CN117384949A

CN117384949A - Method for producing recombinant thaumatin II sweet protein by using rice endosperm cells as bioreactor

Info

Publication number: CN117384949A
Application number: CN202311320741.3A
Authority: CN
Inventors: 张勇; 郑雪莲; 钟超; 向兵
Original assignee: Chengdu Jigu Gene Technology Co ltd
Current assignee: Chengdu Jigu Gene Technology Co ltd
Priority date: 2023-10-12
Filing date: 2023-10-12
Publication date: 2024-01-12

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for producing recombinant thaumatin II sweet protein by using rice endosperm cells as a bioreactor. The technical problem to be solved by the invention is that the existing method for producing thaumatin II sweet protein by taking procaryotic and eucaryotic as host bioreactors has the defects of low expression quantity, poor solubility, no bioactivity, unsafety and the like. The technical scheme of the invention is that the method for producing recombinant thaumatin II sweet protein by using rice endosperm cells as a bioreactor comprises the following steps: constructing a vector for expressing the recombinant thaumatin II gene, and transforming rice to obtain a callus containing the recombinant thaumatin II sweet protein; the recombinant thaumatin II gene has a nucleotide sequence shown as SEQ ID NO. 1. The invention expresses the cord moldy sweet protein through the seed bioreactor, and directionally stores the recombinant protein in a protein body, thereby avoiding the degradation of protease and continuously accumulating in the seed maturation process, and further obtaining higher expression.

Description

Method for producing recombinant thaumatin II sweet protein by using rice endosperm cells as bioreactor

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for producing recombinant thaumatin II sweet protein by using rice endosperm cells as a bioreactor.

Background

Sweeteners are food additives that impart sweetness to food products or are classified as natural sweeteners and artificial sweeteners depending on the source. The traditional sweeteners such as glucose, starch, sucrose, fructose and the like are widely used in foods and other consumer products, have high nutrition and safety, but have extremely high heat and large dosage, are extremely easy to cause cardiovascular and cerebrovascular diseases, hypertension, obesity, diabetes and the like after long-term consumption, become a serious social problem, and seriously threaten human life and health. With the development of organic chemistry, artificial sweeteners are dominant, and although zero-calorie high-power sweeteners such as aspartame, acesulfame potassium, sucralose and stevioside can provide enough sweet taste, the artificial sweeteners can show bad peculiar smell such as metallic taste and after-bitter taste to different degrees, and the artificial sweeteners such as aspartame have questionable safety. In view of this, finding a sweetener that is natural and nontoxic, and has a pure sweetness is of great significance. Since 1968, many tropical plant sweet proteins were discovered successively, such as thaumatin, monellin, mabinlin, miraculin, pentadin, curculin and brazzein. The sweet proteins have the characteristics of high sweetness, low calorie, pure taste, pure nature, no side effect and the like, so that the sweet proteins have very broad application prospects. Sweet protein has no influence on human health, and thaumatin is the most widely studied sweet protein at present and is one of the most promising substitutes of sucrose and artificial sweeteners.

Among various sweet proteins, thaumatin, which is sweet and tasty, has no bad aftertaste or bitter taste, has the effect of enhancing the flavor of the product and masking bad taste, and has a stable sweet feeling in a range of pH2 to 10 and heating at 100 ℃ or below (or ultra-high temperature transient sterilization at 100 ℃ or above), has been the most studied and commercialized. The natural Thaumatin is extracted from tropical rain forest plant Saint Maranta arundinacea (Thaumatococcus daniellii) of western African, and has the characteristics of natural, low calorie, safety, no toxicity, and degradation into amino acids required by human body. At the same level, thaumatin has a sweetness of about 3000 times that of sucrose, and is a low-calorie sweetener. Both major subtypes of Thaumatin (I and II) consist of polypeptide chains of 207 amino acid residues, with a relative molecular weight of about 22kDa. Among them, thaumatin II is most widely studied and used. In addition, the metabolism of thaumatin in vivo is consistent with the metabolism of other edible proteins. The world health organization food additive expert committee reviews its safety toxicology and agrees to use it as a safe food additive without any restrictions on its daily allowable intake (ADI value). The Thaumatin has extremely high sweetness, extremely low sweetness threshold, refreshing taste, no peculiar smell and long duration. In addition, thaumatin has good processing stability, is stable in pasteurized and baked foods, is not affected by common preservatives, is stable in use in food and beverage formulations, and has a shelf life of a dried product of more than two years.

