TW202102528A - Isolated transcriptional enhancer sequence, a modified promoter sequence comprising the same and use thereof - Google Patents

Abstract

The present disclosure provides an isolated transcriptional enhancer sequence for enhancing gene transcription and a modified promoter sequence including a promoter and the transcriptional enhancer sequence. The present disclosure also provides an expression vector including a regulatory sequence having the modified promoter. The present disclosure also provides a plant transformed cell including the expression vector. The present disclosure further provides a method for producing a foreign protein, including culturing the plant transformed cell in a plant culture medium or regenerating the plant transformed cell to plant transgenic lines, and recovering the foreign protein from the plant culture medium or extracting the foreign protein from tissues of the plant transgenic lines.

Description

An isolated transcription enhancer sequence, a modified promoter sequence comprising the isolated transcription enhancer sequence, and uses thereof

The present disclosure relates to a transcription enhancer sequence and a modified promoter sequence including the transcription enhancer sequence; this disclosure also relates to a performance vector including the modified promoter sequence; this disclosure also relates to a plant transgenic The shape cell includes the expression vector; and, the present disclosure also relates to a method of using the expression vector to produce exogenous protein.

At present, molecular farms have been widely used in the production of foreign proteins, such as enzymes, antibodies, and pharmaceutical proteins. Commercially common molecular farm expression systems include bacterial expression systems, yeast expression systems, fungal expression systems, mammalian expression systems, Plant expression systems and insect expression systems, among which plant expression systems have many advantages over other expression systems, including lower capital requirements, lower production costs and plant cultivation costs, large yields, and foreign protein production. Post-translational modifications can be carried out in plants, so that the foreign protein has the function and activity close to the original protein.

In the plant expression system, rice ( Oryza sativa , here or Os for short) is one of the plants that can easily undergo gene transfer. References Kyozuka et al. (1991), Christensen et al. (1992) and Kyozuka et al. (1994) have shown that inducible or persistent promoters commonly used in rice gene transfer technology include rice actin-1 ( actin-1 , Act1 ) and α-amylase (α-amylase, αAmy ) promoters, and promoters from corn ubiquitin ( ubiquitin , Ubi ). Studies have also shown that combining different transcription enhancers into the promoter can enhance the expression activity of the promoter. For example, the research of Olive et al. (1990) confirmed that combining several anaerobic enhancers into the Adh1 promoter of maize can make the Adh1 promoter induced by hypoxia and hypoxia, and enhance the Adh1 promoter. The activity of the son. For another example, the study of Weigel et al. (2000) confirmed that combining four enhancers of cauliflower mosaic virus 35S (CaMV 35S ) into the CaMV 35S promoter can enhance the activity of the CaMV 35S promoter. .

The plant expression system uses molecular biotechnology to join the promoter and the gene of the foreign protein to form an expression vector, and uses such as Agrobacterium gene transfer technology, gene bombardment or pollen electroporation. Isogenic transgenic technology, after the expression vector is introduced into plant cells, the transgenic plants are screened through the selection markers to obtain plant gene transgenic plants. Plant gene transgenic plants can produce exogenous proteins by establishing suspension cells or using tissues such as roots, stems, leaves or seeds of the transgenic plants. However, in order to prevent the production of foreign proteins from reducing the final yield of foreign proteins due to complicated purification steps during the commercialization process, the yield of foreign proteins produced by the plant expression system should at least account for the total soluble protein (total soluble protein) 1%. However, according to the reference document Daniell et al. (2001), the output of foreign protein produced by most plant expression systems accounts for the total soluble protein The quality is less than 1%. Taking the production of recombinant human acidic fibroblast growth factor (haFGF) as an example, the results of Ha et al. (2017) in the reference document show that so far, the highest yield can be obtained by the transient expression system of tobacco. haFGF, and its output accounts for approximately 1.35% of the total soluble protein.

From this, it is obvious that the existing technology uses plant expression system to express foreign protein still has the problem of low yield. Therefore, the development of a plant expression system that can increase the output of exogenous protein is an urgent problem in this field.

In order to achieve the above objective, the present disclosure provides a transcription enhancer sequence comprising at least one copy number of a nucleotide sequence with more than 90% similarity to the nucleotide sequence shown in SEQ ID NO: 13, and the nucleotide sequence The sequence and SEQ ID NO: 13 have the same function of enhancing gene transcription. In a specific embodiment, the nucleotide sequence is at least 92%, 95%, 96%, 97%, 98%, or 99% similar to the nucleotide sequence of SEQ ID NO: 13. In another specific embodiment, the nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:13.

In a specific embodiment, the transcription enhancer sequence disclosed in the present disclosure comprises a nucleus with at least one copy number, at least two copy number, or at least three copy number, which is more than 90% similar to the nucleotide sequence shown in SEQ ID NO: 13 Nucleotide sequence. In another specific embodiment, the transcription enhancer sequence of the present disclosure includes at least three copies of the nucleotide sequence shown in SEQ ID NO:13.

In a specific embodiment, the nucleotide sequence of the present disclosure having more than 90% similarity with the nucleotide sequence shown in SEQ ID NO: 13 includes at least a common sequence, which may be a G box sequence or TA box (TA box) sequence. In another specific embodiment of the present disclosure, The G box sequence is TACGTG from the 5'end to the 3'end, and the TA box sequence is TATCCA from the 5'end to the 3'end.

