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Construction and disease resistance application of a cassava nitrate reductase gene and its overexpression vector

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

Active Publication Date: 2022-04-22
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Construction and disease resistance application of a cassava nitrate reductase gene and its overexpression vector
  • Construction and disease resistance application of a cassava nitrate reductase gene and its overexpression vector

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 the construction and disease resistance application of a cassava nitrate reductase gene and its overexpression vector, belonging to the technical field of plant genetic engineering. The present invention isolates a gene from cassava, the nucleotide sequence of its CDS is shown in SEQ ID No.1. Construction of cassava MeNR1 gene overexpression vector: RNA was extracted from Huanan No. 124 cassava leaves and then reverse-transcribed into cDNA. The full-length gene sequence of the CDS region of MeNR1 gene was used as the insert fragment of the gene overexpression vector, and XbaI and SmaI restriction sites were selected. Homologous primers for pBI121 were designed to obtain cDNA as a template for amplification to obtain the full-length sequence of the MeNR1 gene coding region. After recovery from gel cutting, it was homologously ligated into the plant overexpression vector pBI121 to obtain the pBI121‑MeNR1 gene overexpression vector. By constructing the overexpression vector of MeNR1 gene and injecting cassava leaves, effective transient MeNR1 gene overexpression plants are obtained, which provides a fast and effective method for studying the function of cassava genes.

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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Patent Type & Authority Patents(China)
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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