METHOD FOR PROMOTING EXPRESSION OF FOREIGN GENE IN SOYBEAN, ARABIDOPSIS OR TOBACCO USING GENE PROMOTER PRPS28 OR PRPS28-I

Abstract

A method for promoting expression of a foreign gene in a plant includes: constructing a recombinant vector including a full-length soybean gene promoter pRPS28 or an intron-including soybean gene promoter pRPS28-I, and introducing the recombinant vector into the plant; the full-length promoter pRPS28 being represented by SEQ ID NO: 2, and the intron-including promoter being represented by pRPS28-I SEQ ID NO: 3.

Description

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[0001.1] This application contains a sequence listing, which has been submitted electronically in XML file and is incorporated herein by reference in its entirety. The XML file, created on Feb. 21, 2023, is named ZZLK-03501-UUS.xml, and is 37,550 bytes in size.

BACKGROUND

The disclosure related to the field of plant genetic engineering, and more particularly, to two soybean promoters pRPS28 and pEIF1-1 and applications thereof in soybean, Arabidopsis thaliana and tobacco.

In genetics, a promoter is a sequence of DNA to which proteins bind to initiate the transcription of a single RNA transcript from the DNA downstream of the promoter. The promoter is generally classified into two categories: exogenous promoter and endogenous promoter. An exogenous promoter from viruses, such as the 35S promoter of tobacco mosaic virus, controls the heterologous gene expression, which, however, arises transgenic safety problems. Transgenic silencing is often blamed on the use of the same promoter for expression of different transgenes. The endogenous promoters are more suitable for use in the production of transgenic materials.

Soybean is an important crop that supplies most of protein and oil requirements. Soybean establishes symbiotic relationships with rhizobia, which forms nodules in their roots, leading to biological nitrogen fixation. Rhizobia play a fundamental role in nitrogen supply to ecosystems through their ability to fix nitrogen in symbiosis with legumes and promote the growth of plants. Soybean can also be intercropped or rotated with other plants without fertilizer application, thus providing a safe nitrogen source. Soybean has been genetically modified to produce in larger quantities. In addition to the viral promoter, such as the 35S or CMV promoter for soybean transformation, some endogenous promoters, such as the constitutive promoters, inducible promoters, tissue-specific promoters, have also been reported over the years.

Gmubi promoter is a widely used constitutive promoter with high levels of constitutive expression in soybean. The majority of constitutive promoters are chosen during a single soybean developmental stage, and only rarely during the formation of nodules on the soybean roots. When multiple genes are inserted into the genome of a transgenic organism, transgenic silencing occurs.

SUMMARY

The first objective of the disclosure is to provide two soybean reference genes RPS28 and EIF1, as well as two promoters pRPS28 and pEIF1 thereof; the soybean reference genes RPS28 and EIF1 are used as an internal control to determine the expression levels of a target gene in soybean organs at different developmental stages and the development level of root nodules.

The second objective of the disclosure is to provide a use of the promoters pRPS28 and pEIF1 in regulating the constitution or non-tissue specific expression of soybean genes or other plants (such as tobacco and Arabidopsis) genes; specifically, the full length promoter pRPS28 and intron-comprising promoter pRPS28-I, the full length promoter pEIF1 and intron-comprising promoter pEIF1-I are used to promote the overexpression or universal expression of the target genes in soybean, Arabidopsis, or tobacco.

To achieve the above objectives, the disclosure provides a method for promoting expression of a foreign gene in a plant, and the method comprises:

• constructing a recombinant vector comprising a full length promoter pRPS28 or an intron-comprising promoter pRPS28-I, a full length promoter pEIF1 or an intron-comprising promoter pEIF1-I as a ubiquitous promoter to promote the expression of a foreign gene in soybean organs;
• the foreign gene is beta-glucuronidase (GUS) gene;
• when introduced into soybean, the promotor promotes the foreign gene to express in cotyledons, radicles, plumules, true leaves, compound leaves, buds, petioles, internodes, roots and root nodules of the soybean;
• when introduced into Arabidopsis, the promotor promotes the foreign gene to express in whole plant, flower and pod of the Arabidopsis; and
• when introduced into tobaccos, the promotor promotes the foreign gene to express in leaves of the tobaccos.

Further, the disclosure provides a recombinant vector comprising any one of the promoters.

The recombinant vector is prepared by inserting the one of the promoters into a vector pCAMBIA1391Z-BAR; specifically, inserting the promoters pRPS28, pRPS28-I, pEIF1 and pEIF1-I into the vector pCAMBIA1391Z-BAR to form recombinant vectors pRPS28-GUS-BAR, pRPS28-I-GUS-BAR, pEIF1-GUS-BAR and pEIF1-I-GUS-BAR, respectively.

The disclosure further provides primer sequences for amplification of the genes RPS28 and EIF1.

The gene RPS28 is amplified using the following primers:

RPS28-F: ATGGAGTCTCAGGTGAAGCAC (SEQ ID NO: 8);

RPS28-R: CTAGCGCAATCTTCTTGCTTC (SEQ ID NO: 9).

The gene EIF1 is amplified using the following primers:

EIF1-F: ATGTCTGAATTAGACGATCAAATTCC (SEQ ID NO: 10)
;

EIF1-R: TCAGAAACCATGAATCTTGATATGATC (SEQ ID NO: 11
).

Another objective of the disclosure is to provide primer sequences for amplification of the promoters.

The recombinant vector pRPS28-GUS is amplified using the following primers:

pRPS28-GUS-bar-F:

GACCATGATTACGCCAAGCTTCACCACCCAATCCATAACCACCA (SEQI
D NO: 12)

pRPS28-GUS-bar-R:

CCAGTGAATTCCCGGGGATCCCTGATGCAAAACACGAACAAAGAAAG(SE
Q ID NO: 13)

The recombinant vector pRPS28-I-GUS is amplified using the following primers:

pRPS28-I-GUS-bar-F:

GACCATGATTACGCCAAGCTTCACCACCCAATCCATAACCACCAC(SEQ
ID NO: 14)

pRPS28-I-GUS-bar-R:

CCAGTGAATTCCCGGGGATCC CCTGCTCAAACACAATCAACAG (SEQ
ID NO: 15)

The recombinant vector pEIF1-GUS is amplified using the following primers:

pEIF1-GUS-bar-F:

GACCATGATTACGCCAAGCTT GGAGAGAAGTTGAACTCTGAGTTGTG
(SEQ ID NO: 16)

pEIF1-GUS-bar-R:

CCAGTGAATTCCCGGGGATCC CTGATCGTAAATTTAAGGTTTCG (SEQ
ID NO: 24)

The recombinant vector pEIF1-I-GUS is amplified using the following primers:

pEIF1-I-GUS-bar-F:

GACCATGATTACGCCAAGCTT GGAGAGAAGTTGAACTCTGAGTTGTG
(SEQ ID NO: 16)

pEIF-I-GUS-bar-R: CCAGTGAATTCCCGGGGATCC
AAAACTTGACTCACTAAGACCAAAGG (SEQ ID NO: 17)

The promoter pRPS28 has a nucleic acid sequence comprising SEQ ID NO: 2; and the promoter pRPS28-I has a nucleic acid sequence comprising SEQ ID NO: 3.

The promoter pEIF1 has a nucleic acid sequence comprising SEQ ID NO: 5; the promoter pEIF1-I has a nucleic acid sequence comprising SEQ ID NO: 6.

The seventh objective of the disclosure is to provide a method of using the promoter pRPS28 or pRPS28-I to promote the expression of a foreign gene in soybean.

The eighth objective of the disclosure is to provide a method of using the promoter pEIF1 or pEIF1-I to promote the expression of a foreign gene in soybean.

The following advantages are associated with the promoters of the disclosure.

• 1. The disclosure provides the genes RPS28 and EIF1 that are highly expressed across the soybean tissues at different life cycle stages without being affected by the Bradyrhizobium japonicum strain; the different life cycle stages comprise: 8 days after germination of the aerial and underground parts of the soybean, and the various developmental stages of the roots infect with Bradyrhizobium japonicum strain (USDA 110).
• 2. The promoters pRPS28 and pEIF1 drive the constitutive expression of the target gene in different soybean tissues (comprising the root nodules) of the transgenic plants.
• 3. The promoter pRPS28 promoted the same level of GUS expression as the promoter GmUbi; the promoter pEIF1 comprises an intron sequence that help to maintain the same level of GUS expression as the promoter GmUbi.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are graphs showing the relative expression of the gene RPS28 in soybean tissues at different life cycle stages, as well as in the root nodules;

FIGS. 2A-2C are graphs showing the relative expression of the gene EIF1 in soybean tissues at different life cycle stages, as well as in the root nodules;

FIG. 3 shows two maps of two recombinant vectors comprising a promoter pRPS28 and a promoter pEIF1, respectively;

FIGS. 4A-4E show the GUS activity controlled by the promoters pRPS28 and pEIF1 during genetic transformation;

FIGS. 5A-5G show the relative expression of GUS protein under control of the promoter pRPS28 in T2 transgenic soybean plants;

FIGS. 6A-6G show the relative expression of GUS protein under control of the promoter pEIF1 in T2 transgenic soybean plants;

FIG. 7 is a graph showing the GUS activity controlled by the promoters pRPS28, pRPS28-I, pEIF1, pEIF1-I and pGmUbi in transgenic soybean plants;

FIGS. 8A-8D show the relative expression of GUS protein under control of the promoters pRPS28, pRPS28-I and pGmUbi in T3 transgenic soybean plants;

FIGS. 9A-9B show the relative expression of GUS protein under control of the promoters pEIF1, pEIF1-I and pGmUbi in T3 transgenic soybean plants; and

FIG. 10 shows the transient expression of GUS protein under control of the promoters pRPS28 and pRPS28-I in transgenic tobacco leaves.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The soybean genes RPS28 and EIF1 were amplified; the promoters pRPS28 and pEIF1 were isolated by CTAB method; the promoters pRPS28, pRPS28-I, pEIF1 and pEIF1-I were fused to the GUS gene to form four recombinant vectors pRPS28-GUS, pRPS28-I-GUS, pEIF1-GUS and pEIF1-I-GUS, respectively; the soybean plants were transfected with the vectors; and the GUS protein expression was driven by the promoters pRPS28 and pEIF1. The results showed that the promoters pRPS28, pRPS28-I, pEIF1 and pEIF1-I promoted the expression of the GUS protein in cotyledons, radicles, germs, true leaves, compound leaves, shoots, petioles, internodes, roots and root nodules, which indicated that the promoters pRPS28 and pEIF1 were ubiquitous promoters. The GUS activity was determined in soybean. In addition, the promoters pRPS28 and pEIF1 were used to promote the expression of the GUS protein in Arabidopsis thaliana and tobacco.

If not specified, the reagents and biological material are commercially available.

Example 1

Selection of Genes RPS28 and EIF1

A soybean cultivar Jidou 17 was planted; the true leaves unfolded on 8^th day after planting; at the same time, the soybean roots were inoculated with a Bradyrhizobium japonicum strain (USDA 110); 1, 2, 4, 6, 8, 10, 15, 20, 25 and 30 days after inoculation, tissues including roots, root nodules, hypocotyls, cotyledons, epicotyls, true leaves, true leaf nodes, compound leaves, internodes, petioles and terminal buds were collected for RNA extraction and sequencing by any conventional method. Specifically, the genes RPS28 and EIF1 are selected through the following steps:

• 1. finding the ubiquitous genes that were expressed across the soybean tissues at different life cycle stages with RNA-seq; the RNA-seq data for soybean showed the FPKM expression value of each ubiquitous gene; sorting the ubiquitous genes by the FPKM expression values in a decreasing order; and selecting top 20 ubiquitous genes as candidate genes;
• 2. visualizing the RNA-seq data for the 20 candidate genes; selecting 10 genes that were expressed in the same level across the soybean tissues at different life cycle stages without being affected by the Bradyrhizobium japonicum strain (coefficient of variation CV≤0.3); and
• 3. visualizing the RNA-seq data for the 10 genes; selecting two genes that maintain constant expression levels in all conditions; and analyzing the sequence of the promoter in each gene.

The two genes were named RPS28 and EIF1, respectively; the gene RPS28 encoded a 40S ribosomal protein S2 and has a cDNA sequence comprising SEQ ID NO: 1; the gene EIF1 encoded a eukaryotic initiation factor SUI1and had a cDNA sequence comprising SEQ ID NO: 4.

The results showed that 1-30 days after the true leaves unfolded, the two genes RPS28 and EIF1 were expressed at high levels in different soybean tissues without being affected by the Bradyrhizobium japonicum strain (FIGS. 1A-1C and 2A-2C).

FIGS. 1A-1C were graphs showing the relative expression of the gene RPS28 in the soybean tissues at different life cycle stages, as well as in the root nodules. In FIGS. 1A-1B, when the true leaves unfolded, the soybean roots were inoculated with or without the Bradyrhizobium japonicum strain; after 1, 4, 6, 8, 10 and 20 days of inoculation, RNA sequencing was carried out to reveal the presence and quantity of the gene RPS28 in the soybean tissues comprising hypocotyls, cotyledons, epicotyls, true leaves, true leaf nodes, compound leaves, internodes, petioles and terminal buds. The results showed that the gene RPS28 was stably expressed at high levels across the soybean tissues without being affected by the Bradyrhizobium japonicum strain. In FIG. 1C, when the true leaves unfolded, the soybean roots were inoculated with or without the Bradyrhizobium japonicum strain; after 1, 2, 4, 6, 8, 10, 15, 20, 25, and 30 days of inoculation, RNA sequencing was carried out to reveal the presence and quantity of the gene RPS28 in the roots and the root nodules. The results showed that the gene RPS28 was stably expressed at high levels in the roots and root nodules.

FIGS. 2A-2C were graphs showing the relative expression of the gene EIF1 in soybean tissues at different life cycle stages, as well as in the root nodules.

In FIGS. 2A-2B, when the true leaves unfolded, the soybean roots were inoculated with or without the Bradyrhizobium japonicum strain; after 1, 4, 6, 8, 10 and 20 days of inoculation, RNA sequencing was carried out to reveal the presence and quantity of the gene EIF1 in the soybean tissues comprising hypocotyls, cotyledons, epicotyls, true leaves, true leaf nodes, compound leaves, internodes, petioles and terminal buds. The results showed that the gene EIF1 was stably expressed at high levels across the soybean tissues without being affected by the Bradyrhizobium japonicum strain. In FIG. 2C, when the true leaves unfolded, the soybean roots were inoculated with or without the Bradyrhizobium japonicum strain; after 1, 2, 4, 6, 8, 10, 15, 20, 25, and 30 days of inoculation, RNA sequencing was carried out to reveal the presence and quantity of the gene EIF1 in the roots and the root nodules. The results showed that the gene EIF1 was stably expressed at high levels in the roots and root nodules.

Example 2

Amplification of Promoters pRPS28 and pEIF1

According to the results in Example 1, the genes RPS28 and EIF1 had a genome sequence spanning of 2357 bp and 1640 bp, respectively; and each gene contained an intron sequence between ATG start codon and 5′UTR. Therefore, a full-length promoter and an intron-comprising promoter were designed for each gene and named pRPS28, pRPS28-I, pEIF1 and pEIF1-I.

Genomic DNA was extracted from the soybean cultivar Williams 82 (WS82) by CTAB method, used as a template DNA for amplification of the DNA fragments RPS28, RPS28-I, pEIF1 and pEIF1-I. PCR amplification was performed as follows:

A 50 µL reaction contained 1 µL (about 100 ng) of the template DNA, 25 µL 2×Phanta Max Super-Fertility buffer, 1 µL of 10 mM dNTP, 5 µL of 4 µM primer (i.e. 2 µL of each primer with a concentration of 10 mM), and 1 µL of phanta MAX Super-Fertility DNA enzyme, and 17 µL of ddH2O (sterile deionized water).