In another aspect, the present disclosure provides a modified promoter sequence comprising a promoter and a transcription enhancer sequence, and the promoter system is operably linked to the transcription enhancer sequence. In a specific embodiment, the promoter is actin 1 promoter, ubiquitin promoter, CaMV 35S promoter or rbc S gene promoter.

The present disclosure also provides a performance vector, comprising a left border sequence of the transferred DNA, a regulatory sequence, a nucleotide sequence encoding a multipeptide, and a right border sequence of the transferred DNA, wherein the regulatory sequence is operably linked to The nucleotide sequence encoding multiple peptides is located between the left border sequence of the transferred DNA and the right border sequence of the transferred DNA.

In a specific embodiment, the regulatory sequence comprises the aforementioned modified promoter sequence. In another specific embodiment, the regulatory sequence further comprises an intron sequence, and the intron sequence is operably linked between the modified promoter sequence and the nucleotide sequence encoding the polypeptide . In a specific embodiment, the intron sequence is an actin 1 intron or an alcohol dehydrogenase 1 intron. In another specific embodiment, the Actin 1 intron has the nucleotide sequence shown in SEQ ID NO:15.

In a specific embodiment of the present disclosure, the regulatory sequence further includes a nucleotide sequence encoding a signal peptide, and the nucleotide sequence encoding the signal peptide is operably linked to the aforementioned modified promoter sequence and the encoding polypeptide. Between the nucleotide sequence of the peptide. In another specific embodiment, the nucleotide sequence encoding the signal peptide has the nucleotide sequence shown in SEQ ID NO:17.

The present disclosure also provides a plant transformed cell comprising the aforementioned expression vector.

The present disclosure further provides a method for producing exogenous protein, including: The plant transformed cells are cultivated in plant culture fluid or regenerated into plant transgenic plants; and exogenous protein is isolated from the plant culture fluid or extracted from the roots, stems, leaves or seeds of the plant transgenic plants.

The transcription enhancer sequence of the present disclosure, the modified promoter sequence containing the transcription enhancer sequence, and the expression vector containing the modified promoter sequence can enhance the transcription of exogenous genes transferred into plants , And then improve the expression of exogenous protein in plants. In addition, the implementation of the present disclosure demonstrates that the transcription enhancer sequence of the present disclosure has a better ability to enhance the promoter than αAmy3SRC, and the transcription enhancer sequence of the present disclosure can also enhance the promoter in all tissues of the transgenic rice的activity. Furthermore, with the plant transformed cells and the method for producing exogenous proteins disclosed in the present disclosure, the exogenous protein can be secreted into the culture medium of the plant transformed cells by establishing rice suspension cells, or by regeneration of the plant transgenic plants, Accumulate exogenous protein in the root, stem, leaf or seed of the plant transgenic plant, and obtain the expressed exogenous protein by recovering the culture solution or extracting the protein from the root, stem, leaf or seed of the plant transgenic plant . The implementation of the present disclosure demonstrates that by the method for producing foreign protein of the present disclosure, a significantly higher amount of foreign protein can be obtained.

Figure 1A shows the schematic diagram of the p35mA-Luc plastid structure, where 35Smp represents the CaMV 35S minimal promoter sequence, and Adh1(In) represents the alcohol dehydrogenase 1 intron. Adh1 (In)) sequence, Luc represents the luciferase gene (Luciferase, Luc) sequence, ATG represents the start codon sequence, and Nos 3'represents the NOS terminator sequence.

Figure 1B shows the schematic structure of pMT825-Luc, pMT351-Luc, pMT121-Luc and pMT36-Luc plastids, where 5'UTR stands for 5'untranslated region (UTR), G stands for G box (G box) A common sequence, and TA represents a TA box (TA box) common sequence.

Figure 1C shows a schematic diagram of the structure of pMTSRC-Luc plastid, where MTSRC represents the sugar response complex (SRC) of metallothionein.

Figure 1D shows a schematic diagram of the pAct1-Luc plastid structure.

Figure 1E shows a schematic diagram of the pAct1-MTSRC-Luc plastid structure.

Figure 1F shows a schematic diagram of the pAAct1-MTSRC-haFGF plastid structure, where LB represents the left border sequence of the transferred DNA, RB represents the right border sequence of the transferred DNA, and tml 3'represents the terminator (terminal, tml) sequence, hpt represents the nucleotide sequence of hygromycin phosphotransferase (hygromycin phosphotransferase, hpt), 35S-P represents the CaMV 35S promoter ( CaMV 35S promoter, 35S-P) sequence, and SP represents the signal peptide The nucleotide sequence of signal peptide (SP), and haFGF represents the nucleotide sequence of human acidic fibroblast growth factor (haFGF).

Figure 2 shows the histogram of OsMT2b promoter activity in the transgenic rice suspension cells and rice embryos under sugar and sugar deficiency conditions. +S means in a sugar environment and -S means in a sugar deficiency environment.

Figure 3 shows the histograms of OsMT2b promoters (MT825, MT351, MT121, and MT36) of different lengths conferring luciferase activity under sugar and sugar deficiency conditions in transgenic rice suspension cells.

Figure 4 shows that the OsMT2b full-length promoter (MT825) and only 146 bases with G box and TA box in the transgenic rice suspension cells under sugar and sugar deficiency conditions of the promoter (i.e., OsMT2b promoter transcription start sequence position -266 to -121 of) enzyme activity of imparting fluorescent histogram.

Figure 5 shows a histogram showing that the Act1 promoter and Act1-MTSRC promoter confer luciferase activity in the transgenic rice embryo under the conditions of sugar and sugar deficiency.

