Upstream regulatory factor IbERF73 and application thereof in regulation of expression of purple sweet potato IbWD40

A regulatory factor, the technology of purple sweet potato, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of unclear synthesis regulation mechanism and signal transmission process, etc.

Active Publication Date: 2022-01-14
广东省科学院南繁种业研究所
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the regulation mechanism and signal transduction process of anthocyanin synthesis in non-photodependent or non-photodependent plant roots, tubers or tubers are not well understood

Method used

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  • Upstream regulatory factor IbERF73 and application thereof in regulation of expression of purple sweet potato IbWD40
  • Upstream regulatory factor IbERF73 and application thereof in regulation of expression of purple sweet potato IbWD40
  • Upstream regulatory factor IbERF73 and application thereof in regulation of expression of purple sweet potato IbWD40

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Construction of purple-heart sweet potato yeast one-hybrid cDNA library

[0034] (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.

[0035] (2) The amplified cDNA should be purified with TaKaRa MiniBEST DNA Fragment Purification Kit, so that dH 2 O dissolution.

[0036] (3) The cDNA digested with the restriction endonuclease SfiI was subjected to column treatment, and then purified by PCI / CI to finally make the ddH 2 O dissolution.

[0037] (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.

[0038] (5) Transfer a small amount of the primary library ligation solution into the competent cell E.coli HST08 by electroporation; after the identification is corr...

Embodiment 2

[0040] Example 2: Construction of pAbAi-PIbWD40 bait vector

[0041] (1) Using purple sweet potato tuber DNA as a template, use TaKaRa high-fidelity enzyme Prime Max DNAPolymerase amplifies the IbWD40 promoter DNA fragment with different restriction sites at both ends, and the sequence of the PCR primer pair for amplifying the IbWD40 promoter DNA fragment is PIbWD40-F: 5'-TCCTAGCCAAGAAGAGTGGAGAGA-3' and PIbWD40 -R: 5'-TCTCATACCACCACACCCTAGTGG-3'. The reaction system (20μL) is as follows:

[0042]

[0043] The PCR reaction conditions are:

[0044]

[0045] (2) Construct the promoter IbWD40 (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 the TaKaRa restriction endonuclease QuickCut TM Hind III and QuickCut TM SmaI performed double enzyme digestion on the pAbAi plasmid, and the synthetic primer sequences were shown in PW1F / PW1R in Table 2. The reaction conditions were 37°C, enzyme d...

Embodiment 3

[0067] Example 3: Detection of self-activation of pAbAi-PIbWD40 bait strain

[0068] The pAbAi-PIbWD40 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:

[0069] (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.

[0070] (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.

[0071] (3) Pipette 10 μL of the resuspended bacteria and spot on SD / -Ura, SD / -Ura / AbA (100ng / mL~1000ng / mL)...

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Abstract

The invention discloses an upstream regulatory factor IbERF73 and application thereof in regulation of expression of purple sweet potato IbWD40. According to the invention, a purple sweet potato strain A5 is used as an experimental material, a promoter sequence of IbWD40 is cloned, and the upstream regulatory factor IbERF73 of an IbWD40 gene is successfully obtained through a yeast one-crossing library screening experiment. A yeast single-crossing rotary experiment and a dual-luciferase report system are used for detection to confirm that interaction exists between an IbWD40 promoter and the upstream regulatory factor IbERF73. A subcellular localization result shows that the IbERF73 is localized in a cell nucleus. A self-activation activity experiment result shows that the IbERF73 has self-activation activity. A 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 tuberous roots of purple sweet potatoes.

Description

technical field [0001] The invention relates to the technical field of plant gene breeding, in particular to an upstream regulatory factor IbERF73 and its application in regulating the expression of IbWD40 in purple sweet potato. Background technique [0002] Sweet potato is an important food, feed and industrial raw material crop. Purple heart sweet potato is named for its purple-rich anthocyanin roots, and it is a special type of sweet potato variety. Anthocyanins have antioxidant and free radical scavenging effects, protective effects on the liver and eyes, and therapeutic effects on diabetes. At present, there is a certain understanding of the molecular mechanism of anthocyanin synthesis in above-ground parts of plants such as flowers, fruits, stems, and leaves, and the influence of environmental signal factors, especially the regulation mechanism and signal transduction process of light on anthocyanin synthesis. clear. However, the regulation mechanism and signal tra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/11C12N15/82C12N15/65A01H5/00A01H6/20C12Q1/6895
CPCC07K14/415C12N15/825C12N15/8212C12Q1/6895C12Q2600/13
Inventor 张南南吴嘉云付丹文高峰
Owner 广东省科学院南繁种业研究所
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