Cloning, identification and application of salt tolerance related gene spliceosome

A technology for salt-tolerant genes and transgenic plants, which is applied in the cloning, identification and application of salt-tolerance-related gene splicing bodies, and can solve the problems of increasing the complexity of transcripts and proteomes

Active Publication Date: 2021-01-05
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Through alternative splicing, organisms can generate multiple functionally similar or completely different transcript isoforms, greatly increasing the complexity of transcripts and proteomes
Transcriptome sequencing technology

Method used

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  • Cloning, identification and application of salt tolerance related gene spliceosome
  • Cloning, identification and application of salt tolerance related gene spliceosome
  • Cloning, identification and application of salt tolerance related gene spliceosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1, cloning and identification of spliced ​​body

[0086] 1. Cloning of spliced ​​bodies

[0087] 1. Extract the total RNA of 35 leaf tissues in the process, use the total RNA as a template, and use action primers F1: 5'-ATCCTCCGTCTTGACCTTG-3' and R1: 5'-TGTCCGTCAGGCAACTCAT-3' for PCR amplification to detect whether there is any genomic DNA residue . If PCR amplification yields a band of about 215 bp in size, there is genomic DNA remaining in the total RNA; otherwise, there is no genomic DNA remaining.

[0088] The result is as figure 1 As shown, CK is a PCR product obtained by using cDNA as a template and using action primer PCR amplification, and 1-8 is a PCR product obtained by using total RNA as a template and using action primer PCR amplification. The results showed that there was no genomic DNA residue in the total RNA extracted from leaves of Zhong35.

[0089] 2. Using reverse transcriptase to reverse transcribe the first-strand cDNA from Zhong 35 ...

Embodiment 2

[0108] Embodiment 2, embodiment 1 obtain the salt tolerance analysis of two shear bodies

[0109] 1. Construction of recombinant vector pBI121-s1

[0110] 1. Use the recombinant vector T-s1 as a template, and use F4: 5'-CTAGAGGATCCCCGGGATGGTTTCAGAAATTGGGGCTG-3' and R4: 5'-GATCGGGGAAATTCGAGCTCTTAAAAGTATTTCTGGAAGCATTCCTCTTC-3' as primers to perform PCR amplification to obtain a PCR fragment s1' with a size of about 2000 bp.

[0111] 2. Digest the vector pBI121 with restriction endonucleases XbaI and SacI, and recover the vector backbone 1 with a size of about 12.9 kb.

[0112] 3. Use the In-fusion HD Cloning Kit to connect the PCR fragment s1' and the vector backbone 1 to obtain the recombinant vector pBI121-s1.

[0113] The recombinant vector pBI121-s1 was double digested with XbaI and SacI, and the results were as follows: Figure 5 As shown in A, the recombinant vector pBI121-s1 produced two bands (lane 2) after double enzyme digestion, one of which was similar in size to u...

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Abstract

The invention discloses cloning, identification and application of a salt tolerance related gene spliceosome. The method comprises the following steps: firstly, cloning two GhIRE1 gene spliceosome sequences from a salt-tolerant gene, respectively naming as GhIRE1-s1 and GhIRE1-s2, and performing prokaryotic expression by constructing a prokaryotic expression vector to obtain the GhIRE1-s1 and theGhIRE1-s2. In order to further verify the functions of the GhIRE1-s1 and the GhIRE1-s2, a GhIRE1-s1 transgenic arabidopsis thaliana plant and a GhIRE1-s2 transgenic arabidopsis thaliana plant are respectively constructed, and the stress tolerance of the GhIRE1-s1 transgenic arabidopsis thaliana plant and the stress tolerance of the GhIRE1-s2 transgenic arabidopsis thaliana plant are detected. Results show that compared with wild type arabidopsis thaliana plants, the salt tolerance of s1 transgenic arabidopsis thaliana plants is reduced. Therefore, it shows that the GhIRE1-s1 has the function of regulating and controlling the salt tolerance of the plant.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the cloning, identification and application of a spliced ​​body of a salt-tolerance-related gene. Background technique [0002] In agricultural production, various biotic and abiotic stresses in the external natural environment often affect the growth and development of crops and reduce crop yield. In recent years, with the deterioration of environmental pollution and global climate, adversity stress has become an important factor affecting crop production. Among them, salt stress is one of the main adversity stresses. It is estimated that in the world, the annual agricultural output reduction caused by salt stress can reach more than 20%. According to the statistics of FAO, as of 2015, about 800 million hectares of land and 32 million hectares of dryland in the world are affected by salinization. In my country, there are about 37 million hectares of saline-alkali land...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/60
CPCC07K14/415C12N15/8273
Inventor 叶武威王晓歌陈修贵陆许可王德龙王俊娟王帅郭丽雪陈超阴祖军赵兰杰
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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