Figure 6A shows a histogram of the activity of luciferase conferred by the Act1 promoter and Act1-MTSRC promoter in the transgenic rice suspension cells under the conditions of sugar and sugar deficiency.

Figure 6B shows a histogram showing that the Act1 promoter and Act1-MTSRC promoter confer luciferase activity in the transgenic rice seedlings.

Figure 7A shows a histogram showing that the Act1 promoter, Act1-αAmy3SRC promoter and Act1-MTSRC promoter confer luciferase activity in different tissues (including endosperm, embryo, stem and root) of the seedlings of the transgenic rice plant .

Figure 7B shows a histogram showing that the Act1 promoter, Act1-αAmy3SRC promoter and Act1-MTSRC promoter confer luciferase activity in different tissues (including endosperm, embryo, stem and root) of the seedlings of the transgenic rice plant .

Figure 8A shows that the protein gel ink stain analysis method and the anti-haFGF monoclonal antibody (1:500) are used to detect the haFGF protein expressed in Act1-MTSRC::haFGF transgenic rice, where M represents the molecular weight marker, 1 to 8 respectively represent the number of the lane, that is, the haFGF protein expressed by each transgenic rice cell, haFGF represents the commercially available haFGF protein, and NT represents the non-transformed rice cell.

Figure 8B shows that the protein gel ink stain analysis method is used to detect the rice suspension cells established by Act1-MTSRC::haFGF transgenic rice through the anti-haFGF monoclonal antibody (1:500). The accumulated haFGF protein quality in the culture medium of rice suspension cells.

Figure 9 shows the detection of the biological activity of haFGF protein in ACT-haFGF-3 rice suspension cell culture medium by NIH/3T3 cell expansion analysis method.

Unless otherwise stated herein, the singular forms "a" and "the" used in the specification and the appended patent application include plural entities.

Unless otherwise stated herein, the term "or" used in the specification and the appended claims includes the meaning of "and/or".

A transcription enhancer is a DNA fragment that can positively regulate the expression of a promoter. It can be located upstream or downstream of the promoter, and can bind to a specific protein (enhancer-binding protein) to assist transcription factor (transcription factor). ) Is bound to the promoter to promote transcription and protein expression.

The present disclosure provides a transcription enhancer sequence, comprising the nucleotide sequence shown in SEQ ID NO: 13, which has two consensus sequences, namely G box (5'-TACGTG-3') and TA box (5'-TATCCA-3').

The present disclosure also provides a modified promoter sequence comprising a promoter and a transcription enhancer sequence, and the promoter system is operably linked to the transcription enhancer sequence. In a specific embodiment, the modified promoter sequence contains one, two or three copies of the transcription enhancer sequence. In another specific embodiment, the promoter is actin 1 promoter, ubiquitin promoter, CaMV 35S promoter or rbc S gene promoter. In another specific embodiment, the modified promoter sequence includes the Actin 1 promoter and has a transcription enhancer sequence as shown in SEQ ID NO:13.

The present disclosure also provides a performance vector, comprising the left border sequence of the transferred DNA, the regulatory sequence, the nucleotide sequence encoding the multipeptide, and the right border sequence of the transferred DNA, wherein the regulatory sequence is operably linked to the encoding multiple The nucleotide sequence of the peptide is located between the left border sequence of the transferred DNA and the right border sequence of the transferred DNA. In a specific embodiment, the regulatory sequence comprises a modified promoter sequence.

In a specific embodiment, the regulatory sequence further comprises an actin 1 intron or an alcohol dehydrogenase 1 intron (Alcohol dehydrogenase 1 intron, Adh1(In) ), an actin 1 intron or alcohol dehydrogenation The enzyme 1 intron is operably linked between the modified promoter sequence and the nucleotide sequence encoding the polypeptide. In another specific embodiment, the Actin 1 intron has the nucleotide sequence shown in SEQ ID NO:15.

In a specific embodiment, the regulatory sequence further comprises a nucleotide sequence encoding a signal peptide, and the nucleotide sequence encoding the signal peptide is operably linked to the modified promoter sequence and the nucleotide sequence encoding the polypeptide between. In another specific embodiment, the nucleotide sequence encoding the signal peptide has the nucleotide sequence shown in SEQ ID NO:17.

In a specific embodiment, the nucleotide sequence encoding the multi-peptide encodes human acidic fibroblast growth factor (haFGF), Human epidermal growth factor (hEGF) or Orf1 protein of porcine type II circovirus. In another specific embodiment, the human acidic fibroblast growth factor has the nucleotide sequence shown in SEQ ID NO:18.

The present disclosure also provides a plant transformed cell, which includes an expression vector.

The present disclosure also provides a method for producing exogenous protein, which comprises: culturing plant transformed cells in a plant culture medium, or regenerating the plant transformed cells into a transgenic plant; and recovering the foreign protein from the culture medium or transplanting from the plant The root, stem, leaf or seed of the plant extracts foreign protein.

In a specific embodiment, the plant culture fluid is a plant culture fluid under sugar-deficient conditions.

The following is a specific embodiment to illustrate the implementation of the present disclosure. Those who are familiar with this technique can understand other advantages and effects of the present disclosure from the content disclosed in this specification. However, the exemplary embodiments disclosed in this disclosure are for illustrative purposes only and should not be regarded as limiting the scope of this disclosure. In other words, the present disclosure can also be implemented or applied through other different specific embodiments, and various details in this specification can also be based on different viewpoints and applications, and various modifications and changes can be made without departing from the spirit of the present disclosure.