The PCR cycling and running parameters were described as follows: denaturation at 94° C. for 2 min; 30 cycles of 94° C. for 10 s, 58° C. for 30 s, and 72° C. for 30 s; and the final extension at 72° C. for 5 min.

The gene RPS28 was amplified using the following primers:

F: ATGGAGTCTCAGGTGAAGCAC (SEQ ID NO: 8);

R: CTAGCGCAATCTTCTTGCTTC (SEQ ID NO: 9).

The gene EIF1 was amplified using the following primers:

F: ATGTCTGAATTAGACGATCAAATTCC (SEQ ID NO: 10)

R: TCAGAAACCATGAATCTTGATATGATC (SEQ ID NO: 11)

The promoter pRPS28 was amplified using the following primers:

pRPS28-F: CACCACCCAATCCATAACCACCAC (SEQ ID NO: 25)

pRPS28-R: CTGATGCAAAACACGAACAAAGAAAG (SEQ ID NO: 2
6)

The promoter pRPS28 has a nucleic acid sequence comprising SEQ ID NO: 2;

The promoter pRPS28-I was amplified using the following primers:

pRPS28-I-F: CACCACCCAATCCATAACCACCAC (SEQ ID NO: 2
5)

pRPS28-I-R: CCTGCTCAAACACAATCAACAG (SEQ ID NO: 27)

The promoter pRPS28-I has a nucleic acid sequence comprising SEQ ID NO: 3.

The gene pEIF1was amplified using the following primers:

pEIF1-F: GGAGAGAAGTTGAACTCTGAGTTGTG (SEQ ID NO: 28
)

pEIF1-R: CTGATCGTAAATTTAAGGTTTCG (SEQ ID NO: 29)

The promoter pEIF1 has a nucleic acid sequence comprising SEQ ID NO: 5.

The promoter pEIF1-I was amplified using the following primers:

pEIF1-I -F: GGAGAGAAGTTGAACTCTGAGTTGTG (SEQ ID NO:
28)

pEIF1-I-R: AAAACTTGACTCACTAAGACCAAAGG (SEQ ID NO:
30)

The promoter pEIF1-I has a nucleic acid sequence comprising SEQ ID NO: 6.

Example 3

Transformation of promoters pRPS28 and pEIF1 into soybean

The GUS protein under control of the promoters pRPS28 and pEIF1 was stably expressed during the transformation process, such as co-cultivation, shoot induction and shoot elongation. As shown in FIG. 3, the recombinant vectors pRPS28-GUS, pRPS28-I-GUS, pEIF1-GUS and pEIF1-I-GUSI-GUS were prepared; and a recombinant vector pGmUbi-GUS was constructed as a control.

1. Construction of a Recombinant Vector

In Example 2, the DNA fragments pRPS28, pRPS28-I, pEIF1 and pEIF1-I were used as templates and inserted into a vector pCAMBIA1391Z-BAR by seamless cloning method, and the recombinant vectors pRPS28-GUS-BAR, pRPS28-I-GUS-BAR, pEIF1-GUS-BAR and pEIF1-I-GUS-BAR were prepared; the recombinant vector pGmUbi-GUS-BAR was constructed as a control; the five recombinant vectors were transformed into the soybean cultivar WS82.

PCR amplification was performed as follows:

A 50 µL reaction contained 1 µL (about 100 ng) of the template DNA, 25 µL 2×Phanta Max Super-Fertility buffer, 1 µL of 10 mM dNTP, 5 µL of 4 µM primer (i.e. 2 µL of each primer with a concentration of 10 mM), and 1 µL of phanta MAX Super-Fertility DNA enzyme, and 17 µL of ddH2O (sterile deionized water).

The PCR cycling and running parameters were described as follows: denaturation at 94° C. for 2 min; 30 cycles of 94° C. for 10 s, 58° C. for 30 s, and 72° C. for 30 s; and the final extension at 72° C. for 5 min.

The recombinant vector pRPS28-GUS was amplified using the following primers:

pRPS28-GUS-bar-F:

GACCATGATTACGCCAAGCTTCACCACCCAATCCATAACCACCAC(SEQ
ID NO: 14)

pRPS28-GUS-bar-R:

CCAGTGAATTCCCGGGGATCCCTGATGCAAAACACGAACAAAGAAAG(SE
Q ID NO: 13)

The recombinant vector pRPS28-I-GUS was amplified using the following primers:

pRPS28-I-GUS-bar-F:

GACCATGATTACGCCAAGCTTCACCACCCAATCCATAACCACCAC(SEQ
ID NO: 14)

pRPS28-I-GUS-bar-R:

CCAGTGAATTCCCGGGGATCC CCTGCTCAAACACAATCAACAG (SEQ
ID NO: 15)

The recombinant vector pEIF1-GUS was amplified using the following primers:

pEIF1-GUS-bar-F:

GACCATGATTACGCCAAGCTT GGAGAGAAGTTGAACTCTGAGTTGTG
(SEQ ID NO: 16)

pEIF1-GUS-bar-R:

