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BnSPL14 gene and protein, and application thereof in controlling strain types of Brassica napus L.

A technology of Brassica napus and plant type, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of increased branch number and long cycle of Brassica napus

Active Publication Date: 2019-09-06
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Rapeseed with increased yield can be effectively obtained using traditional breeding methods, but the cycle is longer
Using transgenic technology to control the expression of endogenous genes in Brassica napus to obtain Brassica napus with significantly increased branch number has not been reported yet

Method used

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  • BnSPL14 gene and protein, and application thereof in controlling strain types of Brassica napus L.
  • BnSPL14 gene and protein, and application thereof in controlling strain types of Brassica napus L.
  • BnSPL14 gene and protein, and application thereof in controlling strain types of Brassica napus L.

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: qPCR analysis of Brassica napus endogenous BnSPL14 Gene Expression in Brassica napus-White Mustard Introgression Progeny Multi-branched Materials

[0034]With the help of electrofusion technology, somatic cells of Brassica napus "Yangyou 6" (Y6) were fused with white mustard to obtain hybrid offspring, and then the hybrid offspring were continuously backcrossed with the parent Y6 for multiple generations, and the materials obtained by backcrossing were self-crossed The Brassica napus-White mustard introgression material was obtained. After multiple generations of backcrossing, the genetic background of the introgression material was very similar to that of the parent Y6, and the phenotype of the introgression material could be inherited stably. Among them, W5 and Compared with the recurrent parent Y6, the number of branches in W15 material increased significantly. The samples of Y6, W5 and W15 were taken, quickly placed in liquid nitrogen, and the total RNA ...

Embodiment 2

[0035] Embodiment 2: Brassica napus BnSPL14 molecular cloning of genes

[0036] Brassica napus “Darmor-bzh” seedlings at the three-leaf and one-heart stage were taken, quick-frozen in liquid nitrogen, and stored in a -70°C refrigerator for extraction of total RNA. Total RNA was extracted using the RNAiso Plus kit from Dalian TaKaRa Company. Brassica napus cDNA was synthesized in accordance with the instructions of HiScript 1st Strand cDNA Synthesis Kit of Nanjing Novizan Biotechnology Co., Ltd. for first-strand synthesis.

[0037] The first strand of cDNA synthesized by the above kit was used as the amplification template, and the designed SPL14 F: 5'-GGACGGAGACGAACAAGG-3' (SEQ ID NO: 3) and SPL14 R: 5'-GTCGCAGACTCACAGTAAAGC-3' (SEQ ID NO: 4) as primers, use RT-PCR for cDNA amplification, the amplification conditions are: 94°C for 3 min, 94°C for 15 s, 59°C for 15 s, 72°C for 3 min, a total of 35 cycles; 72°C for 10 min. After PCR, electrophoresis analysis was performed, an...

Embodiment 3

[0038] Example 3: BnSPL14 Construction of gene overexpression vector

[0039] for better analysis BnSPL14 The function of the gene, the applicant overexpressed it in Brassica napus, and studied the function of the gene by observing the phenotype of the transgenic plants.

[0040] The method for constructing the overexpression vector is as follows: the above-mentioned sequence-verified BnSPL14 gene cloning vector plasmid BnSPL14 -T as template, use primer SPL14GF: 5'-ggACTAGTccGGACGGAGACGAACAAGG-3' (SEQ ID NO: 5), sequence-specific primer plus linker Speech I site) and SPL14 GR: 5'-aGGCGCGCCtGTCGCAGACTCACAGTAAAGC-3' (SEQ ID NO:6), sequence-specific primer plus linker Asc Site I) cDNA amplification was carried out by RT-PCR. The amplification conditions were: 94°C for 3 min, 94°C for 15 s, 58°C for 15 s, 72°C for 3 min, a total of 35 cycles; 72°C for 10 min. After PCR, electrophoresis analysis was performed, and the target amplified fragment was recovered using the DNA re...

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Abstract

The invention relates to a BnSPL14 gene and protein, and an application thereof in controlling strain types of Brassica napus L. A DNA fragment including the BnSPL14 gene is separated and applied, andthe fragment gives a function which can increase the number of branches to the Brassica napus L.; and the nucleotide sequence including BnSPL14 gene coding regions is shown as a sequence table SEQ IDNO : 1, the length of the sequence is 3,099 bp, the nucleotide sequence of the protein which coded by the gene is shown as SEQ ID NO : 2, and the number of amino acids is 1,032. The BnSPL14 gene canbe separated from the Brassica napus L.; the biological function of the gene on improving the strain types of the Brassica napus L. can be identified; and therefore, the gene has very important meanings for cultivating new high-yield Brassica napus L. species.

Description

technical field [0001] The invention relates to the field of genetic engineering of Brassica napus. Specifically related to the isolation, cloning and functional verification of a Brassica napus capable of increasing the number of branches BnSPL14 Application of genes in genetic improvement of plant type traits in Brassica napus. The present invention utilizes the method of RT-PCR to isolate the gene controlling the number of branches of Brassica napus BnSPL14 , overexpressed BnSPL14 The gene can increase the branch number of Brassica napus, confirming the function of the gene and its application. Background technique [0002] As an important oil crop in the world, rapeseed is one of the main sources of edible vegetable oil, and can also be used as animal feed and industrial raw materials. Brassica napus ( Brassica napus L.) is currently the main rape cultivar in my country. In recent years, the planting area of ​​domestic rapeseed has decreased, but people's demand ...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N5/10A01H5/08A01H6/20A01H6/46A01H6/82A01H6/54
CPCC07K14/415C12N15/8261
Inventor 方玉洁陆伟王幼平耿玉璐张盼赵璇陈巍秦语悦
Owner YANGZHOU UNIV
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