Plant expression vector for vascular peculiar promoter to control antimicrobial protein gene and method for cultivating greensickness-resistant cotton

A plant expression vector and specific promoter technology, applied in the field of plant genetic engineering, can solve the problems of reduced seed germination rate, reduced number of seeds, reduced formation of lateral roots, etc., to achieve the effect of inhibiting breeding and improving resistance

Inactive Publication Date: 2010-09-01
SOUTHWEST UNIVERSITY
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Problems solved by technology

Fiona Murray et al. (1999) overexpressed the glucose oxidase gene from Talaromyces flavus in cotton, homozygous T 3 Progeny plants have enhanced resistance to cotton Verticillium wilt, but the growth and development of roots and seeds of plants with high expression levels are affected, the roots become shorter, the formation of lateral roots decreases, the number of seeds decreases, and the germination rate of seeds decreases

Method used

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  • Plant expression vector for vascular peculiar promoter to control antimicrobial protein gene and method for cultivating greensickness-resistant cotton
  • Plant expression vector for vascular peculiar promoter to control antimicrobial protein gene and method for cultivating greensickness-resistant cotton
  • Plant expression vector for vascular peculiar promoter to control antimicrobial protein gene and method for cultivating greensickness-resistant cotton

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] The extraction of implementation example 1 plant DNA and RNA

[0062] 1. Extraction of plant genomic DNA

[0063] Select Arabidopsis thaliana, tobacco or cotton (tomato, rice, etc.) fresh plant tissue 0.5g, quickly ground into powder in liquid nitrogen, add 3mL 65 ℃ preheated CTAB extract (100mmol / L Tris-HCl (pH8 .0), 20mmol / LEDTA (pH8.0), 1.5mol / L NaCl, 2% CTAB (W / V), 4% PVP40 (W / V) and 2% mercaptoethanol (V / V), PVP and mercapto Add ethanol before use), shake quickly to mix. Bath at 65°C for 30min, then add 1mL 5mol / LKAc, ice-bath for 20min, extract once with an equal volume of chloroform:isoamyl alcohol (24:1) (10,000r / min, centrifuge at 4°C for 5min), and take the supernatant solution, add 2 / 3 times the volume of -20°C pre-cooled isopropanol, mix well, let it stand for about 30 minutes, pick out the flocculent precipitate with a glass rod, rinse repeatedly with 75% ethanol several times, and then rinse with absolute ethanol 1 time, air-dried and resuspended in 500...

Embodiment 2

[0066] Implementation example 2 Cloning of vascular specific promoter AAP2

[0067] 1. PCR cloning of vascular bundle-specific expression promoter AAP2

[0068] According to the characteristics of the Arabidopsis thaliana Amino acid permease gene2 (AAP2) gene, the Arabidopsis genomic DNA was used as a template, and the sequence 1 and sequence 2 were used as primers to amplify the promoter sequence of the AAP2 gene, and a 25 μL reaction system was constructed: 10×ExPCR buffer (no Mg 2+ 2.5 μL; 2.5 mmol / L dNTPs 2 μL; 25 mmol / L MgCl2 (magnesium chloride) 2 μL; primer 1 (5 μmol / L) 2 μL; primer 2 (5 μmol / L) 2 μL; Ex Taq DNA polymerase 1U; genomic DNA about 60 ng.

[0069] PCR reaction conditions: 94°C for 5min; 94°C for 1min; 50°C for 1min; 72°C for 2min and 30sec (1°C drop for each cycle); 94°C for 1min; 40°C for 1min; 72°C for 2min 30sec (25 cycles); 72°C 10min.

[0070] 2. Recovery of amplified products

[0071] The PCR products were quantified by electrophoresis on agarose ...

Embodiment 3

[0076] Implementation example 3 Construction of vascular specific promoter AAP2 plant expression vector

[0077] 1. Construction of vascular specific promoter AAP2 plant expression vector

[0078] In order to analyze the expression characteristics of the AAP2 vascular-specific promoter, the AAP2 promoter sequence was substituted for the CaMV35S promoter sequence on the pBI121 vector to obtain the plant expression vector pBI121-AAP2. Vector construction flow chart see figure 2 .

[0079] 2. Transformation of the Agrobacterium tumefaciens host integrating the pBI121-AAP2 expression vector

[0080] The pBI121-AAP2 plant expression vector was introduced into Agrobacterium tumefaciens by freeze-thaw method. The specific procedure is: add 0.5-1 μg of plasmid DNA to 100 μL of competent cells, mix well, and ice-bath for 30 minutes. After quick-freezing in liquid nitrogen for 5 minutes, incubate at 37°C for 5 minutes, and immediately ice-bath for 2 minutes. Add 1000μL liquid YEB,...

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Abstract

The invention provides a plant expression vector for a vascular peculiar promoter to control two types of antimicrobial protein gene and a method for cultivating greensickness-resistant cotton. In the method, motherwort antimicrobial protein genes LjAMP2 and LjAMP2 controlled by a vascular tissue peculiar promoter AAP2 are integrated into a cotton genome to realize the high-amount expression of the antimicrobial protein gene in the vascular tissue of the transgenic cotton root and properly express in the vascular tissues of other parts of the plant so as to obviously improve the resistance to greensickness by cotton. After performing homozygosis to defoliating and nondefoliating greensickness bacteria inoculated by a T2 generation plant greenhouse disease pool, the transgenic cotton obtained with the method compares the defoliating and nondefoliating greensickness bacteria with Non-GMO, and disease index can be respectively lowered by 40% and 80%.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. It specifically relates to a plant expression vector of an antibacterial protein gene controlled by a vascular-specific promoter and a method for preparing transgenic cotton containing the expression vector; in addition, the invention also relates to a method for improving cotton resistance to Verticillium wilt by using genetic engineering technology. technical background [0002] Cotton is the most important natural fiber crop in the world. In China, cotton has always been an important material related to the national economy and the people's livelihood. my country's cotton industry involves the income of about 200 million rural population, the employment of 19 million textile workers and the export of more than 100 billion US dollars. pivotal role. However, since cotton verticillium wilt was introduced into my country in 1935, the harm to my country's cotton production has in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N1/21C12N5/10C12R1/01
Inventor 李先碧裴炎李德谋侯磊肖月华罗明宋水清罗小英
Owner SOUTHWEST UNIVERSITY
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