CCAGTGAATTCCCGGGGATCC CTGATCGTAAATTTAAGGTTTCG (SEQ
ID NO: 24)

The recombinant vector pEIF1-I-GUS was amplified using the following primers:

pEIF1-I-GUS-bar-F:

GACCATGATTACGCCAAGCTT GGAGAGAAGTTGAACTCTGAGTTGTG
(SEQ ID NO: 16)

pEIF-I-GUS-bar-R:

CCAGTGAATTCCCGGGGATCC AAAACTTGACTCACTAAGACCAAAGG
(SEQ ID NO: 17)

The recombinant vector pGmUbi-GUS was amplified using the following primers:

pGmUbi-GUS-bar-F:

GACCATGATTACGCCAAGCTT GGGCCCAATATAACAACGAC (SEQ ID
NO: 18)

pGmUbi-GUS-bar-R: CCAGTGAATTCCCGGGGATCC
AAAACTTGACTCACTAAGACCAAAGG (SEQ ID NO: 17)

CCAGTGAATTCCCGGGGATCC ctgtcgagtcaacaatcaca (SEQ ID NO: 19)

The vector pCAMBIA1391Z-BAR was prepared by linearizing the vector pCAMBIA1391Z with the restriction enzyme XhoI, replacing the gene HygR with the gene BAR by the seamless cloning method in the presence of the following primers:

pCAMBIA1391Z-BAR-F:

TACAAATCTATCTCTCTCGAGatgagcccagaacgacgcccg (SEQ ID
NO: 20);

pCAMBIA1391Z-BAR-R:

CATTATTATGGAGAAACTCGAGTCAGATCTCGGTGACGGGCAGGAC(SEQ
ID NO: 21)

The seamless cloning method was performed by conventional techniques and accordingly was not described in detail herein.

2. Soybean Genetic Transformation and GUS Histochemical Staining

The recombinant vectors were transformed into the soybean cultivar WS82 by transformation of the cotyledon nodes with Agrobacterium tumefaciens EHA105 (Luth D, Warnberg K, Wang K. Soybean [Glycine max (L.) Merr]. Methods Mol Biol. 2015; 1223: 275-84. doi: 10.1007/978-1-4939-1695-5_22. PMID: 25300848.)

The transformation process was modified as follows:

Sterilization and germination of soybean seeds: the healthy seeds were separated from damaged and diseased soybean seeds, sterilized with chlorine gas (that was generated by slowly adding 5 mL of concentrated hydrochloric acid to 100 mL of sodium hypochlorite along the wall of a 250 mL beaker, followed by sterilization for 16 hours). The sterilized seeds were transferred in a culture medium and placed in an incubator at 22° C. for 16-24 hours in the dark.

Activation of Agrobacterium and preparation of an infiltration solution: the five recombinant vectors were transformed into the Agrobacterium tumefaciens EHA105 by electroshock method; and the Agrobacterium tumefaciens EHA105 grown on an LB agar plate with kanamycin. Positive clones were inoculated into an LB liquid culture medium comprising kanamycin and incubated in a shaker overnight at 220 rpm and 28° C. 250 µL of the bacterial solution was spread on the surface of the LB agar and incubated overnight at 28° C. The bacterial sample was picked up by an inoculation loop, resuspended in the liquid culture medium, and grown to an optical density at 600 nM (OD600) of 0.5-0.6 that was measured by a spectrophotometer.

Preparation and infection of an explant: after seed germination, the hypocotyl with a length of 3-5 mm was collected, and two cotyledons was separated, followed by removal of the seed coat and the primary bud; the cotyledonary nodes were cut through to form an explant. The explant was subsequently immersed in an infiltration solution and oscillated on a horizontal rotator (at a rotation speed of 50-80 r/min) for 30 min.

Co-cultivation: the explant was transferred from the infiltration solution onto a solid co-culture plate covered with a layer of sterile filter paper, with 15-20 explants in each plate; the explants were then cultivated in the incubator at 22° C. for 3-5 days in the dark; and stained with GUS.

Screening and culture for regeneration (Luth D, Warnberg K, Wang K. Soybean [Glycine max (L.) Merr]. Methods Mol Biol. 2015; 1223:275-84. doi: 10.1007/978-1-4939-1695-5_22. PMID: 25300848.): shoot induction: after co-cultivation for 3-5 days, the explants were transferred in a culture medium, with 5 explants in each plate; the culture was maintained at 25° C. with 16 hours of light and 8 hours of darkness; the repetitive subculture was carried out every two weeks for a total of two times. GUS staining was performed on the bud explants. Shoot elongation: the dead shoots and cotyledons were removed from the explants; the explants were transferred in a culture medium, with 5 explants in each plate; the culture was maintained at 25° C. with 16 hours of light and 8 hours of darkness; and repetitive subculture was carried out every three weeks for a total of 2-4 times. GUS staining was performed on the explants. Root induction: when the seedlings grown to a length of 3 cm, the roots were collected and grown on a culture medium; the culture was maintained at 25° C. with 16 hour of light and 8 hour of darkness.

