositing the cells at the bottom of the tube;

③ removing the supernatant, adding 1000mL of sterilized seawater, keeping out of the sun at 8 ℃, and carrying out transformation culture to obtain the spore liquid. Fluorescence observation is carried out under a fluorescence microscope Blue file of 8h, 24h, 48h, 72h and 168h (7d), and the observation results are respectively shown in FIG. 4, FIG. 5, FIG. 6, FIG. 7 and FIG. 8. More than 20 visual fields are randomly selected for each observation (the total number of cells is ensured to be observed to be more than or equal to 200 cells), the number of dead cells is counted, the death rate is calculated, and the result is shown in table 3.

Cell attachment culture:

seedling curtain: adding 10L of water into each large tray, simultaneously placing 10 seedling curtains (for double-layer culture), 4 glass slides (for detecting the attachment density), totally 1 culture box with 10 seedling curtains, attaching for 2 hours, taking out the seedling curtains, placing the seedling curtains into a culture water tank, and adding sterilized seawater for culture after the number of cells attached to the glass slides reaches 42 cells per field under the field of view of 10 multiplied by 10.

Table 1: pBI221-stGBSS-Bar-GFP transformation results

Table 2: pBI221-Bar-GFP conversion results

Table 3: control group results

As can be seen from tables 1-3, when the foreign plasmid is pBI221-stGBSS-Bar-GFP, the transformation culture time is shortest, the average transformation rate is the greatest, and the cell death rate is the lowest; when the exogenous plasmid is pBI221-Bar-GFP, the transformation culture time is shorter, the average transformation rate is higher, and the cell death rate is relatively lower. The method for breeding kelp molecules based on protoplasts effectively shortens the culture period, improves the conversion rate and reduces the cell death rate.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> China oceanic university

<120> kelp molecule breeding method based on protoplast

<130> 2018

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6930

<212> DNA

<213> Artificial Synthesis (Artificial)

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tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc 4920

gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg 4980

gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat 5040

ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc 5100

acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa 5160

ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa 5220

aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt 5280

gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct 5340

tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat 5400

gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg 5460

cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg 5520

atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt 5580

attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg 5640

ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg 5700

gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg 5760

tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa 5820

aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt 5880

tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 5940

tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 6000

ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 6060

ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 6120

gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 6180

aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 6240

ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 6300

agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 6360

aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 6420

aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 6480

ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 6540

cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat 6600

tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 6660

accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct 6720

ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa 6780

gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct 6840

ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac 6900

acaggaaaca gctatgacca tgattacgcc 6930

<210> 2

<211> 5103

<212> DNA

<213> Artificial Synthesis (Artificial)

<400> 2

aagcttgcat gcctgcaggt ccccagatta gccttttcaa tttcagaaag aatgctaacc 60

cacagatggt tagagaggct tacgcagcag gtctcatcaa gacgatctac ccgagcaata 120

atctccagga aatcaaatac cttcccaaga aggttaaaga tgcagtcaaa agattcagga 180

ctaactgcat caagaacaca gagaaagata tatttctcaa gatcagaagt actattccag 240

tatggacgat tcaaggcttg cttcacaaac caaggcaagt aatagagatt ggagtctcta 300

aaaaggtagt tcccactgaa tcaaaggcca tggagtcaaa gattcaaata gaggacctaa 360

cagaactcgc cgtaaagact ggcgaacagt tcatacagag tctcttacga ctcaatgaca 420

agaagaaaat cttcgtcaac atggtggagc acgacacact tgtctactcc aaaaatatca 480

aagatacagt ctcagaagac caaagggcaa ttgagacttt tcaacaaagg gtaatatccg 540

gaaacctcct cggattccat tgcccagcta tctgtcactt tattgtgaag atagtggaaa 600

aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg aaaggccatc gttgaagatg 660

cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag 720

aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa 780

gggatgacgc acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat 840

ttcatttgga gagaacacgg gggactctag aggatccatt accatgatgg agtttagatt 900

agcgaaaaag tctgatgcta aaagactttt agaaatttat aaaccttatg ttgaaaaaac 960

tgccattact tttgagtatg aagtcccaac gattgcagaa tttgaaaagc gaattgaaaa 1020

aataggaagt cgctatcctt atattgtggc gattgaaaat gacaaaatca ttggttatgc 1080

ttatgctggc gcttatcgag aacgggcggc ttatgattgg gtggttgaat tatcaattta 1140

tttagatgaa aatgaacggc aacatggcgc tggtagtgcc ctttaccaaa agttactgac 1200

agctttatcg gtacttaatt atcagcgtgc ttatgcttgt attacctatc caaatccagc 1260

aagtgttgca tttcataaaa aatttggttt tgaacaaatt ggactttttc caaaagcggg 1320

ttataagttt gaacaatggt atggtattgt ttggttagaa cgctctctcc aaactactga 1380

taaagttact gcgataaaat tactgacaga tttgtcagag aatgaactag aaaaaatctt 1440

ggctaatgat cttcaagagc tccgtcgaca agcttatggt gagcaagggc gaggagctgt 1500

tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca 1560

gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct 1620

gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccttc acctacggcg 1680

tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca 1740

tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga 1800

cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca 1860

tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc 1920

acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc 1980

gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca 2040

tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga 2100

gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg 2160

ggatcactca cggcatggac gagctgtaca agtaaagcgg cccgaatttc cccgatcgtt 2220

caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta 2280

tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt 2340

tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag 2400

aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac 2460

tagatcggga attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt 2520

acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag 2580

gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg 2640

cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt 2700

acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac 2760

gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc 2820

gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc 2880

ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca 2940

ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 3000

tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 3060

aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 3120

tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag 3180

ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt 3240

tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 3300

gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 3360

aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 3420

agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg 3480

acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta 3540

actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 3600

accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt 3660

actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 3720

cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 3780

cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 3840

gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag 3900

ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt 3960

tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 4020

aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 4080

gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 4140

acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 4200

tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 4260

ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 4320

atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 4380

agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 4440

cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 4500

agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 4560

acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 4620

gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 4680

ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 4740

gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 4800

gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 4860

gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa 4920

tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat 4980

gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg 5040

ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac 5100

gcc 5103

Claims

1. A method for molecular breeding of kelp based on protoplast is characterized in that: the method comprises the following steps:

(1) preparation of natural kelp protoplast

Selecting seed herba Zosterae Marinae with well-developed sporangium, washing seed herba Zosterae Marinae with filtered seawater to remove surface impurities, and filtering at 8 deg.CDrying in shade, and filtering sterilized cold seawater cooled after boiling to stimulate the seed Laminaria to release zoospore with the release amount of 0.5-2.0 × 10⁶Obtaining spore liquid, namely the kelp natural protoplast;

(2) protoplast transformation

Adding an exogenous plasmid pBI221-stGBSS-Bar-GFP into the spore liquid obtained in the step (1), adding a PEG4000 solution, uniformly mixing, and standing and culturing at 8 ℃ in a dark place for 30min to obtain a spore culture solution;

adding W5 solution into spore culture solution, standing at 8 deg.C, centrifuging at 2500rpm for 15min, and settling cells at the bottom of the tube; removing supernatant, adding sterilized seawater into the precipitate, performing transformation culture at 8 deg.C in dark place for 8 hr, and performing fluorescence observation under fluorescence microscope at Blue level to obtain transformed and cultured spore solution;

(3) culturing seedlings

Culturing the seedling curtain: and (3) adding the spore liquid transformed and cultured in the step (2) into a large tray, putting a seedling curtain and a slide, performing adhesion culture for 2h, taking out the seedling curtain, putting the seedling curtain into a culture water tank, adding sterilized seawater, and continuing to culture for 30d after the number of cells adhered to the slide reaches 28-70 under the field of view of 10 multiplied by 10, thereby obtaining the sporophyte seedlings.

2. The method according to claim 1, wherein the PEG4000 solution in step (2) is PEG4000 aqueous solution, pH is 5.7, and is prepared from filtered sterilized seawater, and the volume percentage of the PEG4000 is 20-50%;

the W5 solution described in step (2) contained 154mM NaCl and 5mM KCl, pH 5.7, and was prepared from autoclaved seawater.

3. The method of claim 2, wherein the aqueous PEG4000 solution contains 0.4M mannitol, has a pH of 5.7, and is prepared from sterile filtered seawater, and the PEG4000 is present in an amount of 20-50% by volume.

4. The method of claim 2, wherein the PEG4000 is present in an amount of 30 to 40% by volume.

5. The method of claim 4, wherein the PEG4000 is present in an amount of about 35% by volume.

6. The method of claim 3, wherein the PEG4000 is present in an amount of 30 to 40% by volume.

7. The method of claim 6, wherein the PEG4000 is present in an amount of about 35% by volume.