Thaumatin was first approved in 1979 as a natural food additive for sale in japan; thaumatin was approved for use in medicine in the united kingdom in 1981 and as a sweetener in food in 1986; european approval of its use as sweetener and flavor enhancer since 1994; similar approvals have associated regulations in switzerland, israel, canada, mexico, korea, singapore, australia, new zealand and south america, among other countries and regions. Subsequently, the U.S. Food and Drug Administration (FDA) and the U.S. food Flavor and Extract Manufacturing Association (FEMA) consider thamatin to be safe, approving it for use in foods. In 2014, thaumatin is officially approved as a food additive in China, and the maximum usage amount of frozen drinks (except edible ice), processed nuts and seeds, baked foods, table sweeteners and beverages (except packaged drinking water) is 0.025 g/kg, so that the application of thamatin in the food industry in China is greatly promoted.

As Thaumatococcus daniellii Benth is extremely harsh on growth conditions, it is not firm in places other than the original place, and attempts to extract Thaumatin in large quantities by artificial cultivation Thaumatococcus daniellii Benth are not possible, and many researchers around the world have failed to introduce programs. Thus, it is desirable to host the genetic engineering of thaumatin. The transformation of thaumatin genes into different hosts including E.coli, fungi and higher plants has been expected for thirty years to obtain large amounts of recombinant, active thaumatin, but the results are not ideal, and recombinant proteins are either unsweetened or have low yields, especially in higher plants. Recombinant proteins expressed in prokaryotes are typically low in content or inactive because they do not fold properly and there are no glycosylation modifications, etc. Expression systems such as fungi can correctly post-translationally modify the expression product, but still face the problem of focusing on histone separation and purification.

The production of fusion proteins by plant seed bioreactors is an effective solution, and fusion proteins can be stably maintained in the seed endosperm protein body. Because crop seeds are easy to preserve, the seed can be preserved for many years at normal temperature, but can be preserved for decades in a refrigeration house, the continuous production of the egg can be realized, the problems of resource waste and shortage are not caused, and the production cost is reduced.

Disclosure of Invention

The technical problem to be solved by the invention is that the existing method for producing thaumatin II sweet protein by taking procaryotic and eucaryotic as host bioreactors has the defects of low expression quantity, poor solubility, no bioactivity, unsafety and the like.

The technical scheme of the invention is that the method for producing recombinant thaumatin II sweet protein by using rice endosperm cells as a bioreactor comprises the following steps: constructing a vector for expressing the recombinant thaumatin II gene, and transforming rice to obtain a callus containing the recombinant thaumatin II sweet protein; the recombinant thaumatin II gene has a nucleotide sequence shown as SEQ ID No. 1.

In particular, the method further comprises the steps of: and co-culturing, screening, regenerating and rooting the obtained callus to obtain a plant containing the recombinant thaumatin II sweet protein.

Specifically, the element for expressing the recombinant thaumatin II gene in the vector for expressing the recombinant thaumatin II gene comprises ZmUbi-recombinant thaumatin II gene-AtHsp.

In particular, the vector for expressing the recombinant thaumatin II gene also contains a hygromycin resistance screening gene expression element which is started by a CaMV35s promoter and has the structure of CaMV35s-Hyg-CaMV35s polyA.

Wherein the vector skeleton of the recombinant thaumatin II gene is pLSD298.

Specifically, the nucleotide sequence of the vector for expressing the recombinant thaumatin II gene is shown as SEQ ID No. 3.

Specifically, the transformed rice adopts an agrobacterium-mediated method.

Further, the rice variety is Nipponbare.

The invention also provides recombinant thaumatin II sweet protein obtained by the method.

