CN113151266A - Barley endosperm specific expression promoter pHvHGGT and application thereof - Google Patents

Abstract

The present invention relates to biotechnology and plant genetic engineering technology. Specifically, the invention relates to a barley gene expression promoter and application thereof, wherein the promoter can drive a target gene to be specifically expressed in endosperm in a barley transgenic regulation system. The barley endosperm specific expression promoter can specifically drive the expression of a target gene in cereal endosperm, can avoid adverse effects caused by continuous expression of the target gene in other tissues of plants, realizes accurate control of expression of exogenous genes in plant nutritive organs, and particularly drives the specific high-efficiency expression of the target gene in the endosperm of crop grains, thereby having practical significance for improving the quality and increasing the yield of the grains of grain crops such as barley, rice and the like.

Description

Barley endosperm specific expression promoter pHvHGGT and application thereof

Technical Field

The present invention relates to biotechnology and plant genetic engineering technology. Specifically, the invention relates to a barley gene expression promoter and application thereof, wherein the promoter can drive a target gene to be specifically expressed in endosperm in a barley transgenic regulation system.

Background

Barley is one of four grain crops in the world and is mainly used in the fields of food production, animal feeding and the like. The endosperm structure of barley grain is the major edible part of people. The formation and development of endosperm directly affects the yield and quality of barley. The transgenic plant which can make the target gene stably and efficiently expressed in endosperm can be cultivated by biotechnology and plant genetic engineering technology, so that the content of essential amino acid, rare protein and various vitamins in barley grains is increased, and the aim of improving the nutritional quality of the grains is finally achieved.

How to realize the fixed-point, timed and stable high-efficiency expression of exogenous genes in transgenic barley is always the fundamental goal of transgenic breeding work. The key factor for realizing the aim is to select a promoter for expressing the exogenous gene with high efficiency and tissue specificity. The promoter is an important element for regulating gene expression, is a nucleotide sequence at the upstream of the transcription initiation of the gene, and interacts with RNA polymerase and other protein accessory factors to regulate the transcription level of the gene and determine the time-space characteristics of the gene expression.

The promoters with high expression, such as CaMV35S, ACTIN, ubiquitin and the like, which are widely applied at present can express exogenous genes in almost all plant tissues, and the expression of the promoters is not limited by space and time. When the exogenous genes are over-expressed in crop seeds, the promoters can drive the genes to be expressed in other tissues outside the seeds, so that metabolites which can have adverse effects on the growth and development of crops are synthesized, and the waste of biological energy sources is caused. Therefore, the selection of the endosperm specificity high-strength expression promoter is beneficial to the synthesis of the exogenous useful protein in a timing and directional regulation mode, the content of the exogenous useful protein in the grains is improved, and meanwhile, the normal physiological activity of the plant is not interfered. In recent years, a series of seed-specific expression promoters have been discovered. Such as rice endosperm-specific glutelin GluB-4 gene promoter, 26kDa globulin gene promoter, 10kDa and 16kDa prolamin gene promoter, etc. (Qu and Takaiwa, 2004). The hordein Hor3 gene promoter was reported in the literature to be specifically expressed in endosperm of barley transgenic plants (Choi et al, 2003), but in practice, the promoter drives overexpression of the target gene in other parts of the plant as well (Chen et al, 2017).

The barley endosperm-specific expression promoter sequence is derived from the promoter of the Homogentisate yak transfer enzyme (HvHGT) gene. The barley grains are rich in vitamin E. Vitamin E is mainly classified into two types of tocopherol and tocotrienol. The HvHGGT gene is a key rate-limiting enzyme gene in the process of regulating and controlling the synthesis of tocotrienols.

The HvHGGT gene is expressed predominantly in seeds, and mutations in this gene completely block the synthesis of tocotrienols (Zeng et al, 2020).