The sequence of primer pairs used in the embodiments of the present disclosure is shown in Table 1 below:

Preparation Example 1: Construction of pMT825-Luc, pMT351-Luc, pMT121-Luc, pMT36-Luc and pMTSRC-Luc plastids

References Christensen and are based Quail (1996) and Lu et al. (1998 I) of the content, using binding to contain the maize ubiquitin (Ubi) promoter and the NOS terminator β- glucuronidase enzyme son between (- glucuronidase,, GUS) cDNA pUG plastid, and contains CaMV 35S minimal promoter, alcohol dehydrogenase 1 intron ( Adh1(In) ) and luciferase gene (luciferase) , Luc) p35mA-Luc plastid, through polymerase chain reaction (PCR) to amplify the insert to be incorporated into the plastid to construct pMT825-Luc, pMT351-Luc, pMT121-Luc, pMT36-Luc And pMTSRC-Luc plastid.

Download the gene sequence of rice metallothionein (MT) gene of Oryza sativa ( OsMT2b ) from the GenBank database of the National Center for Biotechnology Information (NCBI) (Genebank accession number: AF048750), Design a first primer pair containing the forward primer of the sequence shown in SEQ ID NO: 1 and the reverse primer of the sequence shown in SEQ ID NO: 2, and perform PCR with rice genomic DNA as the template DNA for PCR to Amplify the OsMT2b promoter full-length fragment sequence as shown in SEQ ID NO: 3 containing 825 bp 5'-flanking region and 100 bp 5'non-translated region. The OsMT2b promoter fragment was cut with Hind III and Bam HI restriction enzymes, and then cloned into pUG plastids to obtain pMT-GUS plastids.

Design a second primer pair including the forward primer of the sequence shown in SEQ ID NO: 1 and the reverse primer of the sequence shown in SEQ ID NO: 4, the forward primer of the sequence shown in SEQ ID NO: 5, and the The third primer pair of the reverse primer of the sequence shown in SEQ ID NO: 4, the forward primer of the sequence shown in SEQ ID NO: 6 and the fourth primer pair of the reverse primer of the sequence shown in SEQ ID NO: 4 , The forward primer of the sequence shown in SEQ ID NO: 7 and the fifth primer pair of the reverse primer of the sequence shown in SEQ ID NO: 4, and the pMT-GUS plastid as the template DNA of PCR for PCR to Amplify the full-length fragment sequence of the OsMT2b promoter containing 825bp 5'-flanking region and 100bp 5'non-translated region as shown in SEQ ID NO: 3, respectively, and containing 351bp 5'-flanking region as shown in SEQ ID NO: 8 The OsMT2b promoter partial deletion fragment sequence of the 5'non-translated region of 100 bp, as shown in SEQ ID NO: 9 contains the 121 bp 5'-flanking region and the 100 bp 5'non-translated region of the OsMT2b promoter partial deletion fragment sequence , And as shown in SEQ ID NO: 10, the OsMT2b promoter partial deletion fragment sequence containing a 36 bp 5'-flanking region and a 100 bp 5'non-translated region. Use Hind III and Nco I restriction enzymes to cut the OsMT2b promoter full-length fragment sequence and the OsMT2b promoter partial deletion fragment sequence, and clone them into the p35mA-Luc plastid as shown in Figure 1A to obtain the p35mA-Luc plastid as shown in Figure 1A. The pMT825-Luc, pMT351-Luc, pMT121-Luc and pMT36-Luc plastids shown in the figure.

Design a sixth primer pair containing the forward primer of the sequence shown in SEQ ID NO: 11 and the reverse primer of the sequence shown in SEQ ID NO: 12, and perform PCR with pMT825-Luc plastid as the template DNA for PCR, To amplify the DNA sequence fragment (or MTSRC for short) located at the transcription start position -266 to -121 of the OsMT2b promoter as shown in SEQ ID NO: 13. After cutting the DNA sequence fragments from -266 to -121 at the transcription start position of the OsMT2b promoter with Xho I and Pst I restriction enzymes, they were sub-populated to p35mA-Luc plastids to obtain pMTSRC-Luc as shown in Figure 1C Plastid.

Preparation Example 2: Construction of pAct1-MTSRC-Luc plastid

According to Chen et al. (2002), the pAct plastid was used to construct pAct1-MTSRC-Luc plastid. Firstly, a DNA fragment containing three copies of MTSRC was synthesized by chemical synthesis method (Sanggong Co., Ltd.), and both ends of the DNA fragment had Eco RI restriction enzyme cutting positions, and the DNA fragment was inserted into the pBluescript II SK vector ( Stratagene) to obtain pBS-3xMTSRC plastids. After that, the DNA fragment of three copies of MTSRC was excised from the pBS-3xMTSRC plastid using Eco RI restriction enzyme, and then cloned into the pAct plastid with the Act1 promoter of the sequence shown in SEQ ID NO: 14 to Get pAct1-MTSRC plastid. Finally, the Act1 promoter containing three copies of OsMT2b SRC (MTSRC) was excised from the pAct1-MTSRC plastid using Hind III restriction enzyme, and sub-populated to have the pAct1-MTSRC plastid as shown in Figure 1D. The pAct1-Luc plastid with the actin-1 intron (Actin-1 intron, Act1 In) sequence is shown to obtain three MTSRC repeats and actin in the Act1 promoter as shown in Figure 1E The intron pAct1-MTSRC-Luc plastid, and the Act1 promoter with three MTSRC repeats and the actin intron has the sequence shown in SEQ ID NO:16.