The invention has the beneficial effects that: the invention optimizes the codon of the fumartin protein, is suitable for being expressed in rice, and obtains a transgenic plant. The recombinant thaumatin II protein gene is expressed in all tissues and organs of transgenic rice, the expression level in seeds and leaves is highest, and the expression level in stems and internodes and roots is relatively low. Therefore, the whole rice plant can be used for producing thaumatin II sweet protein, especially seeds, which is very favorable for preserving and transporting sweet protein. Expressing the marmorsweet protein by using a seed bioreactor, and directionally storing the recombinant protein in a protein body, so that degradation of protease is avoided, and the protein is continuously accumulated in the seed maturation process, thereby obtaining higher expression; meanwhile, the recombinant protein enters an inner membrane system and can be correctly processed and folded to generate the recombinant protein with bioactivity, and the recombinant protein can directly taste high sweetness. The invention has the following specific advantages:

1) The expression quantity of sweet protein in each tissue of the whole plant is high;

2) The biological activity is high;

3) High safety: because plant seeds such as rice, wheat, barley and the like are main foods of human beings, protein products produced by the plant seeds are absolutely free from being polluted by any pathogenic bacteria, so that the safety is high;

4) The production cost is low: as the whole rice plant has high expression, and the expression quantity in seeds and leaves, the rice plant is produced in large scale in agricultural production, the production cost is extremely low and is 1/200-1/10 of that of other production systems;

5) The storage and transportation are convenient: recombinant proteins are stored in seeds (especially endosperm cells) in a colloidal state, are extremely stable, and are usually stored for 2-3 years at normal temperature, so that raw materials are not limited by time and space for storage and transportation;

6) The mass production is very easy, and after stable transgenic varieties are formed, the technology can be rapidly expanded when necessary without adding any plant facilities;

7) Low carbon and environmental protection: through agricultural cultivation, plant cells synthesize proteins and accumulate through photosynthesis, so that a large amount of energy and raw materials required by a traditional production system can be saved, and the method accords with the low-carbon and environment-friendly concepts of the modern industry.

Drawings

FIG. 1, PCR amplification product of recombinant thaumatin II protein gene. Lane 1 is a molecular marker, lane 2 is a PCR product, and the arrow indicates the size of the 730 base recombinant thaumatin II protein gene sequence.

FIG. 2, schematic structural diagram of expression vector pLSD298. Wherein Ubi1 promoter is maize Ubiquitin promoter, att HSP terminator is arabidopsis HSP terminator, caMV35S promoter is 35S promoter of cauliflower mosaic virus (CaMV), hyg is hygromycin resistance selection gene hygromycin phosphotransferase gene hpt, caMV35S poly a is terminator, kanamycin is kanamycin resistance selection gene neomycin phosphotransferase gene npt II.

FIG. 3, restriction enzyme map of the constructed rice endosperm expression vector pLSD298-thaumatin II. Wherein Ubi1 promoter is maize Ubiquitin promoter, thaumatin II is 730 bases recombinant thaumatin II protein gene sequence, atHSP terminator is Arabidopsis HSP terminator, caMV35S promoter is 35S promoter of cauliflower mosaic virus (CaMV), hyg is hygromycin resistance screening gene hygromycin phosphotransferase gene hpt, caMV35S polyA is terminator, kanamycin is kanamycin resistance screening gene neomycin phosphotransferase gene npt II.

FIG. 4 PCR amplification product of the thaumatin expression vector pLSD298-thaumatin II. Lanes 1 are molecular markers, lanes 2, 3, 4, 5 and 6 are PCR products, and the arrow indicates the size of 1093 bases of the expression vector pLSD298-thaumatin II sequence.

FIG. 5, callus status during recombinant thaumatin II screening.

FIG. 6, sweetness test of recombinant thaumatin II transgenic rice calli. Wherein, sweet Score is represented by sweet Score, control is Wild Type (WT) rice callus, #1, #2, #3, #4, #5 respectively represent 5 resistant calli obtained.

FIG. 7, analysis of transcript levels in T1 transgenic rice plants by recombinant thaumatin II. Wherein Relative exprression levels represents the expression level, control is Wild Type (WT) rice, #1, #2, #3, #4, #5 represents 5 independent transgenic rice plants, root represents the Root of each plant, stem represents the Stem of each plant, leaf represents the Leaf of each plant, node represents the internode of each plant, and Seed represents the Seed of each plant, respectively.

FIG. 8, western blot detection of expression of recombinant thaumatin II in endosperm of transgenic rice. Wherein, lane 1NC is non-transgenic rice Japanese sunny seed extract, lanes 2, 3, 4, 5 and 6 are 5 independent transgenic rice plants, and lane 7PC is thaumatin II standard.