References referred to:

chen, J., Liu, C., Shi, B., Chai, Y., Han, N., Zhu, M. & Bian, H. (2017). Overexpression of HvHGGT Enhances Tocotrienol Levels and antibiotic Activity in Barley. journal of Agricultural and Food Chemistry,65, 5181-containing 5187 (construction of 28557, Liu Cui, Shibo, Chaiyou, Han Gemini, Zhu Yuan and Bihong Wu et al (2017). barley HvHGGT gene Overexpression increases tocopherol Levels and Antioxidant activity.65, 5181-containing 5187.

Choice, h.w., Lemaux, P.G. & Cho, m.j. (2003) Long-term stability of transport expression drive by barley endosperm-specific hordein promoters in transport barley plant Cell Reports,21, 1108-.

Qu, L.Q. & Takaiwa, F. (2004). Evaluation of tissue specificity and expression intensity of promoters of transgenic rice seed components genes in transgenic rice 125(Qu, L.Q. and Takaiwa, F. (2004). Evaluation of tissue specificity and expression intensity of promoters of transgenic rice seed components genes. plant Biotechnology Journal,2 nd, page 113, 125).

Zeng, z, Han, n, Liu, c, buert, b, Zhou, c, Chen, j, Wang, m, Zhang, Y, Tang, Y, Zhu, m, Wang, j, Yang, Y, and Bian, h.2020.functional separation of HGGT and HPT in barrel vitamin E biosyntheses via CRISPR/Cas9-enabled genome editing.anna of 92tan.126: 929 (great sympathy, korean, Liu cui, boolean, Zhou cheng lu, jian hei, cheng 28557, Wang meng yao, Zhang yu, zhuyi yu, jun, yan, and hong wu (2020/9) through the processes of genome synthesis of genes by Cas/r, hge, ph 11, h.s.r. 92942, and h.r.r. host technologies and ph.r.p. technologies, ph.r.s.r.r.s.r.s.r.s.s.s.r.s.s.e. 3.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a barley endosperm specific expression promoter pHvHGGT, and culture a transgenic plant which can enable a target gene to be efficiently expressed in a specific part, namely endosperm.

In order to solve the technical problems, the invention provides a promoter pHvHGGT specifically expressed by barley endosperm, and the nucleotide sequence of the promoter pHvHGGT is shown as SEQ ID NO: 1.

The invention also provides a recombinant expression vector, which comprises the barley endosperm-specific expression promoter, wherein the promoter can be connected to the upstream of a reporter gene (namely, a gene to be expressed, GUS gene) sequence.

The invention also provides the application of the promoter pHvHGGT specifically expressed by the barley endosperm: starting the specific expression of the exogenous gene (namely, the target gene) in the plant seed endosperm; and (5) cultivating transgenic plants.

Improvement as an improvement of the use of the barley endosperm-specific expression promoter phvhgggt of the invention:

the transgenic plant is a transgenic seed plant, and specifically expresses an exogenous gene in the seed.

The seed of the transgenic seed plant is endosperm seed, the exogenous gene is specifically expressed in the endosperm of the seed of the transgenic seed plant, and the plant is monocotyledon, such as barley, rice and the like.

The HvHGGT gene promoter provided by the invention has the characteristic of specific expression of barley endosperm; the separation and identification of the promoter of the barley endosperm specificity high expression HvHGGT gene have important significance for developing genetic engineering to improve the nutritional quality of barley and other crop grains.

Namely, the scheme provided by the invention is as follows:

in one aspect the invention provides a barley endosperm-specific expression promoter, designated pHvHGGT, derived from wild barley (Hordeum vulgare L. 'Golden premium'). The promoter is a DNA sequence shown in a sequence table 1. SEQ ID NO:1 consists of 1614 nucleotides.

