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E3 ubiquitin ligase gene ospiw regulates the function and application of rice root

A ubiquitin ligase, rice technology, applied in the field of plant genetic engineering, can solve the problems of rice root growth retardation, division frequency affecting meristems, etc.

Inactive Publication Date: 2018-04-24
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These results suggest that the gene may affect the size of the meristem by affecting the frequency of cell division in the root tip meristem, ultimately leading to growth retardation in rice roots

Method used

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  • E3 ubiquitin ligase gene ospiw regulates the function and application of rice root
  • E3 ubiquitin ligase gene ospiw regulates the function and application of rice root
  • E3 ubiquitin ligase gene ospiw regulates the function and application of rice root

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Cloning of OsPIW gene

[0042] The cloning (LOC_Os06g19680) of the gene OsPIW of the present invention is mainly obtained by the method of RT-PCR (method is referred to: J. Sam Brook, EF Fritsch, T Mani Artis, Huang Peitang, Wang Jiaxi etc. translation, molecular cloning experiment Guide (Third Edition), Beijing, Science Press, 2002 edition). The specific operation steps are as follows:

[0043] 1) RNA was extracted from the seedling leaves of the rice variety "Zhonghua 11" (gifted by the Institute of Crop Science, Chinese Academy of Agricultural Sciences), using Invitrogen's Trizol extraction kit for RNA extraction (see the kit's instruction manual for specific steps);

[0044] 2) The steps of RT-PCR reverse transcription to synthesize the first strand of cDNA are as follows: ①Preparation Mixture 1: Total RNA 4μg, DNaseI2U, 10DNaseI buffer 1μl, add DEPC (diethylpyrocarbonate, a strong inhibitor of RNase) for treatment Add water (0.01% DEPC) to 10 μl, mi...

Embodiment 2

[0051] Embodiment 2: Construction of double-strand suppression dsRNAi vector

[0052] According to the full-length cDNA sequence of the OsPIW gene published in the Rice Genome database (http: / / rice.plantbiology.msu.edu / index.shtml), design double-stranded suppression dsRNAi primers, and add SpeI and KpnI to the 5' end of the upstream primer Restriction endonuclease sites, Sac I and BamH I restriction endonuclease sites were added sequentially at the 5' end of the downstream primer. After the target sequence was amplified by PCR, the amplified product was connected to pGEMT-vector (purchased from Promega, USA) by T / A cloning for sequencing verification, and the amplified DNA fragment was shown in the sequence table as SEQ ID NO:3.

[0053] Specific steps are as follows:

[0054] 1) The T / A clone of the RNAi fragment with OsPIW was digested with Kpn I and BamHI restriction endonucleases, and the target band was recovered, and the vector plasmid pDS1301 (provided by Huazhong Agr...

Embodiment 3

[0060] Example 3. The functional verification of OsPIW in rice root growth and development,

[0061] The steps of this embodiment are as follows:

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Abstract

The invention belongs to the technical field of plant genetic engineering, and specifically relates to the function and application of E3 ubiquitin ligase gene OsPIW regulating rice roots. A RING H2 family protein with a high expression level in roots was isolated and cloned, and the gene encoding this protein was named OsPIW, and its nucleotide sequence is shown in SEQ ID NO:1. Through the method of RNAi, the corresponding suppressed expression transgenic rice of the gene was obtained, and it was found that after the gene was suppressed, the length of the main root and the number of crown roots of the transgenic plants were significantly shorter and less than those of the wild-type control plants, and the shortening of the main root was due to division The length of the growing zone and elongation zone shortened, while the number of crown roots decreased mainly due to the delay in the initiation of crown roots. Further analysis showed that the inhibition of the gene expression could slow down the cell division in the root tip meristem. These findings indicate that the gene has the function of promoting rice root growth and development.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. It specifically relates to the function and application of an E3 ubiquitin ligase gene OsPIW in regulating rice root growth and development. The E3 ubiquitin ligase gene OsPIW of the present invention belongs to rice RING-finger H2 family protein gene, we have analyzed the function of this gene in rice root development by transgenic method, and proposed its application in agricultural production value. Background technique [0002] The root of a plant is the most important organ located below the soil surface and capable of responding rapidly to changes in surrounding environmental factors. In addition, the normal growth of roots is closely related to the biomass and adaptability of the above-ground parts of plants. Research on the regulation mechanism of rice root development will help to explore the role of roots in rice yield, mineral elements and nutrient absorption, and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N15/82A01H5/06A01H6/46
Inventor 赵毓蒋玮黄玉兰周道绣
Owner HUAZHONG AGRI UNIV