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Cotton long non-coding RNA-lnc973 and application thereof in plant salt tolerance

A long-chain non-coding, salt-tolerant technology, applied in the field of molecular biology, can solve problems such as influence regulation, and achieve the effect of improving salt-tolerance and wide application value

Active Publication Date: 2018-11-13
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

lncRNA does not have a universal mode of action, it can regulate gene expression and protein synthesis in different ways, such as regulating the expression of coding genes through cis or trans (trans), in addition to acting as The "sponge" of miRNA competitively binds to miRNA, thereby affecting the regulation of miRNA on downstream target genes

Method used

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  • Cotton long non-coding RNA-lnc973 and application thereof in plant salt tolerance
  • Cotton long non-coding RNA-lnc973 and application thereof in plant salt tolerance
  • Cotton long non-coding RNA-lnc973 and application thereof in plant salt tolerance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Detection of the expression level of lncRNA-lnc973

[0040] 1. Materials and Reagents

[0041] 1.1 Materials

[0042] The cotton used in this experiment is G. hirsutum L., a salt-tolerant cotton line Shannong 91-11 (untreated material is denoted as S_CK, 250mmol / L NaCl-treated material is denoted as S_NaCl).

[0043] 1.2 Reagents

[0044] The RNAprep Pure Plant Kit was purchased from Beijing Tiangen Biotechnology Co., Ltd.; the reverse transcription kit (RR047A) and qRT-PCR kit (RR420A) were purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.; primers were provided by Sangon Synthesized by Bioengineering (Shanghai) Co., Ltd.

[0045] 2. Experimental method

[0046] 2.1 Transcriptome sequencing of lncRNA

[0047] Using untreated cotton and cotton samples treated with 250mmol / L NaCl for 24 hours as objects, RNA-seq was entrusted to Beijing Annoroad Gene Technology Co., Ltd. to conduct. Total RNA was isolated from each cotton seedling sample using RNA...

experiment example 2

[0065] Experimental example 2: Cloning and analysis of lncRNA-lnc973 sequence

[0066] 1. Reagents and carriers

[0067] Reverse transcription kit PrimeScript TM 1st Strand cDNA Synthesis Kit, restriction endonucleases Kpn Ⅰ, Pst Ⅰ, cloning vector pMD19-T and DNA A-Tailing Kit were purchased from Baori Medical Biotechnology (Beijing) Co., Ltd.; high-fidelity enzyme 2× Max Master Mix was purchased from Nanjing Nuoweizan Biotechnology Co., Ltd.; Universal DNA Purification and Recovery Kit (DP214) and Plasmid Mini-Extraction Kit (DP103) were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0068] 2. Method

[0069] 2.1 Extraction of total RNA

[0070] The experimental method was the same as 2.2.1 total RNA extraction in Experimental Example 1.

[0071] 2.2 Primer design

[0072] According to the sequence (SEQ ID NO.1) of lncRNA-lnc973 nucleotide, lncRNA-lnc973 amplification primers were designed by Primer premier 5.0 software, specifically as follows: ...

experiment example 3

[0089] Experimental Example 3: Verification of Transgenic Function—Arabidopsis Transformation, Screening and Phenotypic Analysis of Salt Tolerance

[0090] 1. Arabidopsis planting and pre-transformation treatment

[0091] Sprinkle the wild-type Arabidopsis evenly on the seedling tray, and when it grows to four leaves, transplant it into the nutrient pot (mix the nutrient soil and vermiculite in equal proportions), cultivate it at 23°C, and light it for 16 / 8h. cycle. When the lateral moss inflorescences of the plant form flower buds and partly bloom or form 1-2 siliques, they can be used for transformation, and the bloomed flowers and grown siliques need to be cut off before transformation.

[0092] 2. Transformation of Arabidopsis

[0093] Pick the activated monoclonal positive Agrobacterium GV3101 strain containing recombinant plasmid pCAMBIA1300-lnc973-eGFP to 5ml fresh LB liquid medium (containing Kan + and Rif + ), shake culture at 28°C for 24 hours to obtain the bacte...

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Abstract

The invention discloses a lncRNA-lnc973 sequence of cotton long non-coding RNA and application thereof in plant salt tolerance. The nucleotide sequence of the lncRNA-lnc973 is shown by SEQ ID NO.1. According to the cotton long non-coding RNA disclosed by the invention, objective existence of lncRNA-lnc973 is proved by PCR, and the expression amount of the lncRNA-lnc973 is remarkably increased in the condition of salt stress. The full-length transcript of cotton lncRNA-lnc973 is cloned to establish overexpression vector transformed wild type arabidopsis thaliana, and the transgenic arabidopsisthaliana enhances the salt tolerance; by establishing a VIGS (virus-induced gene silencing) knockout vector and transfecting cotton leaves with the VIGS technology, the result proves that the expression amount of cotton lnc973 after VIGS knockout is remarkably lowered, and the salt tolerance is reduced, thus indicating that the plant salt tolerance can be enhanced by overexpression of the cotton lncRNA-lnc973.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a cotton long-chain non-coding RNA-lnc973 and its application in plant salt tolerance. Background technique [0002] Cotton is not only a major fiber crop, but also an important oil crop. Although it is a relatively salt-tolerant crop, soil salinization not only limits the growth of cotton, but also affects its yield and fiber quality, causing great losses to cotton production. Therefore, studying the regulation mechanism of cotton salt tolerance, cloning cotton salt tolerance related genes and regulatory elements, and carrying out cotton transgenic research are the core key technologies to improve cotton salinity breeding. [0003] Long non-coding RNA is a type of RNA molecule longer than 200 bp that does not encode protein. According to the position of lncRNA on the genome relative to protein-coding genes, lncRNA can be divided into five categories: sense, antisense,...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/00
CPCC12N15/113C12N15/8216C12N15/8273
Inventor 沈法富张晓佩王维陈懂懂刘丹程莹莹
Owner SHANDONG AGRICULTURAL UNIVERSITY
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