In another aspect, the present invention provides a recombinant expression vector in which the above-described barley endosperm-specific expression promoter provided by the present invention is ligated upstream of a gene sequence to be expressed. The genes to be expressed are GUS and GFP genes; the recombinant expression vector is prepared by expressing the nucleotide sequence shown in SEQ ID NO:1 is constructed in a plant binary expression vector pHGWFS7-ccdB (containing GUS and GFP reporter genes) to obtain a recombinant expression vector which is called pHGWFS 7-pHvHGGT.

In still another aspect, the invention provides the use of the promoter specifically expressed in barley endosperm in the cultivation of transgenic plants. The cultivation of transgenic plants is to connect the specific expression promoter in the barley endosperm provided by the invention to the upstream of a gene sequence to be expressed (to fuse the promoter and a target gene), and construct the promoter into a recombinant expression vector. The recombinant plasmid is transformed into plant cells for cultivation. The gene to be expressed, i.e. the target gene, can be a gene related to plant nutrient absorption and a gene related to improvement of grain quality and nutrient accumulation, so as to improve the endosperm characteristic of grains.

The invention specifically comprises the following steps:

the invention obtains 1614bp DNA sequence which is a DNA sequence with endosperm specificity and high-efficiency expression promoter function by separating and cloning from wild barley (Hordeum vulgare L. 'Golden Promise'); and designated pHvHGGT (SEQ ID NO:1 in the sequence Listing). The sequence is firstly connected to a pENTRY vector, then a Gateway technology LR is utilized to be added to a plant binary expression vector pHGWFS7-ccdB, the recombinant plasmid is utilized to transform an agrobacterium tumefaciens strain EHA105, and then agrobacterium-mediated barley genetic transformation is utilized to obtain a transgenic barley plant. Histochemical examination of the obtained transgenic barley was carried out. As a result, the transgenic plant only shows blue at the endosperm, so that the 1614bp sequence is proved to have the activity of driving the expression of the GUS gene, and the GUS reporter gene driven by the promoter is only specifically expressed in barley endosperm.

Compared with the prior art, the invention has the beneficial effects that:

the promoter sequence can be linked with a required target gene to construct a recombinant plant expression vector, and drives the specific expression of the target gene in endosperm after transformation, so that the expression quantity of the foreign target gene in plant endosperm is improved, and the influence of the excessive expression of the foreign target gene in other tissues except the plant endosperm on crop traits is relieved.

The barley promoter pHvHGGT cloned by the invention can improve the expression and accumulation level of exogenous genes in plant seed endosperm, and has obvious application value in the improvement of crop quality. The seed endosperm is the main edible part of crops such as barley, rice and wheat. The promoter regulates and controls the specific expression of a target gene in endosperm, so that the content of essential amino acid, rare protein and vitamin in seeds can be increased, the nutritional quality of crop seeds can be improved, and crops with better quality can be cultivated.

In conclusion, the promoter for specific expression of barley endosperm can specifically drive the expression of a target gene in cereal endosperm, can avoid adverse effects caused by continuous expression of the target gene in other tissues of plants, realizes accurate control of expression of an exogenous gene in plant nutritive organs, and particularly drives the specific high-efficiency expression of the target gene in the endosperm of crop grains, thereby having practical significance for improving the quality and increasing the yield of the grain crop grains such as barley, rice and the like.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 shows qRT-PCR analysis of gene expression of HvHGGT gene in wild barley (Hordeum vulgare L. 'Golden Promise') seed embryo, endosperm and husk tissues.

FIG. 2: schematic representation of the construction of the pHvHGGT promoter in the pHGWFS7-ccdB vector plasmid;

in fig. 2:

a is a schematic diagram of pHGWFS7-ccdB vector plasmid;

b is pHGWFS7-pHvHGGT schematic diagram; the pHvHGGT promoter drives the expression of the GFP and GUS genes located downstream of the pHvHGGT promoter.

FIG. 3 is T₀Transferring pHGWFS7-pHvHGGT rice plant positive seedling PCR detection and identification electrophoresis result.

