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Molecular marker co-segregated from burley tobacco control gene and application thereof

A technology of burley tobacco and genes, applied in the biological field, can solve problems such as unfavorable plant growth and development, achieve the effect of deepening understanding and speeding up the process

Active Publication Date: 2018-10-26
TOBACCO RES INST CHIN AGRI SCI ACAD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past, many leaf color mutants were considered unfavorable to plant growth and development and useless in agricultural production

Method used

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  • Molecular marker co-segregated from burley tobacco control gene and application thereof
  • Molecular marker co-segregated from burley tobacco control gene and application thereof
  • Molecular marker co-segregated from burley tobacco control gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1, the positioning and map-based cloning of genes ws1a and ws1b

[0057] 1. Initial mapping of genes ws1a and ws1b

[0058] 1. A chlorophyll-deficient white stem mutant white stem1 (ws1) was screened from the common tobacco variety Zhongyan 100 (ZY) induced by EMS. The method of allele test (see the following literature for the specific method: Mapping of twowhite stem genes in tetraploid common tobacco (Nicotiana tabacco L.)) was used for genetic analysis, and the specific steps were as follows: the white stem mutant ws1 was crossed with the Burley tobacco variety, The F1 generation is obtained, and the performance of the F1 generation is the same as that of the parents; then the F1 generation is self-crossed to obtain self-crossed offspring, and the traits of the self-crossed offspring do not segregate. Genetic analysis showed that the white stem trait was controlled by two recessive nuclear genes ws1a and ws1b, and was allelic with the white stem gene of Bu...

Embodiment 2

[0069] Embodiment 2, application of metalloprotease WS1A or WS1B in regulating plant chloroplast metabolism

[0070] 1. Obtaining WS1A Tobacco and WS1B Tobacco

[0071] 1. Construction of recombinant vector

[0072] (1) Cloning of genes WS1A and WS1B

[0073] (1-1) RNA was extracted from leaves of Dajinyuan safflower, and cDNA was reverse transcribed. RNA extraction and reverse transcription were completed using MiniBEST plant RNA Extraction Kit (Code No.9769) from TaKaRa Company and PrimerScript II 1st Strand cDNA synthesis Kit (Code No.6210) from TaKaRa Company, respectively. The cDNA was used as a template and primers CW-1F / CW-1R were used to amplify to obtain PCR products.

[0074] (1-2) WS1A and WS1B were identified respectively after PCR products were cloned by TA (Mighty TA-cloning Kit (TaKaRa Company, Code No.6028)) and sequenced. The WS1A gene sequence is shown in sequence 3, and the WS1B gene sequence is shown in sequence 4.

[0075] (2) Construction of expressi...

Embodiment 3

[0105] Example 3 Molecular markers co-segregated with Burley tobacco control genes

[0106] 1. Primer design for WS1A, ws1a, WS1B and ws1b genotype identification

[0107] 1. Download the full gene sequence of WS1A and WS1B from the Honghua Dajinyuan Genome Database, and use the online analysis tool MUSCLE (https: / / www.ebi.ac.uk / Tools / msa / muscle / ) for sequence comparison to obtain WS1A For the sequence difference between WS1B and WS1B, intercept the genome sequence of 200-400bp upstream and downstream of ws1a and ws1b mutation sites for future use;

[0108] 2. Since the WS1A and WS1B sequences are highly homologous, use the sequence differences between the two to design specific PCR primers containing gene mutation sites:

[0109] (1) For WS1A and ws1a, a pair of specific primers 1325-2F / 1325-1R (namely M-a) was designed based on the 8-base length difference between the two, and the PCR products were separated by polyacrylamide gel electrophoresis. Those that are similar to ...

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Abstract

The invention discloses a molecular marker co-segregated from a burley tobacco control gene and application thereof. A white stem mutant with chlorophyll metabolism defects is identified from EMS induced common tobaccos; research shows that two invisible control genes in the mutant and a white stem gene of burley tobaccos are allelic; then the two genes are subjected to map-based cloning; the specific molecular markers of the two genes are developed on the basis. An experiment proves that a method and the molecular marker, disclosed by the invention, can be used for simply, rapidly and accurately detecting gene types of two genes, and recognition on the burley tobaccos is easily deepened on the molecular level, so that a tobacco breeding progress is accelerated.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a molecular marker co-separated with a Burley tobacco control gene and an application thereof. Background technique [0002] Chlorophyll is an important pigment involved in photosynthesis in the chlorophyll of green plants, and plays a key role in the energy capture and energy transfer of photosynthesis. After the normal metabolism of chlorophyll is destroyed, plant leaves often appear abnormal phenotypes such as albino, yellow, light green, dark green (Kurata et al.2005). In the past, many leaf color mutants were considered unfavorable to plant growth and development and useless in agricultural production. However, with the advancement of science and technology and the in-depth excavation of related research, some mutants have gradually shown great application potential. For example, the bud-yellow mutant of cotton and the white-to-green mutant of rice can be used as e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686
CPCC12Q1/686C12Q1/6895
Inventor 吴新儒刘贯山张兴伟龚达平薄韶云
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
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