CN101942477A - Method for improving expression level of target protein in transgenic rice endosperm - Google Patents
Method for improving expression level of target protein in transgenic rice endosperm Download PDFInfo
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Abstract
The invention discloses a method for improving expression level of a target protein in transgenic rice endosperm by use of a signal peptide coding sequence of a rice storage protein. The method comprises the following steps: connecting a rice GluA-2 gene signal peptide coding sequence with a target protein coding sequence to form a fusion protein gene; transforming the fusion protein gene into rice cells to obtain transgenic rice; and culturing and ripening the transgenic rice, and then expressing the target recombinant protein in seed endosperm. In the invention, the target protein coding sequence and the GluA-2 signal peptide coding sequence containing an SEQ ID NO. 1 sequence are constructed into the fusion gene and the fusion gene is led into a plant, which can obviously improve the expression level of the target protein in the transgenic rice endosperm.
Description
Technical field
The present invention relates to a kind of method that improves target protein expression amount in transgene paddy rice endosperm.More particularly, the present invention relates to the signal coding sequence of gluten GluA-2 of one of rice paddy seed storage protein and research and the application that utilizes this sequence, this sequence and target protein are merged, can improve the expression amount of target protein in transgene paddy rice endosperm significantly.The invention belongs to the biological gene engineering field.
Background technology
Utilize transgenic technology expression of heterologous genes in plant, to improve resistance, increase output and to improve emphasis and the focus that quality etc. has become the exploitation of modern agriculture biotechnology research.Especially in recent years, utilize this cheap production system of plant as bio-reactor, come heterologous protein, medicinal active polypeptide or vaccine, antibody, industrial enzymes or the protein etc. of the coding tool critical function of mass production reorganization, it is one of key areas of present plant genetic engineering development, its research and development are in the ascendant, might form the emerging biometric technology industry of a low input, high production.But, in plant bioreactor research, there are comparatively distinct issues, the expression amount that is target protein is extremely low, and some albumen can't be stablized and accumulates, as only account for the 0.01-1% of total soluble protein the amount of the recombinant antigen of expressing in the transgenic plant.Therefore, the novel plant bioreactor technology system of setting up efficient stable always is the emphasis and the difficult point of this area research.The mode that cellular metabolism synthetic protein is stored in Plants and Animals is different; Therefore, realize that recombinant protein (the especially target protein in some non-plant sources) efficiently expresses and stable accumulation, need follow the expression consistent with the albumen of plant own and store rule in transgenic plant cells.Except the codon of target protein encoding gene was made amendment, another gordian technique was gene expression regulation element and the target protein target sequence that will use the recipient plant source.Wherein, the promotor of using-system different expression gene and relevant target controlling element thereof, especially the signal coding sequence of the promotor of seed-specific expression gene and amino-end thereof (N-end) is considered to the effective way that realizes that target protein efficiently expresses in the specific tissue of plant.
Seed storage protein is a kind of composition that is rich in that is only second to starch in the rice paddy seed, and the expression of its encoding gene often has extremely strict tissue and space-time specificity, but also contains specific targeting proteins controlling element.When these genes form amyloid protein precursor in translation, its N-terminal all contains a segment signal peptide (signal peptide, SP) sequence, length is different with the difference of storage protein kind, and they play an important role in the location of storage protein and mRNA molecule thereof and transportation.After storage protein gene is transcribed out sophisticated mRNA, at first translation forms the precursor protein that contains signal peptide, this moment, this precursor protein was directionally transferred in the rough endoplasmic reticulum chamber by the signal peptide sequence traction, this signal peptide sequence is wiped out from precursor protein subsequently, so that storage protein can correctly be processed (Coleman etc. 1995).Except having the decisive action in the correct orientation of target protein, signal peptide sequence also has important effect (Boehm etc. 2000) on the regulate gene expression level.The last characteristic of signal peptide sequence has obtained good application in transgenic plant research.For example, utilize Kidney bean Actin muscle endoplasmic reticulum specific signals peptide sequence can obviously strengthen intestinal bacteria ubiquinone transcribing and expression amount subsequently in transgene tobacco.This shows that the signal peptide sequence of storage protein has active effect for the expression of foreign protein in transgenic plant with deposition.
