CN102219843B

CN102219843B - Soybean Dof 17 transcription factor GmDof 17-1 protein as well as coding gene and application thereof

Info

Publication number: CN102219843B
Application number: CN2011101595754A
Authority: CN
Inventors: 喻德跃; 于静静
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2011-06-14
Filing date: 2011-06-14
Publication date: 2012-11-28
Anticipated expiration: 2031-06-14
Also published as: CN102219843A

Abstract

The invention belongs to the field of plant gene engineering and discloses a soybean Dof 17 transcription factor GmDof 17-1 protein as well as a coding gene and an application thereof. The soybean Dof 17 transcription factor GmDof 17-1 protein has an amino acid sequence shown by SEQ ID NO.2; and the coding gene GmDof 17-1 has a sequence shown by SEQ ID NO.1. By means of a plant expression vector, a transgenic cell line with high sulphur-containing amino acid (methionine and cysteine) content and a transgenic plant with high sulphur-containing amino acid content can be obtained by introducing the GmDof 17-1 gene in the invention into a plant cell. The GmDof 17-1 disclosed by the invention has important significance in culturing plant varieties with high sulphur-containing amino acid contents, in particular soybeans with high sulphur-containing amino acid contents, and improving the quality of crops, in particular the quality of soybeans.

Description

A kind of soybean Dof17 class transcription factor GmDof17-1 albumen and encoding sox and application

Technical field

The invention belongs to plant genetic engineering field, relate to a kind of soybean Dof17 class transcription factor GmDof17-1 albumen and encoding sox and application.

Background technology

Soybean [Glycine max (L.) Merr.] is main in the world grain and oil crops, contains the protein of 30-55%, is 4-5 times of cereal, is main source human and animal picked-up plant protein.Soya protein amino acid is formed close with dairy protein, is vegetal complete protein, on nutritive value, can be equal to animal proteinum.But its restricted sulfur-containing amino acid-halfcystine and methionine(Met) content are lower slightly, have influenced the value of soy-protein nutrition.The main target of soy-protein improvement is when improving protein contnt, increases the content of sulfur-containing amino acid, is worth and economic benefit to improve soybean nutritional.

The whole world faces the serious problems of protein resource shortage now; When therefore improving the soybean varieties protein contnt as early as possible; Actively improve proteinic quality, improve its nutritive value, have actively and great realistic significance improving human life quality and herding industry.

Summary of the invention

The present invention provides a kind of soybean Dof17 class transcription factor GmDof17-1 albumen for solving the relatively low technical problem of content of sulfur-containing amino acid in the soybean.

Another object of the present invention provides the proteic encoding sox GmDof17-1 of this soybean Dof17 class transcription factor GmDof17-1.

Another purpose of the present invention provides the application of this albumen and gene.

The object of the invention can be realized through following technical scheme:

A kind of soybean Dof17 class transcription factor GmDof17-1 albumen has the aminoacid sequence shown in the SEQ ID NO.2.

The proteic gene GmDof17-1 of coding claim 1 described soybean Dof17 class transcription factor GmDof17-1.

The gene GmDof17-1 of described soybean Dof17 class transcription factor GmDof17-1 preferably has sequence shown in the SEQ ID NO.1.

The expression vector that contains the gene GmDof17-1 of described soybean Dof17 class transcription factor GmDof17-1.

Described expression vector is to utilize the gateway technology to insert the pMDC83-GmDof17-1 plant overexpression carrier that plant expression vector pMDC32 obtains the soybean Dof class transcription factor gene GmDof17-1 ORFs shown in the SEQ ID NO.1.

The engineering bacteria that contains described soybean Dof17 class transcription factor gene GmDof17-1.

Described engineering bacteria preferably changes described soybean Dof class transcription factor gene GmDof17-1 over to agrobacterium tumefaciens EHA105 gained.

The application of described soybean Dof17 class transcription factor GmDof17-1 albumen in the plant of cultivating high sulfur amino acid content, described plant optimization soybean.

Preferred halfcystine of described sulfur-containing amino acid and methionine(Met).

The application of the gene GmDof17-1 of described soybean Dof17 class transcription factor GmDof17-1 in the plant of cultivating high sulfur amino acid content, described plant optimization soybean, preferred halfcystine of described sulfur-containing amino acid and methionine(Met).

Utilize plant expression vector,, can obtain the transgenic cell line and the transfer-gen plant of high sulfur amino acid content GmDof17-1 gene transfered plant cell of the present invention.

When using GmDof17-1 to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or inducible promoter.For the ease of transgenic plant cells or plant being identified and screening; Can process used plant expression vector, as adding the selected marker's (gus gene, luciferase genes etc.) that in plant, to express or the resistant gene of antibiotic marker thing (qingfengmeisu qiong affinity tag, kantlex affinity tag etc.).From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Carry GmDof17-1 of the present invention plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated through using, and the plant transformed tissue cultivating is become plant.By the plant transformed host both can be monocotyledonss such as paddy rice, wheat, corn, also can be dicotyledonss such as soybean, cucumber, tomato, willow, turfgrass, clover.

