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Nucleic acid molecules from maize event ca09328 and methods for their detection

A technology of CA09328 and nucleic acid molecules, applied in the fields of plant transformation, plant breeding, and plant molecular biology, can solve the problems of lack of evaluation of the impact of corn insect resistance and achieve good results

Active Publication Date: 2022-04-01
广州哈维种业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this patent does not study the impact of the different positions of the Bt insect-resistant gene Cry1Ab-t on the chromosome on the expression level. Assessment of Capacity Impact

Method used

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  • Nucleic acid molecules from maize event ca09328 and methods for their detection
  • Nucleic acid molecules from maize event ca09328 and methods for their detection
  • Nucleic acid molecules from maize event ca09328 and methods for their detection

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1 pCAMBIA3300+35S-CryAb-t expression vector construction

[0055] Using standard gene cloning techniques to construct recombinant expression vectors (such as figure 1 shown). Specific steps are as follows:

[0056] Step 1: Design XbaI and SacI restriction sites (add XbaI restriction site at the 5' end and SacI restriction site at the 3' end) of the artificially modified insect-resistant gene Cry1Ab-t gene (by Sangon Bioengineering Co., Ltd. (Shanghai) company synthesis), wherein the insect-resistant gene adopts the artificially synthesized Bt insect-resistant gene Cry1Ab-t disclosed in the inventor's patent ZL201010552618.0, and its nucleotide sequence is as SEQ described in the patent ZL201010552618.0 As shown in ID NO:1, this patent document is deemed to be fully incorporated herein by reference;

[0057] Step 2: Digest the synthesized Cry1Ab-t gene with XbaI and SacI, then recover the vector fragment, and connect it into the high-efficiency plant expres...

Embodiment 2

[0059] Embodiment 2 adopts Agrobacterium infection method to carry out the transformation of corn plant

[0060] The insertion sequence was introduced into the immature embryos of the recipient plants by Agrobacterium infection method, and the transgenic plants were obtained after screening by the herbicide bialaphos. The specific method is as follows:

[0061]Step 1: Peel off the young embryos of corn: remove the bracts. Cut off about 1 cm from the top of the ear, insert the ear from the top with tweezers, so that the tweezers can be used as a handle, which is convenient for operation, and then put the ear into a beaker containing disinfectant. According to actual needs, you can put 4 in the same beaker. - 6 ears; add about 700mL of disinfectant solution (50% bleach or 5.25% sodium hypochlorite, and add a drop of Tween 20) to the beaker to soak the ears. During the 20 minutes of disinfection, rotate the ears from time to time while gently Pat the beaker to get rid of the ai...

Embodiment 3

[0067] Identification and screening of embodiment 3 transgenic events

[0068] A total of 256 independent transformation events were obtained. The expression levels of each transformant were detected in the greenhouse, and 8 transformants with high expression of Cry1Ab-t insecticidal protein in leaves, stems, filaments, and fruit ears were screened, and these transformants were passed back in the same season. Crossing and selfing were quickly transferred to the parents of Zhengdan 958, Zheng 58 and Chang 7-2, and the genetic stability of their offspring was tested, including the DNA level and protein level of the three generations. According to the copy number of the gene of interest, good insect resistance, bialaphos herbicide tolerance, and agronomic performance, the final selected event CA09328 was excellent, with a single-copy transgene, good genetic stability, high resistance Asian corn borer, bialaphos herbicide tolerance, no effect on non-target organisms, and good agr...

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Abstract

The present invention relates to nucleic acid molecules from corn event CA09328 and detection methods thereof, primer sets, probes and kits for detecting the nucleic acid molecules, and corn products produced from corn event CA09328. The corn event CA09328 of the present invention can stably express the target gene with a strong expression level, has excellent anti-corn borer effect in the whole growth period, and has good safety.

Description

[0001] Cross References to Related Applications [0002] This application claims the priority of the patent application with the application number CN202011021485.4 and the invention title "Nucleic acid molecule from corn event CA09328 and its detection method" filed on September 24, 2020, the entire content of which is incorporated herein by reference . technical field [0003] The present invention relates generally to the fields of plant molecular biology, plant transformation, and plant breeding, and in particular to nucleic acid molecules from maize event CA09328 and detection methods thereof. Background technique [0004] Maize (Latin name: Zea mays L.) is a major food crop in many parts of the world. There have been related studies on improving the agronomic traits and quality of maize through biotechnology. Among them, how to generate insect resistance in corn, especially resistance to corn borer, cotton bollworm, armyworm, etc., without causing harmful effects on ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/158C12Q2600/13
Inventor 铁健雄
Owner 广州哈维种业有限公司
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