Gluten is a topmost storage protein in the paddy endosperm, its generally synthetic in a large number and accumulation (Yamagata etc. 1982) in the middle and later periods of seed development.Though it is different fully with the solubility property of 11S sphaeroprotein in the leguminous plants, (Zhao etc. 1983 but they have many similarities on amino acid composition, protein structure and route of synthesis thereof, Wen and Luthe 1985), therefore the paddy rice gluten also is classified as in the sphaeroprotein extended familys of plant sometimes.The gluten that contains three kinds of different molecular weights in the rice paddy seed, be respectively the alkaline subunit (tool alkalescence iso-electric point) of the precursor gluten of 57KDa, the acid subunit of 37-39KDa (the acid iso-electric point of tool) and 20-22KDa, acid subunit wherein and alkaline subunit are to be sheared through the translation post-treatment by the gluten precursor to form (Yamagata etc. 1982).Sophisticated gluten all is heterogeneous, be made up of 12 polypeptide at least as the acid subunit of gluten, and alkaline subunit also contains 9 polypeptide (Wen and Luthe 1985) at least.According to the aminoacid sequence of gluten, it can be divided into A and two big subfamilies of B (Sub-family abbreviates GluA and GluB respectively as), in each subfamily all by controlled by multiple genes; In GluA or the GluB between each polypeptide the homology of aminoacid sequence between 80-88%, and between the different subfamilies of two classes the amino acid sequence of polypeptide homology less than 65% (Takaiwa etc. 1991).Analyze from glutenin gene or its cDNA deduced amino acid, at the N-terminal (N-end) of gluten precursor by a very typical signal peptide of forming by 24 amino-acid residues, this signal peptide sequence is positioned on the endoplasmic reticulum at guiding gluten precursor, and and then be transported to play an important role in the process in the endoplasmic reticulum chamber (Okita etc. 1989, and Takaiwa etc. 1991).
At present, the existing separated clone of a plurality of glutenin genes (Okita etc. 1989, and Takaiwa etc. 1987, and Takaiwa etc. 1991, Takaiwa and Oono 1991).In the GluA subfamily, have three kinds of different encoding genes at least, Takaiwa and Oono (1991) are with them difference called after GluA-1 (D00584), GluA-2 (Y00687) and GluA-3 (X54313), and they all contain 5-8 copy (Okita etc. 1989) on each paddy rice haploidy genome.Also have a gene to be called GluA-4 in addition, belong to pseudogene (Pseudogene, Takaiwa and Oono 1991).The coding region nucleotide sequence homology of GluA-1 and GluA-2 is higher, reaches 95%, and GluA-1 (or GluA-2) has only 81% with the homology of GluA-3.In the GluA subfamily between each member on gene organization's structure and some distinguished sequences all very conservative (Okita etc. 1989).GluA-2 is one of member of paddy rice gluten family, and the N-terminal of its precursor protein contains the signal peptide sequence of being made up of 24 amino acid.The genome sequence of this gene is by clone's (the GenBank registration number is Y00687).GluA-2 expression of gene characteristic research is shown this gene has only in the paddy endosperm of growth expresses specifically.Contriver's early-stage Study shows that this gene promoter can instruct foreign gene specific efficient in the transgenic paddy rice seed endosperm to express.
Summary of the invention
The purpose of this invention is to provide a kind of method that improves target protein expression amount in the transgenic paddy rice seed endosperm, the signal coding sequence that can be used for improving target protein expression amount in transgene paddy rice endosperm by preparation earlier, again this sequence is transformed and enter regeneration plant in the rice cell, thereby improve the expression amount of target protein in transgene paddy rice endosperm.
Technical scheme of the present invention is such: a kind of method that improves target protein expression amount in transgene paddy rice endosperm, it is characterized in that paddy rice gluten GluA-2 gene signal peptide-coding sequence and target protein gene coded sequence linked together and be built into antigen-4 fusion protein gene, antigen-4 fusion protein gene transformed to enter obtain transgenic paddy rice in the rice cell, transgenic paddy rice is solid through cultivating, and expressing in the seed endosperm has target recombinant protein.