Preparation Example 3: Construction of pAct1-MTSRC-haFGF plastid

Synthesize the full-length haFGF gene containing the signal peptide (SP) of the αAmy3 gene at the 5'end by chemical synthesis method (Shenggong Co., Ltd.), and insert it into the pBluescript II SK vector (Stratagene). Among them, the signal peptide and the haFGF gene are The nucleotide sequence is modified according to the codon usage optimization of rice, and the signal peptide of the αAmy3 gene has the nucleotide sequence shown in SEQ ID NO: 17, and the haFGF gene has the nucleotide sequence shown in SEQ ID NO:17. ID NO: the nucleotide sequence shown in 18 to obtain pBS-haFGF plastid. Afterwards, the synthesized SP-haFGF fusion gene was excised from pBS-haFGF plastid using Sal I restriction enzyme, and then colonized to pAct1-MTSRC-Luc to obtain pAct1-MTSRC-haFGF plastid.

Preparation Example 4: Construction of pA35mA-Luc, pAMT825-Luc, pAMT351-Luc, pAMT121-Luc, pAMT36-Luc, pAMTSRC-Luc, pAAct1-MTSRC-Luc and pAAct1-MTSRC-haFGF plastids

According to the content of Ho et al. (2000), the binary vector pSMY1H plastid was used to construct pA35mA-Luc, pAMT825-Luc, pAMT351-Luc, pAMT121-Luc, pAMT36-Luc, pAMTSRC-Luc, pAAct1 -MTSRC-Luc and pAAct1-MTSRC-haFGF plastids for Agrobacterium transfer.

Hind III restriction enzyme cleavage using a respective p35mA-Luc, after pMT825-Luc, pMT351-Luc, pMT121-Luc and pMT36-Luc plasmid, are all times to both ends of subcloned restriction sites Hind III linearized plasmid pSMY1H, To obtain pA35mA-Luc, pAMT825-Luc, pAMT351-Luc, pAMT121-Luc and pAMT36-Luc plastids.

After cutting pMTSRC-Luc and pAct1-MTSRC-haFGF plastids with Sac II and Kpn I restriction enzymes, respectively, they were treated with Klenow enzyme to form linear pMTSRC-Luc and pAct1 with blunt ends at both ends. -MTSRC-haFGF plastids, and ligate the linear pMTSRC-Luc and pAct1-MTSRC-haFGF plastids with the linear pSMY1H plastids with blunt ends at both ends through ligase to obtain pAMTSRC-Luc and Figure 1F respectively The pAAct1-MTSRC-haFGF plastids shown. After using the Pst I restriction enzyme cleavage pAct1-MTSRC-Luc plasmid, subcloned twice to both ends of Pst I restriction sites are all linearized plasmid pSMY1H, to afford pAAct1-MTSRC-Luc plasmid.

Example 1: Testing the activity of OsMT2b promoter in rice cells and rice embryos under the conditions of sugar and sugar deficiency

According to the content of the reference Chen et al. (2002), using the Agrobacterium transfer method, through the pAMT825-Luc plastid, the OsMT2b promoter and Luc chimeric gene was introduced into the rice gene to obtain an independent transgenic rice strain.

Transformed rice plants were randomly selected, and based on the content of the reference Yu et al. (1991), the rice suspension cells of each transformed rice plant were established. The rice suspension cells were kept at 28℃ in the dark and contained 3% sucrose or lacked 3%. Cultivate for two days in a sucrose culture environment. Rice embryos were cultured at 28°C in the dark for two days in a culture environment containing or lacking 3% sucrose. Use a cell culture lysis reagent (CCLR) buffer solution containing 100mM KH ₂ PO ₄ , pH 7.8, 1mM EDTA, 10% glycerol, 1% Trinitrotoluene X-100 and 7mM β-mercaptoethanol to extract rice Suspension cells and rice embryo total protein, and use Commassie Brilliant Blue R250 protein analysis reagent (Pierce) to determine the protein concentration, and according to the content of the reference Lu et al. (1998), analyze the luciferase in rice suspension cells and rice embryo active.

The results are shown in Figure 2. In the transgenic rice suspension cells, a sugar-deficient environment (-S) can induce the activity of the OsMT2b promoter. In addition, the luciferase activity of transgenic rice embryos in a sugar-deficient environment (-S) was significantly increased by 9.1 times to 14.2 times compared with that in a sugar environment (+S). This result confirms that in rice suspension cells and rice embryos, the activity of the OsMT2b promoter can be induced by a sugar-deficient environment.

Example 2: Confirmation of sugar response complex (SRC) in rice cells that confers sugar response to OsMT2b promoter

Using the Agrobacterium transfer method, pAMT825-Luc, pAMT351-Luc, pAMT121-Luc, and pAMT36-Luc plastids were used to transform the chimeric gene of OsMT2b full-length promoter and Luc and the chimeric gene of OsMT2b partial deletion promoter and Luc, respectively Introduce rice gene into body to obtain independent transgenic rice plant. Establish rice suspension cells of each transgenic rice plant. The rice suspension cells were cultured at 28°C in the dark under 3% sucrose or lacking 3% sucrose for two days, and the activity of luciferase was analyzed.