Beneficial effect

The tobacco that imports overexpression GmDof17-1 gene of the present invention is compared with the non-transgenic tobacco; Its sulfur amino acid content significantly improves, and explains that the soybean Dof17 class transcription factor GmDof17-1 albumen of GmDof17-1 gene and coding thereof plays an important role to the sulfur amino acid content aspect that improves plant.

GmDof17-1 of the present invention is to the plant variety of the cultivating high sulfur amino acid content soybean of high sulfur amino acid content particularly, and particularly the quality of soybean is significant to improve farm crop.

Description of drawings

Below in conjunction with accompanying drawing and embodiment the present invention is further specified.

Fig. 1 is that the GmDof17-1 gene is at soybean chromosome position structural representation.

Fig. 2 is the expression characterization figure of GmDof17-1 in soybean: the expression analysis of GmDof17-1 in the soybean Different Organs.Seed 1-4 wherein represents the seed of blooming back 20 days, 30 days, 40 days, 50 days successively.

Fig. 3 is the part-structure synoptic diagram that contains the plant expression vector of GmDof17-1.

Fig. 4 is the statistical study of transfer-gen plant and wild-type plant sulfur amino acid content.

(*，P≤0.05；**，P≤0.01)

Fig. 5 is the analysis that aminoacids content improves ratio in the transfer-gen plant.

Fig. 6 is the pMDC83 plasmid map.

Embodiment

Below in conjunction with concrete preparation embodiment and application implementation example, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit scope of the present invention by any way.

In following embodiment, various processes and the method do not described in detail are ordinary methods as known in the art.Used primer is all indicated when occurring first, and used thereafter same primers as is all identical with the content of indicating first.

The cDNA clone and the evaluation of embodiment 1, soybean GmDof17-1 and encoding sox thereof

Soybean (Glycine max (L.) Merr.) material south agricultural 94-16 (writing a Chinese character in simplified form NN94-16) (Han Suoyi etc., the structure and the Part Traits analysis of soybean " Nan Nong 94-16 " mutant library.The nuclear agricultural science newspaper, 2008), providing by country of Agricultural University Of Nanjing germ plasm resource research department, modified soybeans center, material is planted in the solarium, conventional field management.

According to soybean Dof17 (ABI16018) gene of having reported; Search soybean gene group DB (http://www.phytozome.net/soybean) finds that the Dof17 transcription factor has two copies in the soybean, lays respectively at No. 7 and No. 16 karyomit(e)s; What the present invention studied is to be positioned at chromosomal Dof17 transcription factor No. 7; Called after GmDof17-1, it has the nucleotide sequence shown in the SEQ ID NO.1, the amino acid residue sequence shown in the coding SEQ ID NO.2.Amino acid sequence analysis shows that this soybean Dof17 class transcription factor GmDof17-1 albumen is made up of 281 amino-acid residues, the Dof structural domain of the high conservative that N-terminal is made up of 1 52 amino acid.

Design primer with the Peimer3 program according to the GmDof17-1 gene order:

Forward primer sequence: ' 5-ATGGAGGGGATGGCTCCCAATTCA-3 ' (SEQ ID NO.3);

Reverse primer sequence: ' 5-CCACGTTCCTTCACCAATCATTCC-3 ' (SEQ ID NO.4).

Use the RT-PCR method, amplification GmDof17-1 gene from the total RNA of soybean, concrete grammar is: get soybean 94-16 blade, place liquid nitrogen to grind, RNA extracts and carries out according to TIANGEN total RNA extraction reagent box RNA Plant Extraction kit DP417.CDNA first chain is synthetic according to the TaKaRa reagent cDNA of the company first chain synthetic agent box TaKaRa PrimeScript ^TM1st strand cDNA Synthesis Kit D6110A, concrete operations see specification sheets for details.With cDNA first chain that obtains is that template is carried out pcr amplification reaction.25 μ l μ l PCR reaction systems are: 1 μ lcDNA, first chain (0.05 μ g), 1 μ l primer (SEQ ID NO.3 and SEQ ID NO.4,10 μ M), 2.5 μ l, 10 * PCR damping fluid, 2.5 μ l Mg ²⁺, 4 μ l dNTP (10mM) and 1.25U LA Taq archaeal dna polymerase, supply 25 μ l with ultrapure water.Be reflected on the BIO-RAD PTC-200 type PCR appearance and carry out, its program is 95 ℃ of sex change 5min; 94 ℃ of 30sec again, 56 ℃ of 50sec, 72 ℃ of 1min30sec, totally 30 circulations; 72 ℃ are extended 10min then; 4 ℃ of preservations.The PCR product reclaims after connect pGEM-T Easy carrier, transformed into escherichia coli DH5 α, the screening of blue hickie, shake bacterium, order-checking, sequential analysis; The result shows that this PCR product has the nucleotide sequence of SEQ ID NO.1 in the sequence table; Called after GmDof17-1 gene; Then SEQ ID NO.1 sequence is put into soybean gene group DB Blast, confirm that the position of SEQ ID NO.1 in soybean karyomit(e) see Fig. 1.