Detailed Description

The method of the present invention is described in detail below:

construction of gene synthesis and expression vector of rice genetic code optimized marmorat protein gene: in order to obtain higher expression of recombinant thaumatin in rice endosperm, rice optimized sequence must be usedThe codon, thus, the amino acid sequence of the thaumatin gene (accession number J01209) was obtained from the American Biotechnology information center (NCBI) gene library by GenSmart ^TM Codon Optimization (Nanjing Jinsri Biotechnology Co., ltd.) the amino acid sequence of the thaumatin gene was converted into a nucleotide sequence containing an optimized rice genetic code by an on-line codon optimization tool, and then the thaumatin gene was artificially synthesized by PCR extension (see SEQ ID No. 1). The nucleotide sequence of the gene of the thaumatin after codon optimization is changed by 22.5 percent compared with the original gene of the thaumatin, but the amino acid sequence is unchanged.

The synthesized pair of PCR primers is shown as SEQ ID NO.4 and SEQ ID NO.5, bsmBI sites are added at two ends of the PCR primers, vector DNA with recombinant thaumatin II protein genes is used as a template, and standard PCR reaction is adopted to amplify a fragment containing recombinant thaumatin II with the length of 730 bp; the fragment with the recombinant thaumatin II protein gene is recovered by agarose gel electrophoresis separation, the fragment of the recombinant thaumatin II protein gene is connected to an expression vector plasmid pLSD298 in a Golden gate mode, and then the escherichia coli strain DH5 alpha is transformed to generate the vector plasmid pLSD 298-thamatin II for expressing the recombinant thaumatin II protein gene. Further, the vector plasmid pLSD 298-thamate II expressing the recombinant thamate II protein gene was transformed into Agrobacterium competent EHA105.

Expression vector pLSD298 describes: the invention provides a pLSD298 expression vector which is self-constructed and not disclosed, but all original papers are disclosed. As shown in fig. 2, the expression vector structure is: zmUbi-BsmBI-BsmBI-AtHsp. Wherein ZmUbi1 is a maize Ubiquitin promoter. The nucleotide sequence of the corn Ubiquitin promoter is shown as 36-934 in SEQ ID No. 3. The BsmBI is a restriction enzyme BsmBI, which recognizes the CGTCTCN site. The AtHSP is an Arabidopsis HSP terminator. The AtHSP coding nucleotide sequence is shown as 2756-3005 in SEQ ID NO. 3.

Furthermore, the expression vector also contains a hygromycin resistance screening gene which is started by a CaMV35s promoter and has the structure of CaMV35s-Hyg-CaMV35s polyA. Wherein, the CaMV35S is a 35S promoter of cauliflower mosaic virus (CaMV). The nucleotide sequence of the CaMV35s promoter is shown in 11426-10657 bits in SEQ ID No. 3. Hyg is hygromycin resistance screening gene hygromycin phosphotransferase gene hpt, and the nucleotide sequence of Hyg is shown in 10650-9625 positions in SEQ ID No. 3. The CaMV35s polyA is a terminator, and the nucleotide sequence of the CaMV35s polyA is shown in 9613-9398 positions in SEQ ID No. 3.

Agrobacterium-mediated genetic transformation of rice: removing shell of Nippon Rice seed, sterilizing with 75% alcohol for 2min, adding 15% sodium hypochlorite, adding 1 drop or 2 drops of Tween, sterilizing at 28deg.C in shaking table or common shaking table for 15 min, repeating the steps (without Tween); after the completion, the seeds are inoculated on a callus induction culture medium, and the callus is produced for genetic transformation by continuous illumination culture for 8-10 d at the temperature of 32 ℃. The induced callus is degerminated and seed-removed, placed into a centrifuge tube, 1mL of AAM liquid culture medium is added into a 1.5mL centrifuge tube, and the agrobacterium vector in a flat plate is scraped into the centrifuge tube and suspended. Adding 20mL of AAM liquid culture medium into an empty 50mL centrifuge tube, sucking the suspended bacterial liquid into 20mL of AAM liquid culture medium, uniformly mixing, measuring an OD value, calculating according to the measured value, diluting the concentration to 0.05-0.08, adding into the centrifuge tube containing the callus, uniformly mixing and infecting for 3 minutes, pouring out the bacterial liquid, and airing for 10 minutes on a cutting disc. The small filter paper is soaked in AAM liquid medium and placed on a co-culture medium for dark culture at 28 ℃ for 3d. After co-cultivation, transferring the calli on the cutting disc into 50mL centrifuge tubes, washing seeds 5 times with 30mL of sterile water for each tube, and washing 2 times with sterile water for a clean 50mL centrifuge tube; soaking in sterile water containing TM antibiotics for 10min. Pouring out the liquid, transferring to a cutting disc, airing for 30min near an alcohol lamp, carefully inoculating the callus to a screening culture medium, and continuously culturing for 2w under illumination at 32 ℃. After the screening culture, the newly-added callus is transferred to a differentiation regeneration medium, and is cultured for 2W at 28 ℃ under 16h/8h photoperiod. Resistant calli were transferred every two weeks until complete plants were formed and transferred to rooting medium. After obtaining transgenic plants, the transgenic plants are detected by PCR, and proved to contain the fumartin gene, and then are transferred to a test field to grow into mature seeds, which are T1 seeds.