FIGS. 4A-4E showed the relative expression of the GUS protein under control of the promoters pRPS28 and pEIF1; where (a) cultivation; (b) shoot induction after 2 weeks; (c) shoot induction after 4 weeks; and (d) shoot elongation; in FIGS. 4A-4E, the results showed that the promoter pRPS28 and pEIF1 promoted the expression of the GUS protein during the transformation process. Seedling refining and transplanting: when the roots regenerated and at least two compound leaves emerged, the cultured medium was removed from the roots; the regenerated plant was planted in a flowerpot comprising sterilized vermiculite and grown in the incubator (at 25° C. with a relative humidity of 85% and a light intensity 90 µM/m2/s, as well as 16 hours of light and 8 hours of darkness) for 5-7 days. The seedlings became robust, were transplanted into a large flowerpot (comprising nutrient soil and vermiculite mixed in a ratio of 1: 1), and moved to a growth room (at 28±2° C. withd a relative humidity of 40%-60%, a light intensity 90 µM/m2/s, as well as 13.5 hours of light and 10.5 hours of darkness) until it reached maturity.

Identification of the regenerated plant by use of the T1 soybean plants: the genomic DNA was extracted from the leaves of the T1 transgenic soybean plant and used as a template for amplification of the BAR gene in the T1 soybean plants; and the specific band was amplified from only the transgenic soybean plants.

PCR amplification was performed as follows: a 10 µL PCR reaction contained 0.5-1 µL of template DNA, 5 µL of 2×Taq Mix, 0.5 µL of primers (i.e. 0.25 µL of BAR-F and 0.25 µL of BAR-R), and ddH2O (deionized water) added to reach a total volume of 10 µL.

The PCR cycling and running parameters were described as follows: denaturation at 94° C. for 2 min; 30 cycles of 94° C. for 10 s, 58° C. for 30 s, and 72° C. for 30 s; and the final extension at 72° C. for 5 min.

The gene BAR was amplified using the following primers:

BAR-F: ATGAGCCCAGAACGACGCCCGGCC (SEQ ID NO: 22)

BAR-R: TTAGATCTCGGTGACGGGCAGGAC (SEQ ID NO: 23)

The BAR gene comprised a nucleic acid sequence comprising SEQ ID NO: 7.

3. GUS Histochemical Assay

Tissues of the homozygous transgenic plants were collected at 5 and 15 days after cultivation and subjected to GUS histochemical assay. Specifically, the plant tissues were fixed with acetone for 30-60 min, washed with a GUS-staining buffer, infiltrated with a GUS-staining solution (comprising GUS buffer and 1 mg/L X-GLuc), placed under vacuum for 30-60 min in darkness, and incubated for 4 h at 37° C. in darkness, dehydrated with ethanol, and examined under a dissecting microscope.

FIGS. 5A-5G showed the relative expression of GUS protein under control of the promoter pRPS28 in different tissues of a T2 transgenic soybean plant. FIG. 5A showed the soybean germinated for 5 days; FIG. 5B showed the embryo; FIG. 5C showed the soybean grown for 15 days; FIG. 5D showed the true leaf, compound leaf, bud, petiole, and internode of the soybean grown for 15 days; FIG. 5E showed the root and root nodule; FIG. 5F showed the immature embryo; and FIG. 5G showed the pod.

FIGS. 6A-6G showed the relative expression of GUS protein under control of the promoter pEIF1 in different tissues of a T2 transgenic soybean plant; FIG. 6A showed the soybean germinated for 5 days; FIG. 6B showed the embryo; FIG. 6C showed the soybean grown for 15 days; FIG. 6D showed the true leaf, compound leaf, bud, petiole, and internode of the plant grown for 15 days; FIG. 6E showed the root and root nodule; FIG. 6F showed the immature embryo; and FIG. 6G showed the pod.

5 days after germination, the GUS protein under the control of the promoters pRPS28, pRPS28-I, pEIF1 and pEIF1-I were expressed in the cotyledon, radicle, embryo of the transgenic soybean plant; 15 days after germination, the GUS protein under the control of the promoters pRPS28, pRPS28-I, pEIF1 and pEIF1-I were expressed in the true leaf, compound leaf, shoot, petiole, internode, root and root nodule, and even expressed in the immature embryo, pod and seed in the transgenic soybean plant; and the results showed that the promoters pRPS28 and pEIF1 are ubiquitous promoters.

4. Comparison of GUS Activities Controlled by Promoters pRPS28, pEIF1 and pGmUbi in Soybean.

The T3 homozygous transgenic plants comprising the recombinant vectors pRPS28-GUS-BAR, pRPS28-I-GUS-BAR, pEIF1-GUS-BAR, pEIF1-I-GUS-BAR and pGmUbi-GUS-BAR were cultivated for 15 days; the tissues such as root, trifoliate leaf, true leaf, and cotyledon were collected and stored in liquid nitrogen. GUS activity was quantified using 4-Methylumbelliferone (4-MU) as a fluorometric standard and 4-MUG as a fluorometric substrate; the fluorescent compound was detected using a fluorimeter with an excitation wavelength of 455 nm; a standard curve of 4-MU fluorescence was generated and used to quantify the GUS activity (pmol MU/min/mg)