Embodiment 2, the GmDof17-1 expression characteristic in the spending of soybean Different Organs and different development stage

Utilize real-time fluorescence quantitative PCR technology, GmDof17-1 is studied at the expression in the spending of each organ of soybean and different development stage.The soybean experiment material is seeded in the Agricultural University Of Nanjing solarium in the beginning of June, and field management is with conventional.When launching, get on the 3rd compound leaf root, stem, leaf; Full-bloom stage is got flower; Take away respectively and spend back 20 days, 30 days, 40 days, 50 days seed.Each tissue takes off back liquid nitrogen flash freezer rapidly, and ℃ preservation is subsequent use afterwards-80.

The extraction of total RNA is with embodiment 1.

The soybean constitutive expression Gene A ctin (GenBank accession No.V00450) that uses, the amplified fragments size is 583bp, its amplimer sequence is:

Forward primer sequence: 5 '-GTTCTCTCCTTGTATGCAAGTG-3 ' (SEQ ID NO.5);

Reverse primer sequence: 5 '-CCAGACTCATCATATTCACCTTTAG-3 ' (SEQ ID NO.6).

Since be template from the cDNA of soybean different tissues or organ, carry out the real-time fluorescence quantitative PCR analysis.The amplimer of GmDof17-1 is:

Forward primer sequence: 5 ' GGGTTTCCCATGCAAGAAGTT-3 ' (SEQ IDNO.7);

The reverse primer sequence:: 5 ' ACCACCCCTCTCTTGAATCTGA-3 ' (SEQ ID NO.8).

Result (Fig. 2) analysis revealed, GmDof17-1 has expression in each tissue, and in root with seed in expression amount higher.

The Function Identification of embodiment 3, GmDof17-1 gene coded protein

Utilize Invitrogen company

Technology with Clonase ^TMThe II test kit is inserted into expression vector pMDC83 (Sokolov et al, 2005, PNAS, 103 with GmDof17-1 gene forward; 9732-9737; Plasmid map is seen Fig. 6) in, obtain pMDC83-GmDof17-1 plant overexpression carrier (Fig. 3), change pMDC83-GmDof17-1 over to agrobacterium tumefaciens bacterial strain EHA105 (Avsian-Kretchmer et al with freeze-thaw method; The Salt-Stress Signal Transduction Pathway That Activates the gpxl Promoter Is Mediated by Intracellular H2O2; Different from the Pathway Induced by Extracellular H2O2,2004, Plant Physiology; 135:1685-1696); Containing on the MS substratum of 50mg/L Totomycin, to cultivate for 28 ℃, preliminary screening obtains having the transfer-gen plant of hygromycin resistance.Extract the genomic dna that preliminary screening obtains having the transgene tobacco of hygromycin resistance, use gene-specific primer PCR detected result to show and obtain the positive GmDof17-1 of commentaries on classics gene plant.

Adopt A200amino acid Nova analyzer amino acidanalyser mensuration commentaries on classics GmDof17-1 gene plant and wild-type plant T1 free amino group acid content for seed; Detection and judgment basis standard are JY/Y 019-1996 amino acid analysis method general rule, and wherein the sulfur amino acid content result is as shown in Figure 4.Change the GmDof17-1 gene plant and compare all extremely significantly increases of sulfur-containing amino acid total content with wild-type tobacco, improved 28%, wherein halfcystine has improved 9.4%, and methionine(Met) has improved 30% (see figure 5).Explain that overexpression GmDof17-1 gene can improve the sulfur amino acid content of transgene tobacco.

Claims

1. the application of the soybean Dof17 class transcription factor GmDof17-1 albumen of aminoacid sequence shown in SEQ ID NO.2 in the plant of cultivating high sulfur amino acid content.

2. application according to claim 1 is characterized in that described plant is a soybean.

3. application according to claim 1 is characterized in that described sulfur-containing amino acid is halfcystine and methionine(Met).

4. the soybean Dof17 class transcription factor GmDof17-1 proteic gene of encoding amino acid sequence shown in SEQ ID NO.2 GmDof17-1Application in the plant of cultivating high sulfur amino acid content.

5. application according to claim 4 is characterized in that the soybean Dof17 class transcription factor GmDof17-1 proteic gene of described encoding amino acid sequence shown in SEQ ID NO.2 GmDof17-1Nucleotide sequence shown in SEQ ID NO.1.

6. application according to claim 5 is characterized in that described plant is a soybean.

7. application according to claim 5 is characterized in that described sulfur-containing amino acid is halfcystine and methionine(Met).