The results are shown in Figure 3. Compared with the OsMT2b full-length promoter (pAMT825-Luc), the 351 base pair upstream fragment of the OsMT2b promoter (pAMT351-Luc) is either in the presence of sugar (+S) or lack of sugar ( Under the environment of -S), the activity of giving fluorescent enzymes is the highest. In addition, the OsMT2b full-length promoter and the OsMT2b partial deletion promoter (pAMT351-Luc) induce similar luciferase activity in a glucose-deficient environment (pAMT825-Luc is 2.8 times, and pAMT351-Luc is 3.1 times). Furthermore, deleting the 5'end of the OsMT2b promoter to the -121 position (pAMT121-Luc) will significantly reduce the promoter activity and sugar reactivity, and when the OsMT2b promoter is further deleted to the -36 position, it will be even more It reduces the activity of luciferase and the reactivity to sugar. Therefore, the results show that the 5'-flanking region of the OsMT2b promoter between -351 and -121 is important for providing a high level of promoter activity in a glucose-deficient environment. The sequence of OsMT2b promoter was further analyzed, and the results showed that there are two consensus sequences between -173 and -160 of OsMT2b promoter, namely G box (5'-TACGTG-3') and TA box (5'-TATCCA-3').

In order to confirm whether the G box and TA box of the OsMT2b promoter are responsible for inducing the activity of the OsMT2b promoter in a sugar-deficient environment, using the Agrobacterium transfer method, through pA35mA-Luc and pAMTSRC-Luc plastids, 35mA-Luc and MTSRC respectively -The Luc chimeric gene is introduced into the rice gene to obtain an independent transgenic rice plant. Establish rice suspension cells of each transgenic rice plant. The rice suspension cells were cultured at 28°C in the dark under 3% sucrose or lacking 3% sucrose for two days, and the activity of luciferase was analyzed. The results are shown in Figure 4. In rice cells, a 146 base pair promoter containing a G box and a TA box (ie, the OsMT2b promoter transcription initiation position -266 to-as shown in SEQ ID NO: 13) The sequence of 121) was significantly induced by the glucose-deficient environment, so the region induced by the glucose-deficient environment was named MTSRC.

Example 3: Confirmation of the activity of MTSRC on Act1 promoter

In order to confirm whether MTSRC can enhance the activity of Act1 promoter, according to the content of the reference Umemura et al. (1998), the short-term performance analysis method of rice embryo particle bombardment was used through the pAct1-Luc plastid shown in Figure 1D and 1E. And pAct1-MTSRC-Luc plastid containing three MTSRC repeat fragments to detect the activity of luciferase in rice embryo. The transfected rice embryos were cultured in MS medium (Murashige and Skoog medium) containing 100 mM glucose or mannitol at 28°C for 24 hours. Each independent experimental system consisted of 8 rice embryos and performed 3 repetitions. According to the content of the reference Chen et al. (2006), the luciferase activity and GUS activity were tested, and the pUG quality system was used as an internal quantitative control (internal quantitative control). control) to standardize data. The results are shown in Figure 5. MTSRC can indeed enhance the activity of Act1 promoter regardless of whether it is in the presence or absence of sugar. In addition, in a glucose-deficient environment, MTSRC significantly enhanced the activity of the Act1 promoter by about 15.5 times. This shows that MTSRC does have the ability to convert the Act1 promoter induced by sugar into a promoter induced by glucose deficiency.

In order to further confirm whether similar results can be obtained in stably transformed rice cells and seedlings, the Agrobacterium transfer method was used to introduce the Act1-MTSRC-Luc chimeric gene into the rice gene through pAAct1-MTSRC-Luc plastids , To obtain an independent Act1-MTSRC-Luc transgenic rice plant, and use Act1-Luc transgenic rice plant as a control group. Randomly select transgenic rice plants and establish rice suspension cells for each transgenic rice plant, or analyze the activity of luciferase after 8 days after the seeds of each T2 transgenic rice plant germinate. The results are shown in Figure 6A. Compared with the Act1 promoter, the average luciferase activity conferred by the Act1-MTSRC promoter increased by up to 8 times and 44 times, respectively, in the presence of sugar and the lack of sugar. In addition, the seeds of 4 T3 transgenic rice plants with Act1-Luc and 6 T2 transgenic rice plants with Act1-MTSRC-Luc seeds were germinated 8 days later, 5 seedlings were selected for each transgenic plant respectively. Analyze the activity of fluorescent enzymes. The results are shown in Figure 6B. In the transgenic rice seedlings, MTSRC can increase the activity of Act1 promoter by about 11 times on average.

Example 4: Comparison of the effect of MTSRC and αAmy3SRC on the activity of Act1 promoter

In order to compare the activities of Act1 promoter, Act1-αAmy3SRC promoter and Act1-MTSRC promoter in different tissues of germinated and transgenic rice seeds , Act1-6-19 with Act1-Luc and Act1-αAmy3SRC-Luc genes, respectively -1-1 and Act1-αAmy3SRC-5-10-4-3 transgenic rice plant T4 seeds, and Act1-MTSRC-Luc gene with Act1-MTSRC-3-12 transgenic rice plant T2 seeds for germination After 14 days of growth, collect the different tissues of the seedlings of each transgenic rice plant and analyze the activity of luciferase. The results are shown in Figure 7A. αAmy3SRC can significantly enhance the activity of Act1 promoter in rice embryos, stems and roots, while MTSRC can generally enhance the activity of Act1 promoter in all tissues of transgenic rice. As further shown in Figure 7B, in rice seedlings, MTSRC has a better ability to enhance the Act1 promoter than αAmy3SRC.

Example 5: Confirmation of the performance of recombinant haFGF mediated by Act1-MTSRC promoter in transgenic rice plants

In order to test whether the Act1-MTSRC promoter in rice can indeed produce high-level foreign proteins, the Agrobacterium transfer method was used to introduce the Act1-MTSRC::haFGF chimeric gene into rice through pAAct1-MTSRC-haFGF plastids Gene body to obtain independent transgenic rice plants. Establish several calli of independently transformed rice plants, and culture each callus in a sugar-deficient N6 medium for 2 days. After extracting the total protein in the cell with CCLR buffer solution, analyze it with Commassie Brilliant Blue protein The reagent (Pierce) measures the protein concentration. Eight transgenic rice plants were randomly selected. According to the content of the reference Lu et al. (2007), the protein gel ink stain analysis method was used to detect the transgenic rice plant through the anti-haFGF monoclonal antibody (1:500, Abcam) The performance of haFGF protein. In addition, the commercially available haFGF (Sigma) was used as a standard to quantify the recombinant haFGF protein through anti-haFGF monoclonal antibodies, and the Quantity One software (Bio-Rad) was used to calculate the cumulative amount of recombinant haFGF protein in rice cells.

The results are shown in Figure 8A. Two forms of recombinant haFGF protein are present in the transgenic rice plants. The higher band is full-length haFGF with a molecular weight of 17.4kDa (as indicated by the triangle mark), while the lower band is full-length haFGF. The band is the same as the commercially available haFGF, with a molecular weight of 15.8kDa (as indicated by the arrow mark), which shows that there is an N-terminal truncated form of haFGF in the transgenic rice cells, and these protein bands do not appear in non-transformed rice cells. Planted in rice cells.

In order to detect whether the recombinant haFGF protein expressed in rice would be secreted into the culture medium, ACT-haFGF-3 rice transgenic plants with the highest haFGF protein expression were selected and regenerated into T0 plants and then selfed to produce T1 seeds. The embryos of independent T1 seeds were induced into callus and cultured into suspension cells. Collect the total protein of the culture fluid from the culture fluid of rice suspension cells, and further detect the recombinant haFGF. The results are shown in Figure 8B. In the rice suspension cell culture medium of the ACT-haFGF-3 rice plant, two forms of recombinant haFGF protein will also accumulate, and the concentration of the recombinant haFGF protein accounts for approximately the total protein in the culture medium. 2% of the amount.

In order to detect whether the recombinant haFGF protein expressed in rice and secreted into the culture medium has biological activity, according to the content of the reference Gospodarowicz (1974), the NIH/3T3 cell expansion analysis method was used to detect and collect from the sugar-deficient treatment 16 The biological activity of the culture solution protein of the wild-type rice line TNG67 and the rice transgenic cell line ACT-haFGF-3 or the commercially available haFGF protein after hours. The results are shown in Figure 9. Compared with the culture medium protein of wild-type rice strain TNG67 and the unstimulated control group, NIH/3T3 cells were significantly stimulated by the recombinant haFGF protein expressed by rice and the commercially available haFGF protein. . These results show that the recombinant haFGF protein expressed by the transgenic rice cell culture system does still have mitogen-stimulating activity and can promote the proliferation of NIH/3T3 cells.

The above is a description of exemplary embodiments of the present disclosure. It should be understood that the scope of the present disclosure is not limited to the disclosed embodiments; on the contrary, it should cover various modifications and similar reorganizations. Together. Therefore, the scope of the patent application should be given the broadest interpretation to cover all such modifications and similar combinations.

The following references are incorporated into this article by reference: Chen PW, Lu CA, Yu TS, Tseng TH, Wang CS, and Yu SM (2002) Rice alpha-amylase transcriptional enhancers direct multiple mode regulation of promoters in transgenic rice. J. Biol. Chem. 277 (16): 13641-13649.

Chen PW, Chiang CM, Tseng TH, and Yu SM (2006) Interaction between rice MYBGA and the gibberellin response element controls tissue-specific sugar sensitivity of alpha-amylase genes. Plant Cell 18 (9): 2326-2340.

Christensen, AH, Sharrock, RA, and Quail, PH (1992) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol. 18: 675-689.

Christensen AH and Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 5(3): 213-218.

Daniell H, Streatfield SJ, and Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci. 6: 219-226.

Gospodarowicz D (1974) Localisation of a fibroblast growth factor and its effect alone and with hydrocortisone on 3T3 cell growth. Nature 249 (5453): 123.

Ho SL, Tong WF, and Yu SM (2000) Multiple mode regulation of a cysteine proteinase gene expression in rice. Plant Physiol. 122 (1): 57-66.

Kyozuka J, Fujimoto H, Izawa T, and Shimamoto K (1991) Anaerobic induction and tissue-specific expression of maize Adh1 promoter in transgenic rice plants and their progeny. Mol. Gen. Genet. 228: 40-48.

Kyozuka J, Olive M, Peacock WJ, Dennis, E.S., and Shimamoto K (1994) Promoter element required for developmental expression of the maize Adh1 gene in transgenic rice. Plant Cell 6: 799-810.

Lu CA, Lim EK, and Yu SM (1998) Sugar response sequence in the promoter of a rice alpha-amylase gene serves as a transcriptional enhancer. J. Biol. Chem. 273 (17): 10120-10131.