EXAMPLE 1 Synthesis of Sumortin Gene containing Rice genetic codon

The amino acid sequence of the thaumatin gene (GenSmart) was obtained from the American Biotechnology information center (NCBI) gene library, accession number J01209, by GenSmart ^TM Codon Optimization (Nanjing Jinsri Biotechnology Co., ltd.) the amino acid sequence of the thaumatin gene was converted into a nucleotide sequence containing the optimized rice genetic code by an on-line codon optimization tool, and then the thaumatin gene was artificially synthesized by a PCR extension method. The nucleotide sequence of the gene of the thaumatin after codon optimization is changed by 22.5 percent compared with the original gene of the thaumatin, but the amino acid sequence is unchanged.

Nucleotide sequence of SEQ ID No.1 thaumatin gene

SEQ ID No.2 Sumay be a sweet protein amino acid sequence

EXAMPLE 2 construction of vectors for expression of thaumatin in Rice

The sequences of the synthesized pair of PCR primers are shown as SEQ ID No.4 (cgtctcacca tatggcggcc actacttgtt tcttttttct ct) and SEQ ID No.5 (cgtctctaag ctcattcatc ctccagctcg agggcggta), bsmBI sites are added at two ends of the PCR primers, so that a vector DNA with a recombinant thaumatin II protein gene is used as a template, the PCR result is shown as figure 1, and a fragment (the arrow indicates a highlight band) with the length of 730bp and containing the recombinant thamatin II is amplified by the PCR reaction; separating by agarose gel electrophoresis, recovering the fragment with the recombinant thaumatin II protein gene, connecting the fragment with the recombinant thaumatin II protein gene to an expression vector plasmid pLSD298 by adopting a Golden Gate mode, and transforming the connection product into escherichia coli competent DH5 alpha to generate a vector plasmid pLSD298-thaumatin II for expressing the recombinant thaumatin II protein gene. Further, the vector plasmid pLSD 298-thamate II expressing the recombinant thamate II protein gene was transformed into Agrobacterium competent EHA105.

The primers are pth-F (SEQ ID No.6: caccctgttg tttggtgtta cttctgcag) and pth-R (SEQ ID No.7: ttctaataaa cgctcttttc tct), the pLSD298-thaumatin II vector is subjected to PCR amplification and sequencing verification, the PCR result is shown in figure 4, and the highlighted band indicated by the arrow is the amplified target band. Comparing the sequencing result with the theoretical sequence by using SnapGene software, wherein the comparison result shows that the actual sequence of the thaumatin II structural gene is consistent with the theoretical sequence, namely the sequence is shown as SEQ ID No.1, no deletion, replacement, insertion and conversion of bases exist, and the vector with correct sequencing is the pLSD298-thaumatin II vector.

EXAMPLE 3 Agrobacterium-mediated genetic transformation of Rice

Removing shell of Nippon Rice seed, sterilizing with 75% alcohol for 2min, adding 15% sodium hypochlorite, adding 1 or 2 drops of Tween, sterilizing at 28deg.C in shaking table or common shaking table for 15 min, repeating the steps (without Tween); after the completion, the seeds are placed on a cutting disc and aired for 10min, inoculated on a callus induction culture medium and continuously cultured for 8-10 d under illumination at the temperature of 32 ℃ for genetic transformation. Firstly, the induced callus is removed from buds and seeds, placed into a centrifuge tube, the callus on each two plates is placed into a tube,

