Identification of enhanced gene expression sequence in cabbage type rape and application thereof

A technology in sequences and sequence listings, applied in the field of identification of enhanced gene expression sequences

Active Publication Date: 2019-01-01
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Variations in crop yield-related traits caused by sequence variation in regulatory regions have been observed in rice (Ishii et al., 2013; Huo et al., 2017), tomato (Frary et al., 2000), corn (Liu et al., 2015) and other crops have been reported in Brassica napus, but have not been reported in Brassica napus

Method used

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  • Identification of enhanced gene expression sequence in cabbage type rape and application thereof
  • Identification of enhanced gene expression sequence in cabbage type rape and application thereof
  • Identification of enhanced gene expression sequence in cabbage type rape and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Drawing of silique growth curve and expression level of qSLWA9 gene at different developmental stages

[0031] In order to visually compare the growth of siliques in the whole development period of Brassica napus, the applicant selected a Brassica napus variety Westar (a known and public variety) with common silique length (about 5-6cm) and thousand-grain weight and production The widely used silique and large-grain variety Zhongshuang 11 (ZS11) was used as a reference, and the growth dynamics of the siliques of S1, S2, ZS11, and Westar were investigated in the experimental field of Huazhong Agricultural University during the spring flowering stage of 2014, and the main inflorescence of the plants was marked. The flowers that bloom on the same day are recorded as 0dpa, and the lengths of at least 8 stigmas or siliques are measured for each material in each period, and the average length is taken as the length of the siliques in this period and the growth curve...

Embodiment 2

[0033] Example 2: Scanning electron microscope analysis of silique peel cells

[0034] When the siliques were no longer elongated, the siliques were cut into small pieces from the field, fixed with 2.5% glutaraldehyde (prepared with 0.1M PBS), and taken back to the laboratory, vacuumed for 30 minutes, and kept overnight at 4°C. Low alcohol dehydration at different levels, the alcohol concentration is 30%, 50%, 70% for 10 minutes each, 90%, 100% for 8 minutes each, after the dehydration is completed, pour off the alcohol, replace it with isoamyl acetate for 20 minutes, and then carry out the sample Critical point drying (CPD 020, Balzers Union), followed by sample gold spraying (Nanotech SEMPrepⅡsputtercoater), and then taking pictures with a JSM-6390 scanning electron microscope, and the operation of the fixed sample was carried out on the public electron microscope platform of Huazhong Agricultural University. By scanning electron microscope observation of S1 and S2 silique p...

Embodiment 3

[0035] Example 3: Sequence analysis of the 3.7-kb upstream regulatory region of the silique parent S1 gene qSLWA9 inserted

[0036] Comparative sequencing of the parents showed that a 3.7-kb DNA fragment was inserted 3.9-kb upstream of the qSLWA9 start codon (ATG) of S1, and the sequence was put into the NCBI database (https: / / www.ncbi.nlm.nih .gov / ) and RepeatMasker website (http: / / www.repeatmasker.org / ), and found that the sequence is an incomplete CACTA-like transposon sequence, which contains the right terminal repeat sequence (TIR) ​​(TGTTCCTTG TAG TG ), a transposase family (Transposase_24) gene and subterminal repeat (STR) (CGTAAAT) (see image 3 ).

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Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to identification of an enhanced gene expression sequence in a cabbage type rape and application thereof. A 3.7-kb DNA fragment in an upstream regulatory region of a qSLWA9 gene of the cabbage type rape is identified, the nucleotide sequence thereof is shown in SEQ ID NO: 1, belongs to incomplete CACTA-like (CACTA-like) transposons, has enhancer function, and can enhance gene expression levels. The combination of markers with the presence of enhancers is developed. Genotype analysis of 245 cabbage type rape materials is conducted, pod length and grain weight phenotype is combined, and the identification of the enhanced gene expression sequence in the cabbage type rape shows that the presenceor absence of the CACTA-like transposons is closely related to phenotype. Further validation shows that the CACTA-like transposons fragment contains enhancer elements, can enhance gene expression inall tissues. The CACTA-like transposons and markers of the invention can be used for improvement of yield-related traits in the cabbage type rape.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to the identification and application of an enhanced gene expression sequence in Brassica napus. Background technique [0002] A change in the coding sequence of a gene is the most important molecular basis for phenotypic variation. However, changes in the regulatory region sequence often affect changes in gene expression levels, which in turn affect phenotypic variation, and are also an important genetic basis for plant trait changes. Variations in crop yield-related traits caused by sequence variation in regulatory regions have been observed in rice (Ishii et al., 2013; Huo et al., 2017), tomato (Frary et al., 2000), corn (Liu et al., 2015) and other crops It has been reported in Brassica napus, but it has not been reported in Brassica napus. Therefore, the study of the variation in the sequence of the transcriptional regulatory region of genes affect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12Q1/6895
CPCC12Q1/6895C07K14/415C12Q2600/13C12Q2600/158
Inventor 刘克德王晶石柳柳
Owner HUAZHONG AGRI UNIV
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