Herbicide-resistant corn HH2823 transformation event capable of efficiently utilizing nutrients and specific identification method and application of herbicide-resistant corn HH2823 transformation event

A transformation event, herbicide-tolerant technology, applied in chemical instruments and methods, biochemical equipment and methods, applications, etc., to achieve the effect of improving agronomic traits

Active Publication Date: 2021-01-29
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Can improve the efficiency of potassium uptake in maize plants

Method used

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  • Herbicide-resistant corn HH2823 transformation event capable of efficiently utilizing nutrients and specific identification method and application of herbicide-resistant corn HH2823 transformation event
  • Herbicide-resistant corn HH2823 transformation event capable of efficiently utilizing nutrients and specific identification method and application of herbicide-resistant corn HH2823 transformation event
  • Herbicide-resistant corn HH2823 transformation event capable of efficiently utilizing nutrients and specific identification method and application of herbicide-resistant corn HH2823 transformation event

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Transgenic maize event and the acquisition of HH2823

[0051] 1. Construction of plant expression vectors for maize transformation

[0052] The plant expression vector used in this study is pCAMBIA3300-Ubi-AlHAK1-Bar (preserved in Dalian University of Technology), see figure 1 , wherein the nucleotide sequence of the T-DNA gene is shown in SEQ ID NO.5. This sequence contains the complete potassium efficient utilization expression cassette and the glufosinate-resistant expression cassette, which specifically consists of the following parts:

[0053] Potassium efficient gene expression frame: composed of maize ZmUbi promoter (nucleotide sequence shown in SEQ ID NO.6), potassium transporter gene AlHAK1 and nos terminator (nucleotide sequence shown in SEQ ID NO.7) . Herbicide-resistant glufosinate-ammonium expression frame: composed of CaMV35S promoter (nucleotide sequence is shown in SEQ ID NO.9), herbicide-resistant glufosinate-ammonium gene bar and CaMVpolyA...

Embodiment 2

[0086] Example 2: Semi-quantitative RT-PCR detection of transgenic corn HH2823

[0087] 1. Extraction of total RNA from maize leaves (RNAiso Plus kit)

[0088] ①Put a certain amount of fresh plant tissue material in liquid nitrogen, grind it into powder in liquid nitrogen, then quickly put it into an ice-precooled centrifuge tube containing 1mL Trizol reagent, shake and mix well, and let it stand at room temperature for 10 minutes to Thoroughly lyse plant tissue.

[0089] ② Add 200 μL of chloroform, slowly invert and mix well, let stand at room temperature for 5 minutes, and then centrifuge at 12000 rpm for 15 minutes.

[0090] ③ Pipette 500 μL of the supernatant into a new centrifuge tube, add the same volume of isopropanol, mix evenly by inversion, let stand at room temperature for 10 minutes, and centrifuge at 12,000 rpm at 4°C for 15 minutes.

[0091] ④ Discard the supernatant, add 1 mL of pre-cooled 75% ethanol solution, shake and wash the RNA pellet, and centrifuge at ...

Embodiment 3

[0115] Example 3: Southern Blot of transgenic maize event HH2823

[0116] 1. Using the CTAB method to extract the genomic DNA of the transgenic maize, amplify the genomic DNA with the internal primers of the AlHAK1 gene / bar gene, and make the PCR product obtained by the amplification into the AlHAK1 probe / bar probe;

[0117] 2. Digest the DNA of positive plants with restriction endonucleases SacⅠ, XbaⅠ and EcoRI respectively, and the digested DNA is precipitated with ethanol and washed with ddH 2 O dissolved, 30V voltage electrophoresis overnight;

[0118] 3. The agarose gel was treated with denaturing solution for 45 minutes and neutralizing solution for 60 minutes, and then transferred to membrane overnight by upward capillary transfer method. Immobilize the DNA on the transferred membrane at 121°C for 30 min;

[0119] 4. Immediately place the probe in the ice-water mixture after denaturing in boiling water for 5 minutes;

[0120] 5. After pre-hybridization at 42°C for 30...

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Abstract

The invention belongs to the technical field of biology, and relates to a herbicide-resistant corn HH2823 corn transformation event for efficiently utilizing nutrients of AlHAK1 and bar genes and a specific identification method and application of the herbicide-resistant corn HH2823 corn transformation event. The transformation event takes a nucleotide sequence shown in SEQ ID NO.1 as a left wingsequence of an exogenous gene and takes a nucleotide sequence shown in SEQ ID NO.2 as a right wing sequence of the exogenous gene. According to the corn transformation event provided by the invention,exogenous gene specificity can be introduced into a corn strain, and the receptor is endowed with efficient utilization of corn potassium nutrition and herbicide glufosinate-ammonium resistance. Theexogenous gene can be stably inherited in the receptor corn; expression of the exogenous gene can improve the potassium absorption and utilization efficiency of the receptor corn. According to the detection method, molecular marker detection can be carried out by utilizing a transformation event, and the breeding work efficiency is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a transformation event of herbicide-tolerant corn HH2823 corn with high nutrient utilization for transgenic AlHAK1 gene and bar gene and its specific identification method and application. Background technique [0002] Corn is an important food crop and feed crop in my country, and it is also the crop with the highest total output in the world. Its planting area and total output are second only to rice and wheat. The level of corn yield and the quality of economic benefits directly affect my country's food security and the development of agricultural production. [0003] Potassium ion is a large amount of elements necessary to maintain the normal physiological and metabolic activities of plants. It participates in the growth and development of plants, photosynthesis, dynamic balance of enzymes, maintenance of intracellular osmotic pressure, protein synthesis, cell extension, regulation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/54C12N15/82C12N5/10C12N1/21C12Q1/6895A01H1/02A01H5/00A01H6/46
CPCC07K14/415C12N9/1029C12N15/8277C12N15/8271C12Q1/6895A01H1/02
Inventor 苏乔王梦姣徐永盛
Owner DALIAN UNIV OF TECH
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