But antigen-4 fusion protein gene is connected with seed-specific expression promotor or composition type expression promoter and is cloned on the carrier of rice transformation in the aforesaid method, transforms to enter in the rice cell, from this transformant plant that regenerates again.
Described signal coding sequence has the nucleic acid of 72 bit bases, and its nucleotide sequence is shown in SEQ IDNo.1.The aminoterminal signal peptide of this nucleic acid encoding paddy rice gluten GluA-2 gene, totally 24 amino-acid residues are shown in SEQ ID No.2.
Described transgenic paddy rice comprises that the transgenic paddy rice cell maybe cultivates into plant with the rice cell that transforms, and by the sexual or vegetative offspring of these transfer-gen plants.
Described target protein can comprise any albumen that using value is arranged or enzyme on producing, and this proteic gene of encoding can be from plant, people, animal or microorganism.
The present invention utilizes transgenic technology to express target gene in paddy endosperm, improves the expression amount of target protein in transgene paddy rice endosperm, and methodological science is simply advanced.The GluA-2 gene signal peptide-coding sequence and the target protein gene coded sequence that will comprise SEQ ID NO.1 link together, be built into antigen-4 fusion protein gene, connect and be cloned into relevant seed-specific expression promotor again and can be used for transforming on the carrier of plant; By transgenic technology the mosaic gene that makes up is transformed and to enter in crop or the vegetable cell, from this transformant plant that regenerates, through selfing or the solid seeding of artificial pollination; Adopt the expression amounts of technology for detection allos recombinant protein in transgenic plant seed such as Northern blot and Western blot, than the structure that does not use GluA-2 gene signal peptide-coding sequence, the expression amount of target protein obviously improves.
Description of drawings
The Northern hybridization analysis of Fig. 1 transgenic paddy rice GG and the total RNA of GSG immature seed.Last figure is the Northern results of hybridization, and figure below shows the 28S ribosome-RNA(rRNA) among total RNA.Total RNA respectively extracting from the immature seeds of GG and GSG class transgenic rice plant or No. 9 unconverted plant of military fragrant round-grained rice (being labeled as WT) (～12DAP), wherein the GUS mosaic gene in the GSG class transgenic rice plant contains the signal coding sequence of gluten GluA-2.Hybridizing used probe is the antisense GUS coding region fragment of digoxigenin labeled.
The Western hybridization analysis of Fig. 2 GG and GSG transgenic rice plant mature seed total protein.Extracting is from the mature seed of GG and GSG class transgenic rice plant or No. 9 unconverted plant of military fragrant round-grained rice (being labeled as WT) respectively for the endosperm total protein, and wherein the GUS mosaic gene in the GSG class transgenic rice plant contains the signal coding sequence of gluten GluA-2.The added total protein concentration unanimity of each swimming lane respectively is 30 micrograms, and albumen is transferred on the nitrocellulose filter after SDS-PAGE separates, and the specific antibody with anti-gus protein carries out hybridization analysis then.The arrow indication is gus protein.
Embodiment
Below divide four optimization procedure further to illustrate the specific embodiment of the present invention.The described content of embodiment does not constitute the restriction to claim scope of the present invention.
1, the clone of paddy rice gluten GluA-2 signal coding sequence
Contain the signal peptide sequence of forming by 24 amino acid at the N-of paddy rice gluten GluA-2 precursor end.Nucleotide sequence (GenBank Y00687) according to the paddy rice gluten GluA-2 gene of having delivered has designed a pair of primer Gt1P
6: 5 ' CA
GGATCCAATGGCATCCATAAATCGCCCC-3 ' (SEQ ID NO.3) and Gt1P
10: 5 '-CA
CCATG GCTAGGGAGCCATCGCACAA-3 ' (SEQ ID NO.4) adds BamHI and NcoI position restriction enzyme site respectively at primer 5 ' end.With No. 8 genomic dnas of rice varieties military fortune round-grained rice is that template is carried out pcr amplification, and the pcr amplification condition is 95 ℃, 5min; 95 ℃, 50sec, 55 ℃, 50sec, 72 ℃, 30sec, 30 circulations; 72 ℃, 5min.The PCR product carries out sequential analysis after being cloned into plasmid pGEM-T (available from Promega company).Through sequencing analysis, clone's GluA-2 signal coding sequence fragment (being called the GluA-2_SP fragment) has comprised the sequence between GluA-2 gene A TG downstream+1 to+72 bit bases, this sequence is the signal coding sequence of this gene, sees shown in the sequence table SEQ ID NO.1.Through order-checking identify correct, to contain the segmental clonal marker of GluA-2_SP be pGtSP.
