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Brassica napus SKIP gene family as well as recombinant vector and application thereof

A technology of Brassica napus and gene family, applied in application, genetic engineering, plant gene improvement, etc., can solve problems such as the number of SKIP gene members in Brassica napus, the evolution relationship of protein characteristics and tissue specificity that have not been reported, and reach the application prospect Good results

Inactive Publication Date: 2015-08-19
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the number of members, protein characteristics, evolutionary relationship, tissue specificity of expression, relationship with drought resistance, and application in genetic engineering of Brassica napus SKIP gene have not been reported so far.

Method used

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  • Brassica napus SKIP gene family as well as recombinant vector and application thereof
  • Brassica napus SKIP gene family as well as recombinant vector and application thereof
  • Brassica napus SKIP gene family as well as recombinant vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the cloning of Brassica napus SKIP gene family

[0038] (1) Extraction of Brassica napus genome total DNA and total RNA

[0039] Take the young leaves of typical plants for each strain, extract the total genomic DNA by cetyltrimethylammonium bromide (CTAB) method, and evaluate the quality and concentration of nucleic acid samples by 1.0% agarose gel electrophoresis and spectrophotometry . At the same time, the root, hypocotyl, cotyledon, stem, true leaf, flower, bud, pod skin, and four-stage seeds (15D, 30D, 45D and 55D) of the typical black seed line of Brassica napus were used as materials. Total RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), extract total RNA according to the kit instructions, and remove DNA impurities contained in total RNA. The quality of total RNA was detected by electrophoresis, and the concentration and purity of total RNA were determined by ultraviolet spectrophotometer. The results of electrophoresis ...

Embodiment 2

[0054] Embodiment 2, the bioinformatics analysis of BnSKIP gene family

[0055] Sequence alignment, open reading frame (ORF) search and translation were performed on Geneious Pro 4.85, in NCBI (http: / / www.ncbi.nlm.nih.gov) and BRAD database (http: / / brassicadb.org / brad / index.php) for BLAST and CDD searches of protein sequences, and for protein structure analysis on bioinformatics sites linked by sites such as www.expasy.org, at http: / / prodes.toulouse.inra.fr / Multiple alignment and cluster analysis of gene and protein sequences are performed on websites such as multalin / multalin.html and http: / / www.ebi.ac.uk / clustalw / . The BnSKIP protein and other biological SKIP protein sequences were compared with ClustalW 2.0, and then MEGA 5.2 software was used to construct a phylogenetic tree using the neighbor-joining (NJ) method, and 1000 bootstrap replicates (Bootstrap replicates) were used to test the confidence of each branch Spend.

[0056] (1) Analysis of the nucleic acid level o...

Embodiment 3

[0065] Example 3, Tissue-organ-specific expression and induced expression detection of BnSKIP gene family

[0066] According to the gene sequences of BnSKIP-1 and BnSKIP-2, fluorescent quantitative primers for detecting the overall BnSKIP family and two BnSKIP gene members in different tissues and organs were designed, and UBC21 was used as an internal reference. The specific primers are shown in Table 2.

[0067] Table 2. Fluorescent quantitative primers for the overall BnSKIP family and two BnSKIP gene members in different tissues and organs

[0068]

[0069] Then, the overall expression of the BnSKIP gene family in 12 different tissues and organs of Brassica napus and the specific expression of two BnSKIP gene members were detected by fluorescent quantitative RT-PCR. The results are as follows Figure 5 shown. The results showed that BnSKIP gene and its family members were expressed in the above tissues and organs, and had similar tissue specificity. After qRT-PCR dete...

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Abstract

The invention relates to a brassica napus SKIP (BnSKIP) gene family as well as a recombinant vector and application thereof. The BnSKIP gene family comprises a BnSKIP-1 gene and a BnSKIP-2 gene, wherein the full-length cDNA (complementary Deoxyribose Nucleic Acid) sequence of the BnSKIP-1 gene is as shown in a SEQ ID No.14, and the genome DNA is as shown in a SEQ ID No.12; the full-length cDNA sequence of the BnSKIP-2 gene is as shown in a SEQ ID No.15, and the genome DNA is as shown in a SEQ ID No.13; the BnSKIP-1 gene and the BnSKIP-2 gene construct the over-expression vector; after oil 821 in the brassica napus variety is converted, a transgenic plant is obtained. Compared with a non-transgenic plant, the transgenic plant has the advantages that the resistance of the transgenic plant under the condition of drought stress is remarkably improved, and important gene resources are provided for the improvement of resistance of the brassica napus through a genetic engineering means.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the brassica napus SKIP (BnSKIP) gene family, and also relates to a recombinant vector containing the BnSKIP gene family and the application of the BnSKIP gene family. Background technique [0002] Drought has an extremely serious impact on agricultural production, and it is the most important natural adversity factor causing crop yield reduction in the world, and its damage is equivalent to the sum of other natural disasters. my country is a country with poor water resources, and its per capita water resources are less than 1 / 4 of the world's average level. It is one of the 13 countries with the poorest per capita water resources in the world. In the past 60 years, the average annual drought-affected area in my country was 21.57 million hectares, and the disaster-affected area was 9.56 million hectares, accounting for 55% of the total meteorological disaster-affected are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00
Inventor 卢坤李加纳张凯曲存民梁颖唐章林
Owner SOUTHWEST UNIV