Chaperonin for enhancing RNP signal communication of plant phloem and application

A technology of chaperone protein and phloem, applied in the fields of application, plant peptides, plant products, etc., can solve the problem of low transport efficiency of macromolecular RNP complexes

Active Publication Date: 2020-12-04
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the defects existing in the prior art, the method provided by the invention solves the problem of low transport efficiency of the macromolecular RNP complex in the phloem

Method used

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  • Chaperonin for enhancing RNP signal communication of plant phloem and application
  • Chaperonin for enhancing RNP signal communication of plant phloem and application
  • Chaperonin for enhancing RNP signal communication of plant phloem and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Embodiment 1 chaperonin gene sequence acquisition process

[0112] 1. Extraction of RNA from the phloem of Du pear

[0113] In order to amplify the full-length sequences of the ten genes of PbWoxT1, PbPTB3 and PbCCT1-PbCCT8, the CTAB method was first used to extract the phloem RNA of Du pear. The specific steps are as follows:

[0114] (1) Preheat CTAB at 65°C, add 20 μL β-mercaptoethanol per 1 mL;

[0115] (2) Grind the sample in liquid nitrogen, take 0.5 g of the sample and put it into a 2 mL RNase-free centrifuge tube, add 1 mL of preheated CTAB, vortex for 30 seconds, and bathe in 65°C water for 10 minutes;

[0116] (3) Add 1mL CI (chloroform:isoamyl alcohol=24:1), and vortex;

[0117] (4) Place in a pre-cooled 4°C centrifuge at 13000rpm for 10min;

[0118] (5) Take the supernatant, add an equal volume of CI, and mix gently;

[0119] (6) 13000rpm in a centrifuge at 4°C for 10min;

[0120] (7) Take the supernatant, add 2 times the volume of isopropanol, and keep...

Embodiment 2

[0153] Example 2 Application of chaperonin-assisted protein phloem long-distance transport

[0154] 1. Vector construction ( figure 1 )

[0155] Taking the core protein PbPTB3 on the RNP complex that can transport long-distance phloem as an example to illustrate the specific application of chaperones. Primers were designed, enzyme sites were added to PbPTB3 through one PCR amplification, and constructed on the pCAMBIA1305 vector; each subunit fragment of the chaperone protein CCT was added with different restriction sites through two PCR amplifications, and the forward and reverse sequences were constructed into on the pFGC5941 vector. The primer sequences are as follows (the first pair of primers and the second pair of primers of PbCCT1-PbCCT8 are respectively the primers of the first PCR amplification and the second PCR amplification):

[0156]

[0157]

[0158] It was detected and recovered by agarose gel electrophoresis, and double-digested with pCAMBIA1305.1 and ...

Embodiment 3

[0236] Example 3 Application of chaperonin-assisted mRNA phloem long-distance transport

[0237] 1. Vector construction ( figure 1 )

[0238] Taking mRNA PbWoxT1 on the RNP complex capable of long-distance transport in phloem as an example to illustrate the specific application of chaperones. Design primers for PCR amplification and add enzyme cutting sites to the above recovered products. The primer sequences are as follows:

[0239]

[0240] 2. Plasmid Extraction

[0241] With embodiment 2.

[0242] 3. Agrobacterium Competent Cell Transformation

[0243] With embodiment 2.

[0244] 4. Genetic Transformation of Agrobacterium rhizogenes

[0245]The above-mentioned vectors PbCCT5 RNAi and pSUC2:GFP-PbWoxT1 were transferred into the Agrobacterium rhizogenes K599 strain respectively, and the 1-2 cm stems of the aboveground part of the pear seedlings with the size of 2 weeks were injected together. After 2 weeks, new roots grew at the injection site. Positive strains wer...

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Abstract

The invention relates to chaperonin for enhancing RNP signal communication of plant phloem and an application. According to the invention, a new phloem RNP signal communication auxiliary factor chaperonin CCT complex is obtained, lack of the chaperonin CCT complex is proved through the transient expression of agrobacterium tumefaciens pear leaves and the transgenic mode of agrobacterium rhizogenes, and the transport efficiency of the RNP complex in phloem is obviously weakened. Through overexpression of the chaperonin CCT complex, communication of the RNP complex in the phloem can be rapidly promoted, besides, long-distance transportation of some signal molecules can be weakened by silencing the chaperonin CCT complex, and long-distance transportation of the RNP complex from a stock to a scion through a grafting opening is promoted by using an auxiliary factor. The scion character is influenced by manually controlling the stock gene, the problem that the transportability of the RNP complex in the phloem cannot be manually regulated is solved, and the method can be universally applied to various types of plants.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for enhancing the communication of signal molecules such as RNA and protein in plant phloem through the expression of chaperone protein. Background technique [0002] The phloem sieve tube is the main transport system of plants, which transports sucrose, amino acids, proteins, hormones, RNA and other substances and signal molecules to the sink organ through the symplast pathway (Lucas et al., 2013). As a kind of asexual reproduction method widely used in fruit trees, grafting mainly connects the transportation system between the rootstock and the scion, and then promotes the loading of nutrients and signal molecules between the rootstock and the scion through the companion cells, and enters the long-distance after the plasmodesmata into the sieve tube. transfer (Duan et al., 2015). The signaling molecules between root and ear mainly include hormones (Mahajan et a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N15/84A01H5/00A01H6/74
CPCC07K14/415C12N15/8205C12N15/8261
Inventor 李天忠王胜男王圣元郝理
Owner CHINA AGRI UNIV
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