FIG. 7 showed the GUS activity controlled by the promoters pRPS28, pRPS28-I, pEIF1, pEIF1-I and pGmUbi in the soybean tissues such as root, compound leaf, true leaf, cotyledon. From the results, the promoters pRPS28, pRPS28-I, pEIF1 and pEIF1-I promoted the ubiquitous expression of the GUS protein across the soybean tissues; the promoter pRPS28 promoted higher levels of GUS expression than the promoter pRPS28-I; and the promoter pEIF1-I promoted higher levels of GUS expression than the promoter pEIF1. The GUS activity in the cotyledon of a soybean line #20 that was controlled by the promoter pRPS28 was 25.93 pmolMU/min/µg; the GUS activity in the cotyledon of a soybean line #35 that was controlled by the promoter pRPS28 was 38 pmolMU/min/µg; the GUS activity in the cotyledon of a soybean line #13 that was controlled by the promoter pEIF1-I was 42.77 pmolMU/min/µg; the GUS activity in the cotyledon of a soybean line #77 that was controlled by the promoter pEIF1 was 26.92 pmolMU/min/µg. From the results, the promoters pRPS28-I, pEIF1 and pEIF1-I promoted high levels of expression of a foreign protein in specific tissues of soybean and hence were suitable for use in genetic transformation.

Example 4

Transformation of vectors comprising GUS gene into Arabidopsis thaliana and tobacco.

1. Construction of Vectors pRPS28-GUS, pRPS28-I-GUS, pEIF1-GUS and pEIF1-I-GUS in Arabidopsis Thaliana and Tobacco.

In Example 2, the DNA fragments pRPS28, pRPS28-I, pEIF1 and pEIF1-I were inserted into the vector pCAMBIA1391Z-GUS-HYG (with hygromycin resistance gene) by the seamless cloning method, and the vector pRPS28-GUS-HYG,

pRPS28-I-GUS-HYG, pEIF1-GUS-HYG and pEIF1-I-GUS-HYG were constructed and transformed into Arabidopsis thaliana and tobacco. PCR amplification was performed as follows:

A 50 µL PCR reaction mixture contained 1 µL of template DNA, 5 µL of 2×Taq Mix, 0.5 µL of primers (i.e. 0.25 µL of BAR-F and 0.25 µL of BAR-R), and ddH2O (deionized water) added to reach a total volume of 50 µL.

The PCR cycling and running parameters were described as follows: denaturation at 94° C. for 2 min; 30 cycles of 94° C. for 10 s, 58° C. for 30 s, 72° C. for 30 s; and the final extension at 72° C. for 5 min.

The recombinant vector pRPS28-GUS-HYG was amplified using the following primers:

pRPS28-GUS-HYG-F:

GACCATGATTACGCCAAGCTTCACCACCCAATCCATAACCACCAC(SEQ
ID NO: 14)

pRPS28-GUS-HYG-R:

CCAGTGAATTCCCGGGGATCCCTGATGCAAAACACGAACAAAGAAAG(SE
Q ID NO: 13)

The recombinant vector pRPS28-I-GUS-HYG was amplified using the following primers:

pRPS28-I-GUS-HYG-F:

GACCATGATTACGCCAAGCTTCACCACCCAATCCATAACCACCAC (SEQ
ID NO:14)

pRPS28-I-GUS-HYG-R:

CCAGTGAATTCCCGGGGATCC CCTGCTCAAACACAATCAACAG (SEQ
ID NO: 15)

The recombinant vector pEIF1-GUS-HYG was amplified using the following primers:

pEIF1-GUS-HYG-F:

GACCATGATTACGCCAAGCTT GGAGAGAAGTTGAACTCTGAGTTGTG
(SEQ ID NO: 16)

pEIF1-GUS-HYG-R:

CCAGTGAATTCCCGGGGATCC CTGATCGTAAATTTAAGGTTTCG (SEQ
ID NO: 24)

The recombinant vector pEIF1-I-GUS-HYG was amplified using the following primers:

pEIF1-I-GUS-HYG-F:

GACCATGATTACGCCAAGCTT GGAGAGAAGTTGAACTCTGAGTTGTG
(SEQ ID NO: 16)

pEIF-I-GUS-HYG-R:

CCAGTGAATTCCCGGGGATCC AAAACTTGACTCACTAAGACCAAAGG
(SEQ ID NO: 17)

2. Analysis of GUS Expression Driven by Promoters pRPS28 and pEIF1 in Arabidopsis Thaliana.

The Agrobacterium strain GV3101 comprising pRPS28-GUS-HYG, pRPS28-I-GUS-HYG, pEIF1-GUS-HYG, pEIF1-I-GUS-HYG recombinant vectors were resuspended in a buffer, transformed into Arabidopsis thaliana Col-0 by inflorescence dip method, and screened to obtain the transgenic Arabidopsis thaliana plants. The GUS protein was expressed in the same level in the transgenic lines #5, #25 and #38 comprising the recombinant vector pRPS28-GUS-HYG; the GUS protein was expressed in the same level in the transgenic lines #2, #5 and #13 transfected with the recombinant vector pRPS28-I-GUS-HYG; the GUS protein was expressed in the same level in the transgenic lines #3, #7 and #12 transfected with the recombinant vector pEIF1-GUS-HYG; the GUS protein was expressed in the same level in the transgenic lines #10, #11 and #12 transfected with the recombinant vector pEIF1-I-GUS-HYG; the seedlings of the T3 transgenic lines were planted in a coculture medium comprising hygromycin and then vernalized in a 4° C. refrigerator for 2 days.

