Application of a soybean E3 ubiquitin ligase family gene gmrnf1a

A technology of soybeans and coding genes, applied in the direction of ligase, application, genetic engineering, etc., can solve the problems of incomplete effects, etc., and achieve the effect of increasing thousand-grain weight, increasing yield, and reducing the phenomenon of plant pod cracking

Active Publication Date: 2021-11-23
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are a large number of E3 ubiquitin ligases, different E3 ubiquitin ligases play different biological functions. Therefore, the function of the new E3 ubiquitin ligase gene still needs further experimental verification

Method used

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  • Application of a soybean E3 ubiquitin ligase family gene gmrnf1a
  • Application of a soybean E3 ubiquitin ligase family gene gmrnf1a
  • Application of a soybean E3 ubiquitin ligase family gene gmrnf1a

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Cloning and identification of soybean GmRNF1a and its coding gene

[0030] Primers were designed according to the sequence information of GmRNF1a predicted by the phytozome website, and the flower cDNA of Willmas82 in full flowering stage was used as a template for PCR amplification.

[0031]Upstream primer GmRNF1a-F: ggatcttccagagatGCTTCTTCACTTCTTCCATTCTCC; (SEQ ID NO.3)

[0032] Downstream primer GmRNF1a-R: ctgccgttcgacgatCCTTATTGTAAACGTCGTTATCAGC. (SEQ ID NO.4)

[0033] The GmRNF1a gene was amplified from the total RNA of soybean floral organs by RT-PCR. Take the soybean flower tissue, grind it with a mortar, add it into a 1.5mL EP tube filled with lysate, shake it fully, and then transfer it into a glass homogenizer. After homogenization, transfer to 1.5mLEP tube, and use plant total RNA extraction kit (TIANGEN DP404) for total RNA extraction. The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA conte...

Embodiment 2

[0034] Example 2 Expression characteristics of GmRNF1a in different organs of soybean

[0035] Extract RNA from roots, stems, leaves, flowers, 7d pods, 15d pods, 25d pods, 35d pods, 45d pods and seeds of Willmas82, reverse to cDNA for RT-PCR analysis.

[0036] The extraction of total RNA was the same as in Example 1. The soybean constitutively expressed gene Tubulin was used as an internal reference gene, and the amplification primers were Tubulin forward primer sequence: GGAGTTCACAGAGGCAGAG (SEQ ID NO.5), and Tubulin reverse primer sequence: CACTTACGCATCACATAGCA (SEQ ID NO.6). Using cDNA from different soybean tissues or organs as templates, real-time fluorescent quantitative PCR analysis was carried out. The amplification primers of GmRNF1a are: GmRNF1a-qPCR-F: CCGTGGATAGAACTCAACTCG (SEQ ID NO.7), GmRNF1a-qPCR-R: GTCCTGAGCCCGTAGAAATC (SEQ ID NO.8). result( figure 2 ) analysis showed that the expression of GmRNF1a in flowers and pods was relatively high, especially in the...

Embodiment 3

[0037] Example 3 Subcellular localization of GmRNF1a

[0038] Subcellular localization adopts the method of transient expression of Arabidopsis protoplasts, the vector used is pAN580, and the primers are GmRNF1a-AN-F: aagtccggagctagctctagATGGCGGCGACGGCGACG (SEQ ID NO.9), GmRNF1a-AN-R: gcccttgctcaccatggatccGCAAGCCGAATCGCCGCCA (SEQ ID NO.10 ). PCR amplification, after the target band is correct, it is recovered by tapping the rubber, and the recovered product of the rubber is connected to the carrier by homologous recombination to construct the subcellular localization vector pAN580-GmRNF1a (the gene is at the N-terminal of GFP), and Arabidopsis protoplasts are transiently After expression, it was cultured in the dark for 16 hours, and after laser irradiation by a laser confocal microscope (Zeiss, LSM780), a green fluorescent signal could be generated, the protein was located, observed and photographed. The result is as image 3 As shown, the transformed empty plasmid is distr...

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Abstract

The invention discloses the application of a soybean E3 ubiquitin ligase family gene GmRNF1a. Soybean GmRNF1a protein coding gene GmRNF1a, its nucleotide sequence is: SEQ ID NO.1. The constructed plant overexpression vector pMDC83‑GmRNF1a was heterologously expressed in the wild type of Arabidopsis, and it was found that the maturation process of the transgenic plants was significantly accelerated, and the fruit pods burst earlier. It shows that the gene can be introduced into plants as a target gene, and by inhibiting the expression of GmRNF1a gene, it can inhibit the early bursting of fruit pods of transgenic plants. It can be seen that the soybean GmRNF1a protein coding gene GmRNF1a of the present invention can be used in promoting early maturity of plants through genetic engineering and regulating the bursting time of plant pods.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering and relates to the application of a soybean E3 ubiquitin ligase family gene GmRNF1a. Background technique [0002] Fruit development and maturity are important links in the growth and development of higher plants. They are closely related to crop yield and agricultural product quality, which in turn affects economic benefits. Timely pod cracking is conducive to seed collection, and premature cracking or non-cracking will affect the quality of agricultural products. reward. When soybeans are harvested, pod dehiscence is an important factor affecting soybean yield. Wang Tingting (2016) in our laboratory screened the soybean pod cDNA library through GmAGL1 and found some proteins that can interact with GmAGL1. One of the interacting proteins, Glyma15g42250.1 (gene number Glyma.15G268100.1 in V2.0 version), belongs to the E3 ubiquitin ligase family and contains a RING-finger domain, which ma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/82A01H5/00A01H5/10A01H6/20
CPCC12N9/93C12N15/8266C12Y603/02019
Inventor 黄方王慧崔艳梅阚贵珍喻德跃
Owner NANJING AGRICULTURAL UNIVERSITY
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