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Gene GmFtsH25 for encoding soybean FtsH metalloproteinase and application of gene GmFtsH25

A technology of metalloprotease and soybean, applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2021-12-07
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are very few research reports on this type of protein in soybean, so studying the FtsH gene in soybean is of great significance for analyzing its biological function in regulating soybean growth and development

Method used

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  • Gene GmFtsH25 for encoding soybean FtsH metalloproteinase and application of gene GmFtsH25
  • Gene GmFtsH25 for encoding soybean FtsH metalloproteinase and application of gene GmFtsH25
  • Gene GmFtsH25 for encoding soybean FtsH metalloproteinase and application of gene GmFtsH25

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, the tissue expression of soybean GmFtsH25 gene in Jack material

[0026] (1) Design primers, extract RNA, reverse cDNA:

[0027] The total RNA of each tissue of soybean Jack was extracted with a plant total RNA extraction kit (DP419, Tiangen), and the integrity of the RNA was detected by 1% agarose gel electrophoresis. cDNA synthesis reference Instructions for the 1st Strand cDNA SynthesisKit kit. Design primers as follows:

[0028] SEQ ID NO.3: GmFtsH25 qPCR primer F'5-CGACTGTGTTTGTGAGTGTGC-3'

[0029] SEQ ID NO.4: GmFtsH25 qPCR primer R'5-ACTCTGATTCCTGCAACCTACTAC-3'

[0030] SEQ ID NO.5: Tubulin qPCR primer F'5-GGAGTTCACAGAGGCAGAG-3'

[0031] SEQ ID NO.6: Tubulin qPCR primer R'5-CACTTACGCATCACATAGCA-3'

[0032] (2) Real-time fluorescent quantitative PCR, the specific steps are as follows:

[0033]Step 1: Dilute the cDNA samples of each tissue obtained in the above (1) 10 times for qPCR reaction, prepare the reaction solution (20 μl system) accord...

Embodiment 2

[0036] Embodiment 2, the cloning of soybean GmFtsH25 gene and the construction of plant overexpression vector

[0037] (1) Using the cDNA of soybean Jack leaves obtained in Example 1 as a template, the CDS fragment was amplified by high-fidelity polymerase chain reaction. Design primers as follows:

[0038] SEQ ID NO.7: Primer F'5- GGATCTTCCAGAGAT TACCCACGTTCCAAGTTCCG-3'

[0039] SEQ ID NO.8: Primer R'5- CTGCCGTTCGACGAT GCTTCGGAGCAATGGTGTCA-3'

[0040] (2) PCR amplification, the specific steps are as follows:

[0041] Step 1: Prepare PCR reaction solution (50 μl system) according to the following components: 25 μl 2×Planta MaxBuffer, 2 μl each of primers F and R, 1 μl dNTP Mix, 2 μl leaf cDNA template, 1 μl Super-Fidelity DNA Polymerase, 17 μl ddH 2 O make up the volume to 50 μl.

[0042] Step 2: The reaction was carried out on a BIO-RAD PTC-200 PCR instrument, and the reaction program was set as follows: pre-denaturation at 95°C for 3min; then 95°C for 15sec, 58°C f...

Embodiment 3

[0046] Embodiment 3, the cultivation of GmFtsH25 gene overexpression transgenic soybean

[0047] The pBA002-GmFtsH25 overexpression vector was transferred into the recipient material Jack by using the Agrobacterium tumefaciens-mediated transformation method of soybean cotyledonary nodes, and the specific method is described as follows:

[0048] (1) Select clean seeds that are mature and plump, without disease spots, and without hard solids, and arrange them in a single layer in a 90*15mm petri dish, and carry out dry chlorine gas sterilization on the surface of soybean seeds, and carry out in a fume hood for total sterilization Bacteria for 6-7 hours. Cover the petri dish and transfer it to a sterile ultra-clean bench, open the lid of the petri dish, and blow with strong wind for 25-40 minutes to remove residual chlorine gas. Sow the sterilized seeds on the germination medium (SG4) with the hilum facing down, stack the petri dishes, wrap them with plastic wrap, place them in ...

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Abstract

The invention discloses a gene GmFtsH25 for encoding soybean FtsH metalloproteinase and an application of the gene GmFtsH25. The nucleotide sequence of an encoding region (CDS) of the GmFtsH25 is as shown in SEQ ID NO.1, the amino acid sequence of the encoded protein is as shown in SEQ ID NO.2, and a GmFtsH25 overexpression transgenic plant is created by using an agrobacterium tumefaciens-mediated soybean cotyledonary node transformation method. The invention discloses the application of the gene GmFtsH25 for encoding soybean FtsH metalloproteinase, and it is found that compared with the net photosynthetic rate of a control plant, the net photosynthetic rate of a transgenic plant can be obviously improved by over-expression of the gene. Therefore, the gene disclosed by the invention can be introduced into a plant as a target gene to regulate the photosynthesis capacity of the transgenic plant, and is of great significance to the cultivation of a new variety of high-photosynthetic-efficiency soybeans.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and relates to a soybean FtsH metalloproteinase gene GmFtsH25 and application thereof. Background technique [0002] Photosynthesis is known as "the most important chemical reaction on earth", using sunlight and inorganic CO 2 with H 2 The organic matter formed by O not only provides the necessary energy for the life activities of plants, but also is the prerequisite for the survival, development and prosperity of almost all forms of life (microorganisms, plants, animals and humans). For plants, about 90%-95% of the dry matter comes from photosynthetic products. It is an important way to increase crop yield by improving the light energy utilization efficiency of crops. There have been many studies to prove its feasibility: such as through The artificial design and modification of the photorespiration process of plants has successfully inhibited the photorespiration of C3 crops, increasi...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N9/50C12N15/84A01H5/00A01H6/54
CPCC12N9/63C12N15/8261
Inventor 喻德跃王莉杨宇明黄方
Owner NANJING AGRICULTURAL UNIVERSITY
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