1mL of AAM liquid medium was added to 1.5mL of the centrifuge tube, and the Agrobacterium vector (one loop) in the plate was scraped into the centrifuge tube and suspended. Adding 20mL of AAM liquid culture medium into an empty 50mL centrifuge tube, sucking the suspended bacterial liquid into 20mL of AAM liquid culture medium, uniformly mixing, measuring an OD value, calculating according to the measured value, diluting the concentration to 0.05-0.08, adding into the centrifuge tube containing the callus, uniformly mixing and infecting for 3 minutes, pouring out the bacterial liquid, and airing for 10 minutes on a cutting disc. The small filter paper is soaked in AAM liquid medium and placed on a co-culture medium for dark culture at 28 ℃ for 3d. After the co-cultivation, the callus on the cutting disc is transferred to a 50mL centrifuge tube, each tube is washed with 30mL of sterile water for 5 times, the clean 50mL centrifuge tube is replaced with sterile water for 2 times, and the seed is soaked in sterile water containing TM antibiotics for 10 minutes. Pouring out the liquid, transferring to a cutting disc, airing for 30min near an alcohol lamp, carefully inoculating the callus to a screening culture medium, and continuously culturing for 2 weeks under illumination at 32 ℃. After the screening culture, a total of 5 resistant calli were obtained as shown in FIG. 5. The newly proliferated calli were transferred to differentiation regeneration medium for the first regeneration and cultured for 2 weeks at 28℃under 16h/8h photoperiod. Only the masses with good callus growth and the growing resistant callus are selected for transfer when the fresh culture medium is replaced for the second regeneration, and the masses with blackish brown and insignificant callus particles are discarded. In the transfer process, consistency of the regenerated callus and the original seeds must be ensured, and cross confusion among different strains is avoided. Resistant calli were transferred every two weeks until complete plants were formed and transferred to rooting medium. After obtaining transgenic plants, the transgenic plants are detected by PCR, and proved to contain the fumartin gene, and then are transferred to a test field to grow into mature seeds, which are T1 seeds.

Example 4 sweetness assay of thaumatin II in transgenic Rice calli

Double-blind experiments were performed using transgenic rice calli. The participants included 10 healthy volunteers, including 5 females and 5 males (between 18 and 35 years of age), all without any abnormality in the taste sensation. Sucrose solutions of 0%, 0.5%, 1%, 2%, 4%, 6% and 8% (w/v) were prepared as controls. 0.001g of transgenic rice callus was taken and placed on the front of the tongue of the subject to bite. Each volunteer tasted the sucrose solution or protein from lowest concentration to highest concentration, and after each test, the subject rinsed 3 times with drinking water to avoid residual taste, and each sample was tested in triplicate. The results showed that the sweetness of the #2, #3 and #4 calli out of 5 resistant calli was equivalent to 6.0% sucrose solution; the sweetness of the #1 callus is slightly lower, which is equivalent to that of 2% sucrose solution; the sweetness of #5 calli was the lowest, essentially equivalent to the sweetness of a 0.6% sucrose solution.

Example 5 sweetness taste of thaumatin II in transgenic Rice calli

Double-blind experiments were performed using transgenic rice calli. The participants included 10 healthy volunteers, including 5 females and 5 males (between 18 and 35 years of age), all without any abnormality in the taste sensation. Wild Type (WT) rice calli served as control. Transgenic rice calli (0.001 g) were taken and placed on the front of the tongue of the subject to bite, and after each test, the subject was rinsed 3 times with drinking water to avoid residual taste, and each sample was tested three times. The test scoring rules are as follows: "not sweet" (0), "slightly sweet" (1.0), "sweet" (2.0), "very sweet" (3.0) and "very sweet" (4.0). The results of the test of 10 volunteers from source are shown in fig. 6, with the sweetness of the calli #2, #3 and #4 being "very sweet" in 5 resistant calli; the sweetness of the #1 callus is slightly lower, and the sweetness of the #5 callus is the lowest, namely the sweetness of the #1 callus is slightly sweet. In the figure, control is Wild Type (WT) rice callus, and #1, #2, #3, #4, #5 are transgenic rice callus; results are expressed in means ± s.d; "" means P <0.001 compared to control, "" means P <0.01 compared to control.

