Upstream regulatory factor IbEBF2 and application thereof in regulating IbbHLH2 expression of purple sweet potatoes

A regulatory factor, purple-heart sweet potato technology, applied in application, genetic engineering, plant gene improvement, etc., can solve problems such as differences in anthocyanin regulation modes

Active Publication Date: 2022-02-08
广东省科学院南繁种业研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, members of the bHLH family of transcription factors involved in the regulation of anthocyanins are dif

Method used

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  • Upstream regulatory factor IbEBF2 and application thereof in regulating IbbHLH2 expression of purple sweet potatoes
  • Upstream regulatory factor IbEBF2 and application thereof in regulating IbbHLH2 expression of purple sweet potatoes
  • Upstream regulatory factor IbEBF2 and application thereof in regulating IbbHLH2 expression of purple sweet potatoes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction of purple-heart sweet potato yeast one-hybrid cDNA library

[0036] (1) RNA was extracted from the roots of purple sweet potato (line A5) by Trizol method, and double-stranded cDNA was synthesized by reverse transcription by SMART technology.

[0037] (2) The amplified cDNA should be purified with TaKaRa MiniBEST DNA Fragment Purification Kit, so that dH 2 O dissolution.

[0038] (3) The cDNA digested with the restriction endonuclease SfiI is subjected to column treatment, and then purified by PCI / CI to finally make the ddH 2 O dissolution.

[0039] (4) The pGADT7-SfiI vector (clontech, Cat. No. 630490) was ligated with an appropriate amount of cDNA after passing through the column with a DNAligation Kit. Purify the refined connection solution to obtain the primary cDNA library.

[0040] (5) Transfer a small amount of the primary library ligation solution into the competent cell E.coli HST08 by electroporation; after the identification is cor...

Embodiment 2

[0042] Example 2: Construction of pAbAi-PIbbHLH2 bait vector

[0043] (1) Using purple sweet potato tuber DNA as a template, using TaKaRa high-fidelity enzyme Max DNAPolymerase amplifies the IbbHLH2 promoter DNA fragment with different restriction sites at both ends, and the PCR primer pair for amplifying the IbbHLH2 promoter DNA fragment is PIbbHLH2-F: 5'-TCCTAGCCAAGAAGAGTGGAGAGA-3' and PIbbHLH2 -R: 5'-TCTCATACCACCACACCCTAGTGG-3'. The reaction system (20μL) is as follows:

[0044]

[0045] The PCR reaction conditions are:

[0046]

[0047]

[0048] (2) Construct the promoter IbbHLH2 (sequence shown in SEQ ID NO.3) into the pAbAi vector (Ke Lei Biotechnology Co., Ltd., product number kl-zl-0879), the steps are: use TaKaRa restriction enzyme QuickCut TM Hind III and QuickCut TM Sma Ⅰ double-digested the pAbAi plasmid, and the synthetic primer sequence was shown in Table 2 as Pb2F / Pb2R. The reaction conditions were 37°C, and the enzyme digestion was over 3 hours....

Embodiment 3

[0069] Example 3: Detection of self-activation of pAbAi-PIbbHLH2 bait strain

[0070] The pAbAi-PIbbHLH2 bait plasmid was transformed into Y1H yeast to obtain the bait strain. Use the self-activation test to test the minimum AbA concentration that inhibits the bait strains, and observe their growth on the SD / -Ura solid medium. The method for determining the self-activation detection and minimum AbA concentration of the bait strains is as follows:

[0071] (1) Preparation of AbA mother solution: 1 mg AbA was dissolved in 1 mL of absolute ethanol to prepare 1 mg / mL AbA mother solution, and stored at 4°C in the dark.

[0072] (2) Pick larger monoclonal colonies from the culture dishes of Y1H[pAbAi-prey] and Y1H[p53AbAi], resuspend the bacterial solution with 10 μL of 0.9% NaCl solution, and dilute the resuspended solution to 10 -1 、10 -2 and 10 -3 Concentration gradient.

[0073] (3) Pipette 10 μL of the resuspended bacteria and spot on SD / -Ura, SD / -Ura / AbA (100ng / mL~1000ng / m...

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Abstract

The invention discloses an upstream regulatory factor IbEBF2 and application thereof in regulating IbbHLH2 expression of purple sweet potatoes. According to the invention, a purple sweet potato strain A5 is used as an experimental material, a promoter sequence of IbbHLH2 is cloned, and an upstream regulatory factor IbEBF2 of an IbbHLH2 gene is successfully obtained through a yeast one-hybrid library screening experiment. A yeast one-hybrid rotary experiment and a dual-luciferase report system are used for detection to confirm that the IbbHLH2 promoter interacts with an upstream regulatory factor IbEBF2. A subcellular localization result shows that IbEBF2 is localized in a cell nucleus. A self-activation activity experiment result shows that the IbEBF2 has self-activation activity. The basic theory of molecular regulation and control of plant anthocyanin biosynthesis can be enriched and deepened theoretically, and meanwhile, new thoughts and clues are expected to be provided for cultivation measures for improving the content of pigments in purple sweet potato tuberous roots.

Description

technical field [0001] The invention relates to the technical field of plant gene breeding, in particular to an upstream regulatory factor IbEBF2 and its application in regulating the expression of IbbHLH2 in purple sweet potato. Background technique [0002] Purple Heart Sweet Potato is a new variety of sweet potato with unique genetic traits successfully bred in Japan. Its roots are dark purple because they contain very rich anthocyanins. The plant type of the purple sweet potato plant is creeping, the vines are relatively long, the branch position is upper middle, the leaves are dark green, there are many fibrous roots, the tubers are enlarged and grow slowly, and they are rich in protein and various amino acids. Compared with ordinary sweet potatoes, the content of trace elements such as selenium, iron and phosphorus is about 25-30% higher. The yield per mu is 1000-1500 kg, and it has strong disease resistance and wide adaptability. In addition, the most important trai...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N15/11A01H5/06A01H6/00
CPCC07K14/415C12N15/825
Inventor 张南南付丹文凌秋平高峰
Owner 广东省科学院南繁种业研究所
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