FIG. 4 is T₀Transferring GUS staining results of roots, leaves, young ears and seeds of pHGWFS7-pHvHGGT rice plants;

in fig. 4:

a is the cross section of the rice seeds 18 days after pollination, and the endosperm is blue after GUS staining; b is rice leaf tissue; c is rice root tissue; d is the young ear of rice. The rice roots, leaves and young ears are not blue.

Detailed Description

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

the methods in the following examples are conventional methods unless otherwise specified.

In the following cases, the reporter gene/gene to be expressed is GUS, GFP, or the like; the exogenous gene/target gene is related to plant nutrient absorption and related genes for improving grain quality and nutrient accumulation, such as HvHGGT gene and the like.

pENTRY vector was purchased from Invitrogen, pHGWFS7-ccdB vector was purchased from the Center for Plant Systems Biology research (VIB-UGent Center for Plant Systems Biology).

Example 1 obtaining of barley endosperm-specific expression promoter (HvHGGT promoter)

According to the cDNA sequence of a barley homogentisic acid yak transfer enzyme HvHGGT gene (Ensembl Plants with the number of HORVU7Hr1G114330), the upstream sequence of the HvHGGT gene is searched in an Ensembl Plants database, and a primer is designed to amplify the HvHGGT promoter. The vector is constructed by Gateway technology, i.e. the CACC is added at the 5' end of the upstream primer when designing the primer. pHvHGGTF: 5'-CACCATCGTGATGGAGGAGAGG-3' is the forward primer of promoter; pHvHGGTR: 5'-AAGTTAACCCGCGGATCTGT-3' is the reverse primer of the promoter.

The CTAB method extracts genomic DNA from leaves of wild barley (Hordeum vulgare L. 'Golden Promise'), and PCR-amplifies the promoter sequence of HvHGGT using the genomic DNA as a template and pHvHGGTF and pHvHGGTR as primers. The reaction system is as follows: phusion super fidelity DNA polymerase (New England Biolabs, NEB) 0.5. mu.L, 5 XBuffer 10. mu.L, dNTP 4. mu.L, genomic DNA 1. mu.L, 10. mu.M forward and reverse primers 2. mu.L each, and triple distilled water to 50. mu.L. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; then denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 2min for 33 cycles; finally, extension is carried out for 10min at 72 ℃.

mu.L of PCR amplification product was collected and detected by 1% agarose gel electrophoresis to obtain a 1.6kb target band. The desired fragment was purified and recovered, and ligated to pENTRY plasmid vector (Thermo Scientific). The connecting body is: salt Solution 1.8. mu.L, Entry vector 0.5. mu.L, target fragment 1. mu.L (about 50ng), and triple distilled water 6.7. mu.L. The ligation system was left at room temperature for 1 hour, 5. mu.L of the ligation system was aspirated for E.coli transformation, 3 to 5 colonies were picked, plasmids were extracted, and sequencing was verified using primers M13F (5'-TGTAAAACGACGGCCAGT-3') and M13R (5'-CAGGAAACAGCTATGACC-3'). The result shows that the HvHGGT promoter sequence obtained by amplification has the size of 1614bp and is a sequence table SEQ ID NO: 1.

The sequencing result of the obtained plasmid is 1614bp, and is shown as SEQ ID NO:1, the pENTRY vector carrying the target fragment.

Example 2 construction of expression vector for barley endosperm-specific expression promoter (HvHGGT promoter) and genetic transformation

1. Expression vector construction of HvHGGT promoter fused GUS gene

Sequencing verification the LR reaction was performed on pENTRY vector carrying the fragment of interest and the vector of interest (pHGWFS7-ccdB, FIG. 2 a). Reaction system: 2. mu.L of Entry vector with the target fragment, 2. mu.L of target vector and 1. mu.L of LR enzyme. The ligation system was allowed to stand at room temperature for 1 hour, and then 1. mu.L of proteinase k was added thereto, followed by water bath at 37 ℃ for 10 minutes to terminate the reaction. mu.L of the transformed E.coli was aspirated, and 3-5 clones were picked for sequencing verification with primers attB1 (5'-CAAGTTTGTACAAAAAAGCAG-3') and attB2 (5'-CCACTTTGTACAAGAAAGCTG-3').

