CN117070529A - Sweet potato transcription factor IbDREB1d and application thereof - Google Patents
Sweet potato transcription factor IbDREB1d and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology, and in particular relates to a sweet potato transcription factorIbDREB1dAnd applications thereof. The sweet potato transcription factorIbDREB1dThe nucleotide sequence is shown as SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 3. It was found that overexpression compared to wild sweet potato plantsIbDREB1dThe accumulation of flavonols in the transgenic sweet potato plants increased, indicatingIbDREB1dForward regulation of sweet potato flavonol synthesis for sweet potatoThe variety improvement of the potato has important significance.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a sweet potato transcription factor IbDREB1d and application thereof.
Background
Sweet potato is an important grain, feed, industrial raw material and energy crop in China, and leaf vegetable type sweet potato is a sweet potato variety which uses fresh and tender stems and leaves as vegetables, and compared with common sweet potato, the yield of the stems and leaves is higher. The sweet potato is a asexual propagation crop, the cross incompatibility between seeds and in-seeds severely limits the resource utilization and parent free combination in the sweet potato breeding, and the breeding practice shows that the conventional cross breeding method is difficult to quickly breed a new sweet potato variety with high quality and high yield. The genetic engineering technology is utilized to cultivate new varieties of sweet potatoes, so that the degradation of sweet potato germplasm can be reduced, the yield and quality of sweet potatoes can be improved, and better environmental benefit and social benefit can be realized.
The flavonol is a unique flavonoid compound with antiallergic, anti-inflammatory and anticancer properties, and mainly comprises substances such as quercetin, kaempferol, myricetin, isoquercitrin, rutin and the like, and the flavonol synthase is the first key enzyme for flavonol synthesis. The flavonol synthase gene (IbFLS) is the first key enzyme gene for flavonol synthesis, no gene for regulating and controlling IbFLS is reported at present, the transcription factor IbDREB1d for regulating and controlling the IbFLS is screened by a yeast single hybridization technology, and the forward regulation and control of the flavonol synthesis of the sweet potato by the IbDREB1d is verified by transgenic sweet potato.
Disclosure of Invention
The invention aims to provide a sweet potato transcription factor IbDREB1d and application thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the nucleotide sequence of the sweet potato transcription factor IbDREB1d is shown as SEQ ID NO. 2.
The amino acid sequence of the protein coded by the sweet potato transcription factor IbDREB1d is shown as SEQ ID NO. 3.
A vector containing the sweet potato transcription factor IbDREB1d.
An engineering bacterium containing sweet potato transcription factor IbDREB1d.
The primer pair for amplifying the sweet potato transcription factor IbDREB1d has the sequence as follows: 5'-ATGGATTACTCGACTTCG-3' and 5'-CTAGGTTCGCTCCTCACAAA-3'.
The application of the sweet potato transcription factor IbDREB1d in promoting the synthesis and accumulation of sweet potato flavonols.
The sweet potato transcription factor IbDREB1d, the sweet potato IbFLS gene promoter or the interaction of the sweet potato transcription factor IbDREB1d and the sweet potato IbFLS gene promoter is applied to cultivation of transgenic sweet potato with altered flavonol compound content, wherein the nucleotide sequence of the sweet potato transcription factor IbDREB1d is shown as SEQ ID NO.2, and the nucleotide sequence of the sweet potato IbFLS gene promoter is shown as SEQ ID NO. 1.
The invention has the remarkable advantages that:
compared with wild sweet potato plants, the research of the invention discovers that the accumulation amount of flavonols compounds of transgenic sweet potato plants which overexpress IbDREB1d is increased, which indicates that IbDREB1d positively regulates the synthesis of the flavonols compounds of sweet potato, and has important significance for variety improvement of sweet potato.
Drawings
Fig. 1: agarose gel electrophoresis of sweetpotato IbFLS gene promoter.
Fig. 2: self-activation detection of recombinant bait vector IbFLSpro-pAbAi.
Fig. 3: interaction verification of IbFLS gene promoter and IbDREB1d.
