Maize drought induced expression gene ZmRD101 promoter and application thereof

A gene expression, drought-induced technology, applied in applications, genetic engineering, recombinant DNA technology, etc., can solve problems such as plant energy waste, excessive accumulation of gene products, metabolic processes, and plant death.

Active Publication Date: 2021-09-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, most of the promoters used in maize transgenic technology are constitutive promoter Ubi, and the target gene will be expressed at a high level throughout the life cycle of the plant and in all tissue parts, which will lead to ex

Method used

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  • Maize drought induced expression gene ZmRD101 promoter and application thereof
  • Maize drought induced expression gene ZmRD101 promoter and application thereof
  • Maize drought induced expression gene ZmRD101 promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Obtaining the promoter sequence of the drought-induced expression gene ZmRD101 in maize

[0050] The mature leaves of the B73 inbred line at the adult plant stage without diseases and insect pests were picked, and the plant genomic DNA was extracted by CTAB method. The RNA-seq data of the maize B73 inbred line under different drought degrees in the early stage of the laboratory showed that the expression of the maize ZmRD101 gene was induced by drought, and the 2000 bp upstream of the translation start site was selected as the promoter sequence for research. According to the genome sequence of maize B73, ZmRD101 gene promoter-specific amplification primers were designed, and its sequence is:

[0051] Forward primer P1: 5'-TTCACTTTTTTAGTCTGGCAAT-3'

[0052] Reverse primer P2: 5'-GCTCACGGTTGCCTTG-3'

[0053] The PCR reaction system is as follows:

[0054] 2x phanta buffer 10ul dNTP 0.4ul forward primer P1 0.8ul reverse primer P2 0....

Embodiment 2

[0057] Example 2 Investigation of expression pattern of maize ZmRD101 gene under different drought degrees

[0058] Since the expression level of the maize ZmActin5 gene is basically the same in different tissues and physiological environments of maize, the ZmActin5 gene is generally used as an internal reference gene to detect the relative expression level of the maize gene.

[0059] Design the following specific RT-PCR primers:

[0060] ZmRD101-qPCR-F: 5'-GAATCGTAGCAGCAGCCAAGGCA-3'

[0061] ZmRD101-qPCR-R: 5'-CTTCTTGTACCGCGGGTAGTG-3'

[0062] ZmActin5-F: 5'-GCCGAGCGAGAAATTGTAAG-3'

[0063] ZmActin5-R: 5'-TGGTGATTACTTGGCCATCA-3'

[0064] The leaves of the B73 inbred line at the seedling stage under different drought degrees were picked and quick-frozen in liquid nitrogen. The method of obtaining total RNA is as follows: take an appropriate amount of sample for grinding with liquid nitrogen, add 1ml Trizol to mix, and place at room temperature for 5 minutes; add 200ul of c...

Embodiment 3

[0078] Embodiment 3 Construction of plant ZmRD101 gene promoter expression vector

[0079] 1) A primer pair was designed using the promoter sequence of the drought-induced expression gene ZmRD101 of the maize B73 inbred line as shown in SEQ ID NO.1 as a template, as follows: RD101-F: 5'-CCTGTCAAACACTGATA GTTT TTCACTTTTTTAGTCTGGCAAT-3', RD101-R: 5'-CCGGGATACTAGTGC GTTTAAAC GCTCACGGTTGCCTTG-3' (the underline is the PMEI restriction site);

[0080] 2) PCR was performed using the sequence of the corn drought-induced expression gene ZmRD101 as a template, and the PCR amplification conditions were as follows:

[0081] reaction system:

[0082] 2x phanta buffer 10ul dNTP 0.4ul Forward primer RD101-F 0.8ul Reverse primer RD101-R 0.8ul Phantas max 0.4ul cDNA 1.5ul wxya 2 o

6.9ul

[0083] The PCR reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 sec; annealing at 60°C for 30 sec; extension at 7...

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Abstract

The invention discloses a corn drought induced expression gene ZmRD101 promoter and application thereof. The nucleotide sequence of the corn drought induced expression gene ZmRD101 promoter is shown as SEQ ID NO. 1. The promoter of the gene ZmRD101 has the characteristics of induced high expression under drought and low expression in normal growth, and can be used in the field of transgenic breeding. A plant expression vector containing the promoter is constructed and transformed into a genome of a plant such as monocotyledon corn, and a target gene is driven to be specifically and highly expressed under drought and to be low expressed under a normal state. The strain can be used for balancing the contradiction between yield and stress resistance, is used for cultivating a new breeding material, and provides a basis for further stress-resistant breeding of corn.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a promoter of a corn drought-induced expression gene ZmRD101 and an application thereof. Background technique [0002] Corn is an important food crop as well as an important feed and industrial raw material. Maize production is seriously threatened by drought. Data show that the drought during the silking period can cause about 50% reduction in corn production. Identifying drought-resistant genes and cultivating drought-resistant varieties is the fundamental way to relieve the threat of drought. In recent years, a series of maize drought resistance genes have been identified and cloned, but the application of these genes in drought resistance breeding is still very limited. To a certain extent, there is an antagonistic relationship between drought resistance and yield, and simple aggregation of stress resistance genes may result in a decrease in maize yield i...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8273
Inventor 代明球何能向艳丽
Owner HUAZHONG AGRI UNIV
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