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Salt stress inducible promoter as well as primers, expression vector and application thereof

A technology of expressing vectors and promoters, applied in the field of genetic engineering, can solve problems such as adverse side effects, energy consumption, and yield reduction, achieve wide application value, and improve salt tolerance

Active Publication Date: 2019-03-22
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past 20 years, many plants have been transformed by genetic engineering technology, which has enhanced the tolerance of the transformed plants to salt stress; however, some transgenic plants have produced adverse side effects after transformation, such as delayed growth, Problems such as stunted plants and reduced yields, which may be the result of energy consumption driven by constitutive promoters, high levels of transgene expression, and ectopic expression of transgenes

Method used

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  • Salt stress inducible promoter as well as primers, expression vector and application thereof
  • Salt stress inducible promoter as well as primers, expression vector and application thereof
  • Salt stress inducible promoter as well as primers, expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The action element analysis of embodiment 1 promoter fragment

[0042] The promoter sequence of the LHR1 gene obtained from the Tair website was analyzed by http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / , and it was found that in addition to the core promoter elements, such as TATA-BOX, CAAT- In addition to elements related to BOX, light response and circadian rhythm, there are also promoter elements related to biotic and abiotic stress, such as ARE elements are cis-acting elements necessary for anaerobic induction, LTR elements are involved in low temperature induction, HSE elements and Related to heat stress, TC-rich repeats participate in stress and stress response, CGTCA-motif participates in jasmonic acid response, GARE-motif responds to gibberellin-induced response, etc.

[0043] According to the distribution of the active elements, the fragment is further divided into two segments, the lengths of which are 1380bp and 821bp respectively.

Embodiment 2

[0044] Example 2 Cloning of Salt Stress Inducible Promoter Fragments of Different Lengths

[0045] Using Arabidopsis genomic DNA as a template, the promoter fragment was amplified by PCR with high-fidelity enzymes (Takara, Japan) (10X buffer 5 μL, dNTP 4 μL, primer-F primer 1 μL, primer-R primer 1 μL, template 1 μL, enzyme 1 μL , RNase-Free water to make up 50 μL; program: 98°C 10″, 55°C 15″, 72°C 30″, 30circle; 16°C, hold). Perform agarose gel electrophoresis on the amplified PCR product to recover the target fragment Afterwards, it was connected to the pEASY cloning vector, transformed into Escherichia coli and incubated at a constant temperature of 37°C for 12 hours.

Embodiment 3

[0046] Example 3 Construction of the vector of the promoter fragment

[0047] Using the vector pBI101 connected with the GUS reporter gene, the plasmid and the fragment were digested with endonucleases Sal Ⅰ and Xba Ⅰ, and the promoter fragment was ligated to the polymorphism of the vector fragment with T4 ligase (Thermo, USA) at 22°C. The cloning site (MCS) region is used to construct recombinant plasmids. The promoter fragment was inserted into the MCS and introduced into Escherichia coli, a large number of recombinant plasmids were amplified, a single colony was picked, and the colonies were verified by PCR using the primers designed in the upstream and downstream of the pBI101::GUS vector multiple cloning site to detect whether there was a successful promoter fragment The positive colony inserted into the pBI101::GUS vector was generated, and the positive colony was picked and cultured overnight in LB liquid medium with shaking, and the plasmid of the positive bacterial li...

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Abstract

The invention belongs to the field of genetic engineering, and in particular relates to a salt stress inducible promoter as well as primers, an expression vector and application thereof. A promoter fragment is cloned, a vector of the promoter fragment is constructed, arabidopsis genetic transformation is performed, an arabidopsis resistant material is screened and detected, GUS staining is carriedout, and RT-qPCR is used for detecting the expression situation of a GUS gene. The results show that the promoter fragment provided by the invention can drive the expression of the GUS gene and can be expressed in roots, stems, leaves, flowers and other parts; under the condition of abiotic stress induction, the activity and driving efficiency of the promoter are also higher than those under thenormal conditions; the qPCR results also show the similar results, so that the promoter provided by the invention can improve the salt tolerance of transgenic crops; therefore, the salt stress inducible promoter has a wide application value.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a salt stress inducible promoter, a primer, an expression vector and application thereof. Background technique [0002] In the north of the Yangtze River and most of the coastal areas in my country, the saline-alkali content in the soil is often too high, which is easy to harm the growth of plants, especially the yield of crops. In recent years, with the rapid development of biotechnology, crop genetic engineering has gradually become a fast method to improve crop performance and improve crop quality. In the past 20 years, many plants have been transformed by genetic engineering technology, which has enhanced the tolerance of the transformed plants to salt stress; however, some transgenic plants have produced adverse side effects after transformation, such as delayed growth, Problems such as stunted plants and reduced yields can be the result of energy consumption ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/20C12N15/11
CPCC12N15/11C12N15/8273C07K14/415
Inventor 黄勇皮博艺阮颖刘博宇谭成方何星辉
Owner HUNAN AGRICULTURAL UNIV
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