The obtained pHGWFS7 vector has NO ccdB sequence and carries a sequence table SEQ ID NO:1 is pHGWFS 7-pHvHGGT.

2. Acquisition of pHGWFS7-pHvHGGT (FIG. 2b) transgenic rice

The correct vector pHGWFS7-pHvHGGT plasmid was verified to electroporate Agrobacterium EHA105 competent cells. Mature embryos of wild rice (Oryza sativa L.ssp. japonica 'Nipponbare') are used as explant materials, Agrobacterium EHA105 is used for infection and transformation, and hygromycin resistance screening is carried out, and resistant callus is differentiated and regenerated to obtain transgenic plants. Extracting the genome DNA of the transgenic plant, and performing molecular detection on the hygromycin gene of the transgenic plant by using a PCR method. The forward primer was HygF: 5'-CGCCGATGGTTTCTACAA-3', respectively; the reverse primer is HygR: 5'-GGCGTCGGTTTCCACTAT-3' are provided. The reaction system is as follows: 2 Xtag DNA polymerase (Vazyme) 0.5. mu.L, genomic DNA 0.5. mu.L, 10. mu.M forward and reverse primers 1. mu.L each, and Tri-distilled water to 20. mu.L. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 3 min; then denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 25s, and extension at 72 ℃ for 1min for 33 cycles; finally, extension is carried out for 8min at 72 ℃.

The target band of 836bp obtained by PCR amplification is the positive detection. The result of agarose gel electrophoresis is shown in FIG. 3, wherein Lane 1 is DL2000 DNA Marker (M), Lane 2 is positive control, that is, pHGWFS7-pHvHGGT plasmid is used as template for amplification; lane 16 is a negative control, i.e., a wild-type rice DNA template is added to the PCR system; lanes 3 to 15 are the T of the pHGWFS7-pHvHGGT plant expression vector₀And (5) plant generation.

From fig. 3, it can be seen that: obtaining 13 PCR detection positive strainsSexual trans-pHGWFS 7-pHvHGGT rice T₀And (5) plant generation.

Example 3 functional verification of barley endosperm-specific expression promoter (HvHGGT promoter)

Counter-rotating pHGWFS7-pHvHGGT rice T₀The plants were histochemically stained for GUS protein. The method comprises the following specific operations: the pHGWFS7-pHvHGGT transformed rice T₀Partial roots, leaves, young ear tissues of the plants and seeds 18 days after flowering were transected, immersed in GUS reaction solution (1mg/mL X-gluc, 1mM potassium ferricyanide, 0.1% Triton X-100,0.1M phosphate buffer, 10mM EDTA, pH 7.0), and reacted at 37 ℃. The stained tissue was decolorized with 70% ethanol until chlorophyll disappeared and recorded by taking a picture with a stereoscope. The result shows that no GUS expression is observed in roots, leaves and young ears of pHGWFS 7-pHvHGGT-transferred rice; the endosperm of seeds flowering for 18 days was dark blue (fig. 4). The staining results indicated that the HvHGGT promoter allows the beta-Glucuronidase (GUS) reporter gene to be specifically expressed only in the endosperm of rice seeds.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Sequence listing

<110> Zhejiang university

<120> barley endosperm specific expression promoter pHvHGGT and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1614