3) the seedlings were transferred in the incubator (at 22±2° C. with 16 hour of light and 8 hour of darkness) for 7 days; and GUS staining was performed on five positive seedlings of T3 generation of each line. The five positive seedlings of T3 generation of each line were planted in the same pod and grow at 22±2° C. with 16 hour of light and 8 hour of darkness. The flower and pod of Arabidopsis thaliana were stained for GUS activity.

FIGS. 8A-8D showed the relative expression of GUS protein under control of the promoters pRPS28, pRPS28-I and pGmUbi in different tissues of the T3 transgenic soybean plants. FIG. 8A showed the fluorescence results of GUS protein expression driven by the promoter pRPS28 in the whole plant, flower and pod of Arabidopsis thaliana; and FIG. 8B showed the fluorescence results of GUS protein expression driven by the promoter pRPS28-1 in the whole plant, flower and pod of Arabidopsis thaliana. The results showed that the promoter pRPS28 promoted the heterologous expression of the target gene (GUS gene) in Arabidopsis thaliana.

FIGS. 9A-9B showed the relative expression of GUS protein under control of the promoters pEIF1, pEIF1-I and pGmUbi in different tissues of the T3 transgenic soybean plants; where FIG. 9A showed the fluorescence results of GUS protein expression driven by the promoter pEIF1 in the whole plant, flower and pod of Arabidopsis thaliana; and FIG. 9B showed the fluorescence results of GUS protein expression driven by the promoter pEIF1-I in the whole plant, flower and pod of Arabidopsis thaliana. The results showed that the promoter pEIF1 promoted the heterologous expression of the target gene (GUS gene) in Arabidopsis thaliana.

3. Analysis of GUS Expression Driven by Promoters pRPS28 and pEIF1 In Tobacco Leaves.

The Agrobacterium strain GV3101 comprising the recombinant vectors pRPS28-GUS-HYG, pRPS28-I-GUS-HYG, pEIF1-GUS-HYG and pEIF1-I-GUS-HYG were resuspended in an infiltration solution; the infiltration solution was injected via a syringe into the leaves of N. benthamiana plants; the leaves were subsequently co-cultivated for 2 days and immersed in a GUS staining solution.

FIG. 10 showed the transient expression of GUS protein under the control of the promoters pRPS28 and pEIF1 in transgenic tobacco leaves; in the mock, no GUS activity was detected in the tobacco leaves transfected with an empty vector pCAMBIA1391Z; and GUS expression was observed in the leaves transfected with the vector comprising the promoters pRPS28 and pEIF1. From the results, the promoters pRPS28 and pEIF1 promoted the transient and heterologous expression of GUS (target) protein in the tobacco leaves.

Claims

1. A method for promoting expression of a foreign gene in a plant, the method comprising: constructing a recombinant vector comprising a full-length soybean gene promoter pRPS28 or an intron-comprising soybean gene promoter pRPS28-I, and introducing the recombinant vector into the plant; the full-length promoter pRPS28 being represented by SEQ ID NO: 2, and the intron-comprising promoter pRPS28-I being represented by SEQ ID NO: 3.

2. The method of claim 1, wherein the foreign gene is beta-glucuronidase (GUS) gene.

3. The method of claim 1, wherein when introduced into soybean, the promotor promotes the foreign gene to express in cotyledons, radicles, plumules, true leaves, compound leaves, buds, petioles, internodes, roots and root nodules of the soybean.

4. The method of claim 1, wherein when introduced into Arabidopsis, the promotor promotes the foreign gene to express in whole plant, flower and pod of the Arabidopsis.

5. The method of claim 1, wherein when introduced into tobaccos, the promotor promotes the foreign gene to express in leaves of the tobaccos.

6. A recombinant vector comprising a soybean gene promoter, the recombinant vector being prepared by inserting a soybean gene promoter pRPS28 or pRPS28-I into a vector pCAMBIA1391Z-BAR, the method comprising using a PCR amplification fragment of pRPS28 or pRPS28-I as a template, and cloning the PCR amplification fragment into a soybean stable transformation vector pCAMBIA1391Z-BAR by a seamless cloning method to obtain soybean stable transformation vectors pRPS28-GUS-BAR, pRPS28-I-GUS-BAR; wherein: the vector pCAMBIA1391Z-BAR is obtained by linearizing a vector pCAMBIA1391Z with a restriction enzyme XhoI, replacing a gene HygR with a gene BAR by the seamless cloning method;the recombinant vector pRPS28-GUS-BAR is amplified using the following primers: pRPS28-GUS-BAR-F: GACCATGATTACGCCAAGCTTCACCACCCAATCCATAACCACCAC(SEQ ID NO: 14);pRPS28-GUS-BAR-R: CCAGTGAATTCCCGGGGATCCCTGATGCAAAACACGAACAAAGAAAG (SEQ ID NO: 13);the recombinant vector pRPS28-I-GUS-BAR is amplified using the following primers: pRPS28-I-GUS-BAR-F: GACCATGATTACGCCAAGCTTCACCACCCAATCCATAACCACCAC (SEQ ID NO: 14);pRPS28-I-GUS-BAR-R: CCAGTGAATTCCCGGGGATCC CCTGCTCAAACACAATCAACAG(SEQ ID NO: 15);the promotor pRPS28 has a nucleotide sequence of SEQ ID NO: 2, and the promoter pRPS28-I has a nucleotide sequence of SEQ ID NO: 3.