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Corn transformation event 'double resistance 12-5' and specificity identification method thereof

A transformation event and specific technology, applied in chemical instruments and methods, biochemical equipment and methods, and botanical equipment and methods, etc., can solve problems such as affecting the expression of endogenous genes, and achieve the effect of stable inheritance

Inactive Publication Date: 2015-09-30
HANGZHOU RUIFENG BIOTECH LIMITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the insertion of exogenous genes may also affect the expression of endogenous genes

Method used

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  • Corn transformation event 'double resistance 12-5' and specificity identification method thereof
  • Corn transformation event 'double resistance 12-5' and specificity identification method thereof
  • Corn transformation event 'double resistance 12-5' and specificity identification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Acquisition of Conversion Events

[0044] (1) Obtaining the plasmid vector containing the foreign gene

[0045] The vector map of the present invention for corn transformation is as follows figure 1 As shown, the nucleotide sequence of the transforming plasmid vector is SEQ ID NO. 13, and the names and positions of specific vector components are shown in Table 1. The nucleotide sequence of the T-DNA gene is shown in SEQ ID NO:2. SEQ ID NO: 2 contains a complete glyphosate-resistant expression frame and an insect-resistant expression frame, and is specifically composed of the following parts, a glyphosate-resistant expression frame: a complex formed by a 35S promoter derived from CaMV and a maize Polyubiqutin-1 promoter Promoter (the nucleotide sequence is shown in SEQ ID NO. 9), the composite promoter drives a glyphosate-resistant gene G10evo (EPSPS) that encodes the chloroplast signal peptide of the AHAS gene at the 5' end (the nucleotide sequence is SEQ ...

Embodiment 2

[0058] Example 2: Screening of transformation events

[0059] 240 independent transformants of insect-resistant and glyphosate-resistant maize were obtained by Agrobacterium (Example 1). Primers were designed according to the vector sequence (SEQ ID NO. 13, and the PCR method was used to screen the genes containing glyphosate resistance gene G10eve (nucleotide sequence shown in SEQ ID NO. 5) and insect resistance gene cry1Ab-cry2Aj (nucleotide sequence shown in SEQ ID NO. 5) SEQ ID NO. 4) and maize transformants without the vector backbone sequence at the same time, and the preliminary test of insect resistance and glyphosate resistance was carried out in the greenhouse: by spraying 0.4wt% glyphosate, glyphosate was removed For transformants with poor phosphine resistance, the remaining transformants were connected to corn borer, and the transformants without corn borer damage were screened. A total of 12 transformation events including "double antibody 12-1" ~ transformation ...

Embodiment 3

[0085] Example 3: Identification of sequences flanking foreign gene insertion sites

[0086] Use the TAIL-PCR (Thermal asymmetric interlaced PCR) method reported by Liu et al. (Liu, Plant Journal 1995, 8(3): 457-463) to double-antibody the regions flanking the insertion point of the exogenous transgene DNA of 12-5 for the excellent transformation event. sequence was determined. In this method, three nested specific primers are respectively combined with degenerate primers for continuous PCR amplification, and target fragments are selectively amplified by different annealing temperatures. The amplified DNA fragments were cloned, sequenced and compared with the maize online database (http: / / www.maizegdb.org).

[0087] T-DNA left flank

[0088] The fragment identified as containing the 3' flanking region was sequenced (SEQ ID NO. 1. The sequence between nucleotides 1-576 bp corresponds to maize genomic DNA and the sequence between nucleotides 577-826 bp corresponds to exogenous...

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Abstract

The invention discloses a corn transformation event 'double resistance 12-5' and a specificity identification method thereof. The corn transformation event 'double resistance 12-5' is characterized in that a nucleotide sequence shown in SEQ ID No.1 is taken as a left side wing region of a foreign gene and a nucleotide sequence shown in SEQ ID No.3 is taken as a right side wing region of the foreign gene. The transformation event '12-5' provided by the invention is good, the foreign gene can be specifically introduced into a corn line, and insect resistant and glyphosate resistant capability is given to a corn acceptor; an insect-resistant and glyphosate-resistant gene can be stably inherited in the corn acceptor; and expression of the insect-resistant and glyphosate-resistant gene can not produce adverse effects on agronomic characters of the corn acceptor.

Description

(1) Technical field [0001] The invention relates to a maize transformation event and a specific identification method thereof, in particular to a maize transformation event "double antibody 12-5" and a specific detection method thereof. (2) Background technology [0002] Maize (Zea mays) is an important crop and a major source of food in many parts of the world. With the development of plant genetic engineering, genetic modification by introducing foreign genes has become one of the main means of genetic breeding. Both insect resistance and herbicide resistance are highly desirable agronomic traits in maize production. Insect-resistant genetically modified corn can greatly reduce the use of chemical pesticides, thereby reducing production costs and reducing the pollution of pesticides to the environment and crop products. Herbicide-resistant corn can greatly reduce the agricultural labor required for weed control, reduce labor input, and reduce the impact of weeds on corn ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N5/10C12Q1/68C12R1/91
CPCA01H1/02C07K14/325C07K2319/00C12N9/1092C12N15/8275C12N15/8286C12Q1/6895C12Y205/01019C12N5/14C12N15/11C12N15/82C12Q1/68
Inventor 沈志成林朝阳张先文徐晓丽
Owner HANGZHOU RUIFENG BIOTECH LIMITED
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