Simultaneously, according to the GluA-2 gene order of having delivered, designed and synthesized a pair of primer Gt1P again
1: 5 '-GGC
AAGCTTCACTGCTACCTTTAAGTAAC-3 ' (SEQ ID NO.5) and Gt1P
3: 5 ' CGA
G GATCCGTTGTTGTAGGACTAATGAA-3 ' (SEQ IDNO.6) is by means of round pcr long promoter region of amplification GluA-2 gene translation initiator codon ATG upstream 1.3kb from No. 8 total DNA of rice varieties military fortune round-grained rice.5 ' end at two primers has added Hind III and Bam HI restriction endonuclease sites respectively, with convenient clone subsequently.Before the pcr amplification, in 50 μ l reaction solutions, mix 1 * reaction buffer, 1.5mM MgCl
2, 0.2mMdNTPs, 1 μ M primer Gt1P
1, 1 μ M primer Gt1P
3, 2. western 5 Taq of unit archaeal dna polymerases (Promega); The pcr amplification program is as follows: 95 ℃, 5min; 95 ℃, 1min, 50 ℃, 1min, 72 ℃, 1min 30sec, 30 circulations; 72 ℃, 10min.The dna fragmentation of amplification is called GluA-2_Pro.
2, the structure of GluA-2 signal coding sequence and GUS fusion gene
For detecting the influence that above-mentioned GluA-2 signal coding sequence of cloning (GluA-2_SP fragment) is expressed in the transgenic paddy rice seed endosperm for target gene, respectively GluA-2_Pro is become fusion gene with the GluA-2_SP fragment with GUS (beta galactose thuja acid enzyme) reporter gene coding region, nopaline (NOS) gene terminator sequence construct.At first GluA-2_Pro and GluA-2_SP fragment are linked together, form the GluA-2_Pro+SP fragment.Then, this fragment through Hind III and Nco I double digestion, is substituted the 35S promoter of GUS upstream of coding region among the binary vector pCAMBIA1301 (being so kind as to give by Australian CAMBIA mechanism), promptly be built into binary vector p13GSG.In this carrier, the signal coding sequence that between GluA-2_Pro promotor and gus gene coding region (remaining with the ATG sequence of oneself), promptly contains the GluA-2 gene, signal coding sequence and GUS coding region sequence are formed a fusion gene, all do not change through both reading frames of order-checking proof.
Simultaneously, for comparing the influence that GluA-2 gene signal peptide-coding sequence is expressed gus gene, made up the GUS mosaic gene that only contains GluA-2 gene promoter (GluA-2_Pro) again.The structure flow process is as follows.At first downcut the long NOS terminator of 270bp with restriction enzyme Sac I and Eco RI from plasmid pBI121, the clone advances in the corresponding site of binary vector pCAMBIA1300 (being so kind as to give by Australian CAMBIA mechanism), is built into plasmid pC1300/NOS.Downcut and reclaim the long gus gene coding region sequence of 2.0kb from binary vector pIG121Hm (being so kind as to give) (Hiei etc. 1994) with Bam HI and Sac I again by Japanese Hiei etc., it is cloned in the corresponding site of plasmid pC1300/NOS into, form plasmid pC1300/GN.Further use Hind III and BamHI double digestion GluA-2_Pro fragment, connect in the corresponding restriction enzyme site of plasmid pC1300/GN, be built into and contain the plasmid p13GG that GluA-2_Pro promotor and gus reporter gene merge mutually.