Fig. 4: plasmid map of the overexpression vector pEGOEP-35S-DREB1D (namely pEGOEP35S-G418-DREB 1D).
Fig. 5: the IbDREB1d over-expression transgenic sweetpotato is obtained through rooting transformation.
Fig. 6: and (3) carrying out PCR verification on the IbDREB1d over-expressed transgenic sweet potato.
Detailed Description
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
Example 1
Extracting total RNA of leaf tissue of 'Fucai potato No. 18', reversely transcribing into cDNA as a template, and amplifying by a PCR method with 5'-GGCAGCTCTTCCAATAGCGGCG-3' and 5'-TTTTTAGCAAGATAATGGAG-3' as primers to obtain the IbFLS gene promoter with the base sequence of 2746bp and the nucleotide sequence shown as SEQ ID No. 1.
Example 2
The cloned gene promoter sequence of the 'Fu potato No. 18' IbFLS is constructed in a bait vector pAbAi (Clontech) by a homologous recombination method, and the recombinant bait vector is named IbFLSpro-pAbAi. According toThe recombinant bait vector IbFLSpro-pAbAi was integrated into the Yeast Y1H Gold genome, resulting in a bait strain, coated with the following media, as described in Gold Yeast One-Hybrid Library Screening System (Clontech): SD-Ura/AbA (0 ng/mL), SD-Ura/AbA (100 ng/mL), SD-Ura/AbA (150 ng/mL), SD-Ura/AbA (200 ng/mL), SD-Ura/AbA (250 ng/mL), and culturing at 28℃for 3d. The results show that the recombinant bait vector IbFLSpro-pAbAi is not self-activated in the presence of 200ng/mLAbA, and that the vector can be used for the next single-hybrid yeast screening library experiment.
Extracting total RNA of leaf tissue of' Fucai potato No. 18The Gold Yeast One-Hybrid Library Screening System (Clontech) constructed a cDNA library and loaded into the prey vector pGADT7-Rec to obtain a sweetpotato cDNA Yeast library plasmid. According to->Yeast single hetero screening library experiments were performed by the method described in Gold Yeast One-Hybrid Library Screening System (Clontech), the sweet potato cDNA library plasmid and recombinant bait vector IbFLSpro-pAbAi were co-transformed into Yeast strain Y1 HGold, and the transformed resuspended bacteria were plated on SD-Leu solid plates containing AbA at the corresponding concentration and cultured for 3d at 28 ℃. Candidate transcription factor IbDREB1d was obtained by PCR detection, sequencing analysis and sweetpotato genome database alignment annotation. The nucleotide sequence of the transcription factor IbDREB1d is shown as SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 3.
To further verify the interaction of the IbFLS gene promoter with IbDREB1d, the following media were applied after dilution by constructing the transcription factor IbDREB1d in the prey vector pGADT7-AD (Clontech), the IbFLS promoter in the decoy vector pAbAi, and then co-transforming the yeast strain Y1 HGold: SD-Leu/AbA (0 ng/mL), SD-Leu/AbA (400 ng/mL), and incubation at 28℃for 3d. The verification procedure used pGADT7-AD empty vector as control. The results show that IbDREB1d can bind to IbFLS promoter to develop AbA resistance, indicating that IbDREB1d can interact with IbFLS promoter.
Example 3
Extracting total RNA of leaf tissue of 'Fucai potato No. 18', reversely transcribing the total RNA into cDNA as a template, and amplifying the cDNA by a PCR method by using 5'-ATGGATTACTCGACTTCG-3' and 5'-CTAGGTTCGCTCCTCACAAA-3' as primers to obtain a transcription factor IbDREB1d. The transcription factor IbDREB1d sequence was ligated with the super-expression vector pEGOEP-35S (available from Ai Dijing Biotechnology Co., ltd.) to obtain the super-expression vector pEGOEP-35S-DREB1d. The transgenic sweet potato is obtained by a rooting transformation method, and the specific steps are as follows:
1) Single colony of Agrobacterium rhizogenes K599 was picked up and cultured in 3ml LB liquid medium containing antibiotics (50 mg/L kanamycin+50 mg/Lstreptomycin) at 28℃with shaking for 1 d%OD 600 >0.4)。
2) 0.5ml of the bacterial liquid is taken, 30ml of LB liquid medium containing antibiotics (50 mg/L kanamycin+50mg/L streptomycin) and 0.5% glucose is added, and the culture is carried out at 28 ℃ for 16-18h in a shaking way.
