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Cassava nitrate reductase gene and construction and disease-resistant application of overexpression vector of cassava nitrate reductase gene

A technology of overexpression and reductase, which is applied in the field of plant genetic engineering to achieve the effect of enhancing the expression level

Active Publication Date: 2020-02-04
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the functional research of MeNR1 gene in cassava

Method used

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  • Cassava nitrate reductase gene and construction and disease-resistant application of overexpression vector of cassava nitrate reductase gene
  • Cassava nitrate reductase gene and construction and disease-resistant application of overexpression vector of cassava nitrate reductase gene

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1: Construction of cassava MeNR1 gene overexpression vector

[0026] (1) Cloning of cassava MeNR1 gene CDS sequence

[0027] Take an appropriate amount of cassava leaves of Huanan No. 124 and place them in a mortar cooled by liquid nitrogen, add an appropriate amount of liquid nitrogen, and grind the cassava leaves into fine powder. Root Biochemical Technology (Beijing) Co., Ltd., the same below), extract cassava total RNA. A protein detector (DM650BECKMAN, MSA) was used to measure the absorbance value of RNA at 260nm and 280nm and the concentration of RNA, and the purity of RNA was detected by 1.5% (mass volume ratio) agarose gel electrophoresis. According to the instructions of the reverse transcription kit (purchased from Thermo Fermentas, the same below), RNA was reverse-transcribed into cDNA and stored in a -40°C refrigerator for later use.

[0028]By designing primers, we obtained the full-length sequence of the cassava MeNR1 gene for the first time. ...

Embodiment 2

[0033] Example 2: Application of pBI21-MeNR1 Gene Overexpression Vector in Cassava Disease Resistance

[0034] (1) cassava material preparation:

[0035] A. Seed selection: Choose a variety with good adaptability and high yield, and choose Huanan 124 for planting. Generally, it is better to choose cassava stems that are fully mature, suberized, thick and thick, with complete epidermis and axillary buds, thick and obvious bud eyes, no pests and diseases, no damage, fresh and solid main stems. Old and young stems should be avoided.

[0036] B. Acceleration of germination: before planting, cut off the stem with a sharp knife. The incision should be kept flat so as not to detach the epidermis and not damage the axillary buds. Each section is 13-20cm long, leaving 3-5 strong axillary buds. Put the selected stem with axillary buds facing upwards, and immerse the bottom end in water for 1-2 cm to ensure that the bottom end is moist, and place it for 3-4 days until new buds emerge. ...

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Abstract

The invention provides a cassava nitrate reductase gene and construction and disease-resistant application of an overexpression vector of the cassava nitrate reductase gene, and belongs to the technical field of plant genetic engineering. The gene is separated from cassava, and a nucleotide sequence of CDS of the gene is shown as SEQ ID No. 1. Construction of the cassava MeNR1 gene overexpressionvector includes the steps that RNA is extracted from South China 124# cassava leaves and then is subjected to reverse transcription to form cDNA, a full-length gene sequence of a CDS region of the MeNR1 gene is used as a gene overexpression vector insertion fragment, XbaI and SmaI enzyme cutting sites are selected to design a pBI121 homologous primer, amplification is performed by taking the obtained cDNA as a template to obtain a full-length sequence of a MeNR1 gene coding region, and after gel cutting recovery, homologous connection is performed for connection into a plant overexpression vector pBI121 to obtain the pBI121-MeNR1 gene overexpression vector. By constructing the overexpression vector of the MeNR1 gene and injecting the vector into cassava leaves, an effective transient MeNR1gene overexpression plant is obtained, and a rapid and effective method is provided for researching cassava gene functions.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a cassava nitrate reductase (NR1) gene that enhances the resistance of cassava to pathogenic bacteria Xam (Xanthomonas axonopodis pv.manihotis), and also relates to a cassava NR1 gene overexpression Vector construction method, and the use of a cassava NR1 gene overexpression vector in enhancing plant resistance to pathogenic bacteria Xam. Background technique [0002] Transient expression technology is a technology that transfers the target gene into target cells in a relatively short period of time, establishes a temporary and efficient expression system in the cells, and obtains a short-term high-level expression of the target gene. When exogenous genes are introduced into plant cells, there are two ways of expression: transient expression and stable expression. Compared with stable expression, transient expression takes a shorter time and does not ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/06C12N15/84C12N15/66A01H5/00A01H6/38
CPCC12N9/0044C12Y107/01001C12N15/8281C12N15/66
Inventor 施海涛王鹏闫语
Owner HAINAN UNIVERSITY
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