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atcgtgatgg aggagaggac gaggtcgccg atcaggagcg gcagaggaca agtttgagga 60

gacgacgccg catagtggat tcggcatgga ggaaggaggc tggcggcgtt aacctagacg 120

ccacggtgta gggcagcggg agtcgatcgg gaaacgcgag aagagcatgg aggtggaggt 180

gtgtttgtgg cgtgggtcgt gtgggctggt ctctctctgg aaggaagaat tgccctggtt 240

ttcttagggc agttttttgg ttcgaaggac acgggtgtcg gctcaaaaaa atcggtgact 300

agcgaggagg tcgtgcgagt agaggggagc gaacaagcaa acgaacgacg acatacagac 360

tcctccttta ggagtagaga aatgttagga gtgagattac gcaacaatca attttgaatc 420

ctaactttcc taacacatgg attgccacat aattggattt cattatttga taaggtatta 480

ttggttttct ccatttgtaa taatgtggca ttcaatttcg aatcctaact ttcctaacac 540

atggattgcc acataattgg atttcattat ttggtaacgt attattggtt tcctgccatt 600

tgtaattata tggcaatcaa tttcgaatcc taactttcct aacacatgga ttgccacata 660

aatggatttc attatttggt aatgtattat tagtttcctg ccatttataa catgactggt 720

tagaaaaagt ttgaaagatt aagaaagttt ttattgaaag gtgaaaaaac aaatgtgaag 780

ggatggtggt gcgaggtgag acgaaaaaac tggctgaaaa aaaccggtta aaaaaaccta 840

gaagttattc taccattagg agtagagatt atccaaaatt accaaggtac tcaggtaatt 900

tgttcgacct cgacccattc cacgaccgta aatcaacctg taattgaatg gttagaggga 960

caatggtatc cccagaccat caggatttaa gtcctggtgt ttacattatt cctgcattta 1020

tttcaggatt tccggtgata cgctttcagt ggaaggagac gtttccgtca actacgaggc 1080

gcctacgata actttgtaaa tctcaagatg acatgtcgtc tcaatctctc ggagatgctc 1140

ataaggatag ggtgtacatg tgtacgttca tagagataaa tgtatgtgca ggtatatgga 1200

cgtttgcgcc tgcataaaaa aaagacccat tccacgaccg ggtcgtgacg cacacgcaca 1260

cccgaaaaat gatgcatggc tcgctccacc gaggatcgtg tcgttttggc aagtcatcga 1320

ctttgccaaa acgtctgtgc gtcacaccgg cagcgagtcc accgcactgc accatcacca 1380

gctagctagc gcgcgcgtcc atctttttct tcagaatcac atgccgcctc cgtctttttc 1440

tttacaatca cacggcgcat ccgcctgcat gcagccggat tgcattacat tacgcacgca 1500

cccacgccat gcagtaatca atcggcacat aaaccccctc ctccccctga aggcctgaac 1560

ctcccgtccc gtctgctcct ccccctcctt tcacacagat ccgcgggtta actt 1614

Claims (6)

1. The promoter pHvHGGT specifically expressed by barley endosperm is characterized in that the nucleotide sequence is shown as SEQ ID NO: 1.

2. A recombinant expression vector characterized by: a promoter phvhgggt comprising barley endosperm-specific expression according to claim 1, which is linked upstream of a reporter gene sequence.

3. Use of the barley endosperm-specific expression promoter phvhgggt according to claim 1, characterized in that: the specific expression of the exogenous gene in the plant seed endosperm is started.

4. Use of the barley endosperm-specific expression promoter phvhgggt according to claim 1, characterized in that: and (5) cultivating transgenic plants.

5. Use of the barley endosperm-specific expression promoter phvhgggt according to claim 4, characterized in that: the transgenic plant is a transgenic seed plant, and specifically expresses an exogenous gene in the seed.

6. Use of the barley endosperm-specific expression promoter phvhgggt according to claim 5, characterized in that: the seed of the transgenic seed plant is endosperm seed, the exogenous gene is specifically expressed in the seed endosperm of the transgenic seed plant, and the plant is monocotyledon.

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