3) The bacterial liquid was centrifuged at 5000Xg for 10min (4 ℃ C.), the supernatant was discarded, 30ml of a medium (1/2MS+0.5% glucose+0.1 mM AS) was added to the pellet, and the pellet was subjected to shaking culture at 28℃for 6 hours to activate the infectivity of the bacteria (OD was adjusted) 600 ＝0.2)。
4) The stem node of 'Fucai potato No. 18' is put into activated bacterial liquid, and is infected by shaking at 125rpm for 10-20min, then is washed with sterile water for multiple times, and then is sucked dry by filter paper.
5) The pedicles (cut next to medium) were placed in medium (1/2MS+3% sucrose+0.7% agar+0.01 mM AS) and incubated for 3d in the dark.
6) The stem nodes were transferred to medium (1/2MS+125ppm Timetin+125mg/L cefotaxin+50 ppm kana+3% sucrose+0.7% agar) and rooting was performed.
7) After rooting, the root system is transferred to a 1/2MS (medium containing 200ppm Titin, 200mg/L cefotaxine) +3% sucrose+0.7% agar to induce sprouting, and plantlets are formed.
8) Transgene validation using PCR, PCR reaction procedure: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 55℃for 45s, extension at 72℃for 1min,35 cycles, and extension at 72℃for 10min. Obtaining IbDREB1d over-expression transgene positive plants.
And (3) taking transgenic plants and wild plants ('Fucai potato No. 18') of 30d years old, and measuring the flavonol content of stems and leaves.
TABLE 1 comparison of IbDREB1d over-expressed transgenic sweetpotato and wild type control flavonol content
| Sequential order | Substance (B) | IbDREB1d overexpressing transgenic sweetpotato (mg/kg) | Wild sweet potato (mg/kg) |
| 1 | Rutin | 12.21 | 2.01 |
| 2 | Isoquercitrin | 83.46 | 30.97 |
| 3 | Quercetin | 3.15 | 0.49 |
| 4 | Kaempferol | 0.31 | 0.05 |
| Total flavonol content | 99.13 | 33.52 |
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (7)
1. Sweet potato transcription factorIbDREB1dWhich is provided withIs characterized in that: the nucleotide sequence is shown as SEQ ID NO. 2.
2. The sweet potato transcription factor of claim 1IbDREB1dA protein encoded therein, characterized in that: the amino acid sequence is shown as SEQ ID NO. 3.
3. A transcription factor comprising sweet potato as claimed in claim 1IbDREB1dIs a carrier of (a).
4. A transcription factor comprising sweet potato as claimed in claim 1IbDREB1dIs an engineering bacterium.
5. A method for amplifying the sweet potato transcription factor of claim 1IbDREB1dIs characterized in that: the sequence is as follows: 5'-ATGGATTACTCGACTTCG-3' and 5'-CTAGGTTCGCTCCTCACAAA-3'.
6. The sweet potato transcription factor of claim 1IbDREB1dThe application of the sweet potato flavonol compound in promoting the synthesis and accumulation of sweet potato flavonol compounds.
7. Sweet potato transcription factorIbDREB1dSweet potatoIbFLSGene promoter, or sweet potato transcription factorIbDREB1dWith sweet potatoIbFLSUse of gene promoter interactions in the cultivation of transgenic sweetpotato with altered flavonol content, characterized in that: sweet potato transcription factorIbDREB1dThe nucleotide sequence is shown as SEQ ID NO.2, sweet potatoIbFLSThe nucleotide sequence of the gene promoter is shown as SEQ ID NO. 1.
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