Construction method for anti-insect transgene corn with stable heredity

A technology of transgenic corn and its construction method, which is applied in the field of corn biotechnology breeding, can solve problems such as unsuitable for long-term use, easy residue, environmental pollution, etc., and achieve the effect of excellent agronomic traits and good resistance

Inactive Publication Date: 2014-03-26
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is a lack of effective natural resistance genes in corn materials, and it is difficult to use traditional breeding techniques to screen corn materials with excellent insect resistance. Traditional pest control measures are mainly chemical control, but chemical pesticides have high costs, environmental pollution, Disadvantages such as easy residue, poisonous beneficial insects and natural enemies of pests are not suitable for long-term use

Method used

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  • Construction method for anti-insect transgene corn with stable heredity
  • Construction method for anti-insect transgene corn with stable heredity
  • Construction method for anti-insect transgene corn with stable heredity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] A method for constructing stable hereditary insect-resistant transgenic corn, the steps of which are as follows:

[0031] (1) Preparation of embryogenic maize callus:

[0032] By artificially controlling pollination, the callus induction material Hi-II (A188*B73) material seeds were prepared, and the F 1 The generation seeds are planted in the field, strictly self-pollinated, and the pollination materials and dates are recorded on the signboard. Take back the young ears 10-14 days after pollination, sterilize the surface with 75% alcohol, pick immature embryos (1.2-1.8mm in diameter) and place them in N6 induction medium, culture them in the dark at 28°C to induce callus, and wait for type I callus After formation, transfer to subculture medium, culture in dark at 28°C, and subculture once every 20 days to induce embryogenic callus (loose, dry, brightly colored, fast-growing somatic embryos) as transformation recipients.

[0033] Specific steps are as follows:

[003...

Embodiment 2

[0085] Integration and expression verification of offspring of insect-resistant transgenic plants, the specific steps are as follows:

[0086] T 0The generation seeds were planted in the field of Huazhong Agricultural University in Wuhan, and T 1 generation single plant. At the jointing stage, fresh leaf DNA was extracted using the CTAB method, PCR primers (Cry1C*-F and Cry1C*-R) were designed using the target gene sequence for PCR detection, and individual plants carrying the target gene were selected for Southern hybridization detection, and those with fewer copies were selected Individual plants were tested by RT-PCR and ELISA.

[0087] (1) Transgenic plant seeds are planted in ear rows, using T 0 The same primers and PCR detection method were used to screen the T cells carrying the target gene. 1 Subsequent verification of individual plants.

[0088] (2) Southern hybridization detection of cry1C* gene integration:

[0089] 1) Using the CTAB method to extract fresh le...

Embodiment 3

[0116] Field resistance and phenotype analysis of new materials carrying cry1C*, the specific steps are as follows:

[0117] (1) Field resistance detection

[0118] In 2011, the T3 generation transgenic materials ZmKc-2-3, ZmKc-3-2, ZmKc-3-5 strains and ZmKc-2-3 backcrossed materials in the background of Zheng 58 were planted in the field, and the inbred line Zheng 58 was used as the control , without using pesticides during the whole growth period for field resistance identification, and to evaluate the damage of Asian corn borer under natural conditions. On the 15th day after the insect infestation appeared, the proportion of damaged leaves was counted. 43.71% of the leaves of the control Zheng 58 were severely damaged, with an average of 5.75 leaves damaged, while the damage to a single transgenic plant did not exceed 0.02. After growing to the stage of milk maturity, the proportion of dead tassels caused by the borer of the stem was counted. The proportion of the control ...

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Abstract

The present invention discloses a construction method for anti-insect transgene corn with stable heredity, wherein embryonic corn callus is adopted as a transformation acceptor material. According to the present invention, a gene gun-mediated genetic transformation method is adopted, a transformation vector pCAMBIA3300 carrying exogenous gene is wrapped with gold powder, and then is adopted to bombard the corn embryonic callus to complete transformation, tissue culture, screening, differentiation, seedling exercising and transplantation are sequentially performed to obtain the new anti-insect transgene corn material with stable heredity, and excellent performances of the resistance gene in different genetic backgrounds are verified through the backcross and molecular marker-assisted selection method so as to provide excellent germplasm resources for subsequent breeding of new anti-stem borer corn varieties.

Description

technical field [0001] The invention belongs to the technical field of corn biotechnology breeding, and specifically relates to a method for constructing insect-resistant transgenic corn that can be stably inherited. The insect-resistant gene is transferred through the transformation technology mediated by a gene gun, and a series of homozygous genes are performed on the transgenic offspring. Identification, expression verification, agronomic trait screening, resistance identification, genetic stability research; and the introduction of insect resistance genes into breeding materials through backcrossing and molecular marker-assisted selection methods to obtain excellent corn germplasm materials with high resistance to corn borer. Background technique [0002] Corn (Zea mays L.), scientific name maize, is an important food crop, as well as an important feed and industrial raw material, and plays a vital role in national livelihood and economic construction. There are about 3...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00
Inventor 邱法展张祖新杜邓襄张小波张方东郑用琏
Owner HUAZHONG AGRI UNIV
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