3, GluA-2 signal coding sequence and GUS fusion gene import paddy rice and obtain transfer-gen plant
Constructed binary vector p13GSG and p13GG in above-mentioned 2 imported respectively in the agrobacterium tumefaciens bacterial strain EHA105 competent cell through freeze-thaw method; The program (Liu Qiaoquan etc., plant physiology journal, 1998) of the agrobacterium mediation converted paddy rice of having set up by the applicant imports two constructed GUS fusion genes in the paddy rice respectively.
Concrete Transformation Program is as follows.Take away and spend No. 9 immature seeds of the military fragrant round-grained rice of 12～15 days the rice varieties in back behind 70% ethanol surface sterilization 2min, in the NaClO solution that contains 2% reactive chlorine, sterilize more than the 90min, and shake frequently, with aseptic water washing 4～5 times, strip out rataria with scalper and tweezers then and be incubated at evoked callus on the callus inducing medium, the nascent callus that induced after pre-the cultivation through 4-7 days is used for agriculture bacillus mediated transformation experiment.Will be after activation on the YEB semisolid medium that contains the 50mg/l kantlex at agrobacterium tumefaciens bacterial classification that very low temperature is preserved, picking list colony inoculation contains to 3ml in the YEB liquid nutrient medium of 50mg/l Km, in 28 ℃ of jolting overnight incubation; Contained in the AB liquid nutrient medium of 50mg/l Km by the 1% inoculum size 40ml that transfers in the 2nd day, and continued to cultivate 6～8hr in 28 ℃, 250rpm.In 6000rpm, 4 ℃ of centrifugal 5min, the collection thalline also is resuspended in AAM (containing 100～400 μ mol/l Syringylethanones) liquid nutrient medium of 10～15ml with fresh nutrient solution, is used for immediately transforming with the common cultivation of paddy rice acceptor material.Various suitable paddy rice acceptor materials are immersed in 15～30min in the fresh AAM Agrobacterium bacterium liquid, or shake several times.Then rice material is shifted out, on aseptic filter paper, inhale and remove too much bacterium liquid, transfer to N immediately
6D
2C is total on the culture medium, cultivates altogether 3 days under 26-28 ℃ of dark condition.After 3 days, cut plumule and change over to and select to select on the substratum to cultivate.The fresh callus that grows after 10～14 days is transferred to and is continued screening 2 generations (10～14 days/generation) on the new selection substratum, select eugonic resistant calli to transfer to then to break up on the division culture medium seedling (12hr illumination/sky), regenerated seedling behind strong plantlets and rootage, to move into the solarium or phytotron potted plant.During two fusion genes made up, each had obtained the independent transformant more than 20.The transfer-gen plant that contains the GluA-2_Pro+GUS fusion gene is called GG, and number consecutively is GG1, GG2 and GG20 etc., and the transfer-gen plant that contains the GluA-2_Pro_SP+GUS fusion gene is called GSG, and number consecutively is GSG1, GSG2 and GSG20 etc.Each transformant contains 2-6 transgenic rice plant.After transgenic rice plant is transplanted into the land for growing field crops, most can normal growths, bloom and solid.
All transfer-gen plants have been integrated in the genome of transgenic paddy rice through PCR and Southern hybridization analysis proof GUS fusion gene, and from 1 to 4 of integration site does not wait, and GG is similar to the integration situation in the GSG two class transgenic rice plants.
4, GluA-2 signal coding sequence and the expression of GUS fusion gene in transgene paddy rice endosperm
Bloomed back about 12 days at transgenic paddy rice, collect 40 left and right sides immature seeds from each plant, extract total RNA, and use with the special antisense DNA probe of gus gene coding region sequence and carry out the Northern hybridization analysis, the result is presented at the transcript (Fig. 1) that higher gus gene is arranged in the two class transgenic rice plant immature seeds.Judge from the power of hybridization signal, though the expression amount between different transformants has bigger difference, but from the general status analysis, in the GSG transgenic paddy rice, the transcriptional level of the expression level of the GUS mosaic gene that merges with GluA-2 signal peptide sequence GUS mosaic gene (promptly do not contain signal peptide sequence, only contain the GUS mosaic gene of GluA-2 promoter sequence) in the GG class transgenic paddy rice seed, the existence of GluA-2 signal peptide sequence is described, can improves the transcriptional level (Fig. 1) of target gene in transgene paddy rice endosperm.
After the transgenic paddy rice maturation, collect mature seed, from its endosperm, extract seed protein, carry out the Western hybridization analysis with the specific antibody of anti-GUS, the result is also shown in the accumulation (Fig. 2) that gus protein is arranged in the two class transgenic paddy rice mature seeds.The expression amount of gus protein is different in different transgenic paddy rice seeds, and the result that this and Northern are hybridized is similar.But analyze the accumulation volume of gus protein (Fig. 2) in the GG class transgenic paddy rice in GSG class transgene paddy rice endosperm from general trend.
Comprehensive The above results when adding the signal coding sequence that connects the GluA-2 gene before the gus gene coding region, not only can obviously promote the efficient of transcribing of GUS fusion gene, and can make also showed increased of final protein product expression amount.Each hybridization signal in Northern and the Western hybridization is scanned the quantitative comparison analysis, and the expression amount of gus protein than the high 5-10 in the GG class transgenic paddy rice doubly in GSG class transgene paddy rice endosperm.
Claims (5)
1. method that improves target protein expression amount in transgene paddy rice endosperm, it is characterized in that paddy rice GluA-2 gene signal peptide-coding sequence and target protein encoding sequence linked together and be built into antigen-4 fusion protein gene, antigen-4 fusion protein gene transformed to enter obtain transgenic paddy rice in the rice cell, transgenic paddy rice is solid through cultivating, and expressing in the seed endosperm has target recombinant protein.
2. the method for raising target protein according to claim 1 expression amount in transgene paddy rice endosperm is characterized in that described signal coding sequence shown in SEQ ID No.1, is the nucleic acid that 72 bit bases are arranged; The aminoterminal signal peptide of this nucleic acid encoding paddy rice gluten GluA-2 gene, the aminoacid sequence of signal peptide is shown in SEQ ID No.2.
3. the method for raising target protein according to claim 1 expression amount in transgene paddy rice endosperm, it is characterized in that but said antigen-4 fusion protein gene is connected with seed-specific expression promotor or composition type expression promoter and is cloned on the carrier of rice transformation, transform again and enter rice cell.
4. according to the method for claim 1,2 or 3 described raising target proteins expression amount in transgene paddy rice endosperm, it is characterized in that said transgenic paddy rice comprises that the transgenic paddy rice cell maybe cultivates into plant with the rice cell that transforms, and by the sexual or vegetative offspring of these transfer-gen plants.
5. according to the method for the described raising target protein of claim 1 expression amount in transgene paddy rice endosperm, it is characterized in that said target protein comprises any albumen that using value is arranged or enzyme on producing, this proteic gene of encoding is from plant, people, animal or microorganism.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105907780A (en) * | 2016-04-29 | 2016-08-31 | 华南农业大学 | Transgenic breeding method producing astaxanthin in crop seed endosperm |
| CN114592000A (en) * | 2020-12-03 | 2022-06-07 | 上海市农业科学院 | Application of six-gene combination in improving VB2 content in rice seeds and method |
| CN114634559A (en) * | 2018-10-12 | 2022-06-17 | 武汉禾元生物科技股份有限公司 | Method for improving expression level of recombinant protein in endosperm bioreactor |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105907780A (en) * | 2016-04-29 | 2016-08-31 | 华南农业大学 | Transgenic breeding method producing astaxanthin in crop seed endosperm |
| CN105907780B (en) * | 2016-04-29 | 2020-02-21 | 华南农业大学 | Transgenic breeding method for producing astaxanthin in crop seed endosperm |
| CN114634559A (en) * | 2018-10-12 | 2022-06-17 | 武汉禾元生物科技股份有限公司 | Method for improving expression level of recombinant protein in endosperm bioreactor |
| CN114592000A (en) * | 2020-12-03 | 2022-06-07 | 上海市农业科学院 | Application of six-gene combination in improving VB2 content in rice seeds and method |
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