EXAMPLE 6 analysis of transcriptional level of thaumatin II in T1 transgenic Rice

Trizol (Beijing Soy Bao technology Co., ltd.) is used to extract total RNA from roots, stems, leaves, internodes and seeds of T1 generation transgenic rice, and the expression level of recombinant thaumatin II protein gene at different tissue sites of transgenic rice plant is detected by RT-qPCR after reverse transcription. The primers are pth-F1 (SEQ ID No.8: gtagttacac ggtgtgggca) and pth-R1 (SEQ ID No.9: gtccttgccg tactggttga). As positive controls, the rice actin gene (actin) was used, with primers AC-F (SEQ ID No.10: accattggtg ctgagcgttt) and AC-F (SEQ ID No.11: cgcagcttcc attcctatga a). The results are shown in FIG. 7, wherein #1, #2, #3, #4, #5 represent 5 independent transgenic rice plants, root represents the Root of each plant, stem represents the Stem of each plant, leaf represents the Leaf of each plant, node represents the internode of each plant, and Seed represents the Seed of each plant, respectively. As shown in the figure, the recombinant thaumatin II protein gene is expressed in all 5 rice tissues and organs, the expression amount is highest in seeds and leaves, and then the expression amount is relatively low in roots after stems and internodes in comparison with non-transgenic plants.

Example 7 analysis and identification of thaumatin II

In this example, western blot hybridization was used to examine whether the recombinant thaumatin II protein expressed by the obtained transgenic rice was accumulated in rice endosperm cells. As a positive control, a recombinant thaumatin II protein from E.coli (MBS 1226455, myBioSource, USA) was used. 100mg of T1 seeds of transgenic rice plants identified as positive by PCR are respectively taken, 1mL of extraction buffer (50 mM Tris-HCl (pH 7.2), 2% (w/v) SDS, 0.6% (v/v) beta-mercaptoethanol and 4M urea) is ground for 60 minutes at 4 ℃, the mixture is uniformly mixed by vortex, and then the mixture is placed in a refrigerated centrifuge 10000rpm and centrifuged for 10 minutes at 4 ℃ to obtain crude protein extract. Samples were mixed with SDS-PAGE loading buffer and cooked at 100℃for 10 minutes and separated by 15% SDS-PAGE. The proteins on the membrane were then electrotransferred to PVDF membrane. The transferred film is decolorized by 5% skimmed milk (TBST preparation) on a shaking table at room temperature, and is sealed for 1h; adding diluted rabbit anti-thaumatin II polyclonal antibody (diluted with TBST 5% skimmed milk 1:2000, self-made), and incubating at 4deg.C overnight; washing with TBST on a decolorizing shaking table at room temperature for 5min for 3 times; HRP-labeled goat anti-rabbit IgG secondary antibody (diluted 1:10000 in 5% skim milk formulated with TBST, multi sciences, usa) was diluted, incubated for 1 cell at room temperature, and then washed 3 times with TBST on a decolorizing shaker at room temperature for 5min each. And finally, detecting target protein by using ECL luminous solution (biosharp, china) as a substrate. As a result, as shown in FIG. 8, there was a large accumulation of recombinant thaumatin II protein in endosperm of all 5 transgenic rice plants, compared with non-transgenic seeds.

SEQ ID No.3

Claims

1. A method for producing recombinant thaumatin II sweet protein using rice endosperm cells as a bioreactor, comprising the steps of: constructing a vector for expressing the recombinant thaumatin II gene, and transforming rice to obtain a callus containing the recombinant thaumatin II sweet protein; the recombinant thaumatin II gene has a nucleotide sequence shown as SEQ ID No. 1.

2. The method of claim 1, further comprising the step of: and co-culturing, screening, regenerating and rooting the obtained callus to obtain a plant containing the recombinant thaumatin II sweet protein.

3. The method of claim 1, wherein the thaumatin II gene expression element in the vector expressing the recombinant thaumatin II gene comprises ZmUbi-recombinant thaumatin II gene-AtHsp.

4. The method of claim 1, wherein the vector for expressing the recombinant thaumatin II gene further comprises a hygromycin resistance selection gene expression element, which is promoted by a CaMV35s promoter, and has a structure of CaMV35s-Hyg-CaMV35s polyA.

5. The method of claim 4, wherein the vector backbone for expressing the recombinant thaumatin II gene is pLSD298.

6. The method according to claim 5, wherein the nucleotide sequence of the vector expressing the recombinant thaumatin II gene is shown in SEQ ID No. 3.

7. The method of claim 1, wherein said transformed rice is agrobacterium-mediated.

8. The method of claim 1, wherein the rice variety is japan.

9. The method of any one of claims 1-8 resulting in a recombinant thaumatin II sweet protein.