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Method for in-vitro recombination of rice SCF (D3) E3 ligase and application thereof

A ligase, rice technology, applied in the biological field, can solve the problem of difficult in vitro ubiquitination system

Pending Publication Date: 2022-08-09
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It is very difficult to establish an in vitro ubiquitination system for multi-subunit E3 ligases. So far, the research on the activity of recombinant multi-subunit SCF E3 ligases in plants is still blank, and obtaining active multi-subunit E3 ligase complexes Body composition is one of the bottlenecks in the establishment of the system

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  • Method for in-vitro recombination of rice SCF (D3) E3 ligase and application thereof
  • Method for in-vitro recombination of rice SCF (D3) E3 ligase and application thereof
  • Method for in-vitro recombination of rice SCF (D3) E3 ligase and application thereof

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Embodiment 1

[0083] Example 1. Rice multi-subunit SCF D3 Establishment and application of E3 ligase activity recombination system in vitro

[0084] 1. In vitro recombinant SCF D3 Conditions necessary for E3 ligase activity

[0085] 1. Identification of rice SCF D3 Components of the complex

[0086] To identify rice SCF D3 For the components of the complex, 10 g each of rice-derived Act:D3-GFP / d3 and Act:GFP transgenic callus were collected, quick-frozen and ground in liquid nitrogen.

[0087] Among them, the accession number of the gene encoding D3 protein is LOC_Os06g06050 (Table 1). The transgenic construction of Act:D3-GFP / d3 and Act:GFP is from the literature published by our laboratory "Jiang, L., Liu, X., Xiong, G., Liu, H., Chen, F., Wang, L. , Meng, X., Liu, G., Yu, H., Yuan, Y., et al. (2013). DWARF 53 acts as arepressor of strigolactone signalling in rice. Nature 504:401-405.”. The potential interacting proteins of D3 protein were analyzed and identified by LC-MS / MS mass...

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Abstract

The invention discloses a method for in-vitro recombination of rice SCF (D3) E3 ligase and application of the rice SCF (D3) E3 ligase. According to the invention, an SCFD3E3 ligase complex obtained from a transgenic callus tissue is used for screening and identifying an E2 protein: OsUBC14 which works together with the SCFD3E3 ligase in an efficient and specific manner; according to the present invention, an insect baculovirus expression system is utilized to obtain the active D3 protein and the active Cullin1 protein; the two aspects of the invention are to overcome the biggest difficulty in the establishment of a plant multi-subunit SCF E3 ligase in-vitro active recombination system. The establishment of an in-vitro recombinant rice SCFD3E3 ligase active system provides an efficient and reliable method platform for deeply researching strigolactones signal sensing, transmission and the like. The invention has important effect and wide potential application prospect for illuminating the molecular mechanism of the plant hormone signal pathway and the molecular mechanism of the multi-subunit E3 ligase.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an in vitro recombinant rice SCF (D3) E3 (also known as SCF D3 E3) The method of ligase and its application. Background technique [0002] Protein ubiquitination modification requires the sequential cooperation of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin ligase (E3). In plants, the multi-subunit SCF E3 ligase is one of the largest E3 ligase subfamily, consisting of SKP1 protein, Cullin1 protein, RBX1 protein and a switchable F-box protein. Multi-subunit SCF E3 ligase is involved in the regulation of important plant growth and development processes, and plays a key role in plant hormone signaling pathways such as jasmonic acid, gibberellin, ethylene, and strigolactone. Therefore, the establishment of a multi-subunit SCF E3 ligase in vitro activity recombination system is crucial for deciphering the molecular mechanism of key regu...

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Application Information

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IPC IPC(8): C12N9/00G01N33/68C07K1/107
CPCC12N9/93G01N33/6845C07K1/107
Inventor 李家洋王冰刘会会孟祥兵余泓刘贵富陈明江荆彦辉
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI