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Cassava CAMTA (calmodulin-binding transcription activator) gene as well as construction and disease-resistant application of inhibitory expression vector of cassava CAMTA gene

A cassava, carrier technology, applied in the application, genetic engineering, plant genetic improvement and other directions, to achieve the effect of fast and efficient silencing

Active Publication Date: 2019-12-20
HAINAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the function of CAMTA gene in cassava

Method used

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  • Cassava CAMTA (calmodulin-binding transcription activator) gene as well as construction and disease-resistant application of inhibitory expression vector of cassava CAMTA gene
  • Cassava CAMTA (calmodulin-binding transcription activator) gene as well as construction and disease-resistant application of inhibitory expression vector of cassava CAMTA gene
  • Cassava CAMTA (calmodulin-binding transcription activator) gene as well as construction and disease-resistant application of inhibitory expression vector of cassava CAMTA gene

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Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1: Construction of cassava MeCAMTA gene expression silencing vector

[0026] (1) Cloning of the specific nucleic acid silent fragment of cassava MeCAMTA gene 5' end 1-600bp:

[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 Co., Ltd., hereinafter the same), extract cassava total RNA. A protein detector (DM 650BECKMAN, MSA) was used to measure the absorbance value of RNA at 260 nm and 280 nm 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 cloned and obtained the full-lengt...

Embodiment 2

[0033] Example 2: Application of pTRV2-MeCAMTA gene silencing expression 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 ...

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Abstract

The invention provides a cassava CAMTA (calmodulin-binding transcription activator) gene as well as construction and disease-resistant application of an inhibitory expression vector of the cassava CAMTA gene. One gene is obtained by separation from cassava, and the nucleotide sequence of CDS (coding sequence) of the gene is represented as SEQ ID No.1. The construction of the inhibitory expressionvector of the cassava CAMTA gene comprises steps as follows: RNA in leaves of Huanan 124 cassava is extracted and subjected to reverse transcription into cDNA, a 5'-end 1-600bp segment of the CAMTA gene is taken as a specific nucleic acid silent segment of a virus-induced gene silent system, the 5'-end 1-600bp segment of the CAMTA gene is obtained by amplification with obtained cDNA as a templateand is connected to a TRV (tobacco rattle virus) induced gene silent vector pTRV2 after gel recovery, and a pTRV-MeCAMTA gene silent expression vector is obtained. The silent expression vector of thegene is constructed and injected to the leaves of cassava, effective instantaneous MeCAMTA gene silent cassava is obtained, and an effective method is provided for study of gene functions.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering and technology, in particular, relates to a MeCAMTA gene that enhances the resistance of cassava to pathogen Xam (Xanthomonas axonopodis pv.manihotis), and also relates to the construction of a cassava CAMTA gene silencing system and expression vector The method also relates to the use of a cassava CAMTA gene silencing expression vector in enhancing plant resistance to pathogenic bacteria Xam. Background technique [0002] Virus-induced gene silencing system (Virus-induced gene silencing, VIGS) is a gene transcription suppression technology used to characterize the function of a certain gene in plants. In recent years, a variety of VIGS vectors have been widely used in plants. Among them, tobacco rattle virus (TRV) is currently the most widely used carrier, which is composed of two viral chains, TRV1 and TRV2, and TRV1 is used to assist TRV2 carrying target gene fragments to move in plant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/83C12N15/66A01H5/12A01H6/38
CPCC07K14/415C12N15/8218C12N15/8281C12N15/66
Inventor 施海涛昌燕李
Owner HAINAN UNIVERSITY
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