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Corn Zm-APX gene molecular marker, obtaining method and application in stalk rot control

A corn stalk rot and molecular marker technology, applied in application, horticultural methods, genetic engineering and other directions, can solve the problems of not finding resistance genes, limiting the improvement of corn varieties, etc., and achieving the effect of increasing stalk rot resistance

Inactive Publication Date: 2019-04-19
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Specific genes are an effective means to identify plant resistance, but so far no resistance genes that can effectively identify maize stalk rot have been found, which limits the improvement of maize varieties

Method used

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  • Corn Zm-APX gene molecular marker, obtaining method and application in stalk rot control
  • Corn Zm-APX gene molecular marker, obtaining method and application in stalk rot control
  • Corn Zm-APX gene molecular marker, obtaining method and application in stalk rot control

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 A kind of cloning method of Zm-APX gene, concrete steps are as follows:

[0046] Step 1, using RNA extraction reagent TRIZOL Reagent to extract total RNA from leaves of maize inbred line B73, and obtain maize total RNA for future use. Detected by 1.5% agarose gel electrophoresis, the results are shown in Figure 1A, figure 1 Lanes 1 and 2 in A are the total RNA of corn, including 28S and 18S, which are the large and small subunits of the ribosome, and the bottom band is 5.8S and 5S (because the length difference is not large, 5.8S and 5S are difficult to distinguish in electrophoresis), indicating that the RNA integrity is good and can be used for follow-up experiments.

[0047] Step 2, extraction of maize genomic DNA

[0048] Maize genomic DNA was extracted with a plant genomic DNA extraction kit (Tiangen) for later use.

[0049] Step 3, cDNA first-strand synthesis and detection

[0050] (1) Synthesis of the first strand of cDNA

[0051] According to t...

Embodiment 2

[0071] Example 2 Preparation of the Zm-APX transgenic strain whose nucleotide sequence is shown in SEQ ID NO.10

[0072] Utilize the molecular marker Zm-APX gene whose nucleotide sequence is shown in SEQ ID NO.10 to construct an overexpression vector capable of stably expressing Zm-APX, and then transform the overexpression vector into Agrobacterium competent cells to obtain transformation Bacteria, and finally use the transformed bacteria to transform corn plants to obtain positive transgenic lines with resistance to corn stalk rot. Specific steps are as follows:

[0073] S1, preparation of Agrobacterium competent cells, Zm-APX gene solution

[0074] Take a single colony of Agrobacterium GV3101 (commercially available) in 3ml of YEB liquid medium, shake and culture overnight at 28°C; take 2ml of the overnight culture solution and inoculate it in 100ml of YEB liquid medium, shake at 28°C until the OD600 is 0.5-0.8 (about 3-4h); 4°C, centrifuge at 5000rpm / min for 10min, colle...

Embodiment 3

[0086] Real-time fluorescence quantitative PCR detection of embodiment 3Zm-APX transgenic strain

[0087] Get 243, 241 negative strains and 235, 236 positive strains obtained in Example 2, extract corn total RNA with reference to the method in Example 1 step 1, then synthesize 243, 241 negative strains and 241 negative strains and The cDNA first strands of 235 and 236 positive strains were then used for fluorescent quantitative PCR.

[0088] Fluorescent quantitative PCR target gene primers:

[0089] Zm-APX F7: 5'-CATCGCCGAGAAGAGCT-3', as shown in SEQ ID NO.15, Zm-APX R7 5'-GGGAACTCCTCCTTGAT-3', as shown in SEQ ID NO.16; PCR amplification fragment 183bp;

[0090] Fluorescence quantitative PCR internal reference gene 18S primer:

[0091] 18S R8: 5'-CCTGCGGCTTAATTGACTC-3', as shown in SEQ ID NO.17,

[0092] 18S F8: 5'-GTTAGCAGGCTGAGGTCTCG-3', as shown in SEQ ID NO.18; the PCR amplified fragment is 174bp.

[0093] The fluorescent quantitative PCR system is as follows: Premix T...

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Abstract

The invention belongs to the technical field of corn resistance genes, particularly relates to a corn Zm-APX gene molecular marker, an obtaining method and application in stalk rot control, and provides a molecular marker capable of identifying corn stalk rot and an obtaining method thereof. The molecular marker is a Zm-APX gene shown as SEQ ID NO.1 or SEQ ID NO.10. The invention also provides a method for constructing a transgenic corn plant by using the molecular marker and using the molecular marker for controlling corn stalk rot. By detecting the Zm-APX transgenic material and identifyingthe stalk rot resistance thereof, it is found that the Zm-APX gene can improve the resistance of corn to stalk rot, and a genetic resource is provided for corn stalk rot resisting breeding.

Description

technical field [0001] The invention belongs to the technical field of maize resistance genes, and in particular relates to a maize Zm-APX gene molecular marker, an obtaining method and an application in the prevention and treatment of stem rot. Background technique [0002] Maize is one of the most widely cultivated food crops in the world. On the one hand, maize provides a large food source for humans and animals, but on the other hand, the occurrence of maize diseases can cause huge economic losses. Especially in recent years, corn diseases have occurred more and more frequently, and the types of diseases have increased day by day, which has seriously affected the development of the corn industry. The use of chemical pesticides has improved the control of corn diseases to a certain extent, but the use of pesticides not only pollutes the environment, but also causes drug resistance in corn after long-term use. threat. Therefore, in the study of corn disease resistance, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/53C12N15/82C12N15/66A01H5/00A01H4/00A01H6/46
CPCA01H4/00C12N9/0065C12N15/66C12N15/8281C12N15/8282C12Q1/6895C12Q2600/13
Inventor 吴刘记王顺喜陈赞张雪海
Owner HENAN AGRICULTURAL UNIVERSITY
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