Lu CA, Lin CC, Lee KW, Chen JL, Huang LF, Ho SL, Liu HJ, Hsing YI, and Yu SM (2007) The SnRK1A protein kinase plays a key role in sugar signaling during germination and seedling growth of rice. Plant Cell 19 (8): 2484-2499.

Olive MR, Walker JC, Singh K, Dennis ES, and Peacock WJ (1990) Functional properties of the anaerobic responsive element of the maize Adh1 gene. Plant Mol. Biol. 15: 593-604.

Umemura T, Perata P, Futsuhara Y, and Yamaguchi J (1998) Sugar sensing and alpha-amylase gene repression in rice embryos. Planta 204(4): 420-428.

Weigel D, Ahn JH, Blazquez MA, Borevitz JO, Christensen SK, Fankhauser C, Ferrandiz C, Kardailsky I, Malancharuvil EJ, Neff MM, Nguyen JT, Sato S, Wang ZY, Xia Y, Dixon RA, Harrison MJ, Lamb CJ , Yanofsky MF, and Chory J (2000). Activation tagging in Arabidopsis. Plant Physiol. 122: 1003-1013.

Yu SM, Kuo YH, Sheu G, Sheu YJ, and Liu LF (1991) Metabolic derepression of alpha-amylase gene expression in suspension-cultured cells of rice. J. Biol. Chem. 266 (31): 21131-21137.

<110> National Chiayi University

<120> The isolated transcription enhancer sequence, the modified promoter sequence comprising the isolated transcription enhancer sequence and its use

<130> Chen Pengwen, Chen Jianlong, Li Zhendong

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Claims (20)

An isolated transcription enhancer sequence, comprising at least one copy number of a nucleotide sequence with more than 90% similarity to the nucleotide sequence shown in SEQ ID NO: 13, and the nucleotide sequence is the same as SEQ ID NO: 13 also has the function of enhancing gene transcription. The isolated transcription enhancer sequence described in the first item of the scope of patent application, wherein the nucleotide sequence having more than 90% similarity with the nucleotide sequence shown in SEQ ID NO: 13 contains at least one common sequence. According to the isolated transcription enhancer sequence described in item 2 of the scope of patent application, the at least one common sequence is a G box sequence or a TA box sequence. According to the isolated transcription enhancer sequence described in item 3 of the scope of patent application, the G box sequence is TACGTG from the 5'end to the 3'end, and the TA box sequence is TATCCA from the 5'end to the 3'end. The isolated transcription enhancer sequence described in item 1 of the scope of patent application includes at least one copy number of the nucleotide sequence shown in SEQ ID NO:13. The isolated transcription enhancer sequence described in item 5 of the scope of patent application includes at least three copies of the nucleotide sequence shown in SEQ ID NO:13. A modified promoter sequence, comprising a promoter and the isolated transcription enhancer sequence as described in any one of items 1 to 6 of the scope of patent application, wherein the promoter is operably linked to the isolated Transcription enhancer sequence. The modified promoter sequence described in item 7 of the scope of patent application, wherein the promoter is actin 1 promoter, ubiquitin promoter, CaMV 35S promoter or rbc S gene promoter. An expression vector comprising a left border sequence of a transferred DNA, a regulatory sequence, a nucleotide sequence encoding a multi-peptide, and a right border sequence of the transferred DNA, wherein the regulatory sequence is operably linked to the coding sequence The nucleotide sequence of the peptide is located between the left border sequence of the transferred DNA and the right border sequence of the transferred DNA, and wherein the regulatory sequence includes the modified as described in item 7 or 8 of the scope of patent application Promoter sequence. The expression vector according to item 9 of the scope of patent application, wherein the regulatory sequence further comprises an intron sequence, and the intron sequence is operably linked to the modified promoter sequence and the encoded polypeptide Between the nucleotide sequence. The expression vector described in item 10 of the scope of patent application, wherein the intron sequence is the actin 1 intron or the alcohol dehydrogenase 1 intron. The expression vector described in item 11 of the scope of patent application, wherein the actin 1 intron has the nucleotide sequence shown in SEQ ID NO:15. The expression vector according to claim 9, wherein the regulatory sequence further comprises a nucleotide sequence encoding a signal peptide, and the nucleotide sequence encoding the signal peptide is operably linked to the modified promoter Between the subsequence and the nucleotide sequence encoding the multipeptide. The expression vector described in item 13 of the scope of patent application, wherein the nucleotide sequence encoding the signal peptide has the nucleotide sequence shown in SEQ ID NO:17. The expression vector described in item 9 of the scope of patent application, wherein the nucleotide sequence encoding the multi-peptide encodes human acidic fibroblast growth factor, human epidermal growth factor or Orf1 protein of porcine type 2 circovirus . The expression vector described in item 15 of the scope of patent application, wherein the human acidic fibroblast growth factor has the nucleotide sequence shown in SEQ ID NO: 18. A plant transformed cell comprising the expression vector as described in any one of items 9 to 16 in the scope of the patent application. A method for producing exogenous protein, comprising: culturing the plant transformed cell as described in item 17 of the scope of patent application in a plant culture medium; and separating the foreign protein from the plant culture medium. The method according to item 18 of the scope of patent application, wherein the plant culture medium is a plant culture medium under sugar-deficient conditions. A method for producing exogenous protein, comprising: regenerating the plant transformed cell as described in item 17 of the patent application into a plant transgenic plant; and extracting the foreign protein from the root, stem, leaf or seed of the plant transgenic plant Source protein.

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