CN111333706A

CN111333706A - Soybean GmSPLE gene and encoding protein and application thereof

Info

Publication number: CN111333706A
Application number: CN202010167942.4A
Authority: CN
Inventors: 夏正俊; 翟红; 焦爽; 周静文; 吴红艳; 朱金龙; 徐坤
Original assignee: Northeast Institute of Geography and Agroecology of CAS
Current assignee: Northeast Institute of Geography and Agroecology of CAS
Priority date: 2020-03-11
Filing date: 2020-03-11
Publication date: 2020-06-26
Anticipated expiration: 2040-03-11
Also published as: CN111333706B

Abstract

A soybean GmSPLE gene, a coded protein and application thereof, relating to a soybean gene, a coded protein and application thereof. The invention provides a soybean GmSPLE gene and a coded protein and application thereof. The nucleotide sequence of the soybean GmSPLE gene is shown as SEQ ID NO: 1 is shown. The amino acid sequence of the coded protein is shown in a sequence table SEQ ID NO: 2, respectively. According to the invention, the soybean GmSPLE gene is overexpressed in soybeans, so that the plant height of the soybean GmSPLE gene is regulated and controlled, the number of main stem nodes of the soybeans is reduced, and the soybean internode distance is promoted to be reduced, thereby dwarfing the soybeans. The gene has important application in lodging resistance or increasing soybean yield by regulating plant height.

Description

Soybean GmSPLE gene and encoding protein and application thereof

Technical Field

The invention relates to a soybean gene and a coded protein and application thereof.

Background

The contradiction between the annual increase of global population and the gradual decrease of arable area is solved, and the yield of unit area can only be improved. Soybeans are important food and economic crops, and provide important plant protein and oil for human beings. Plant height is one of the main traits affecting soybean yield, and is closely related to lodging resistance, yield and the like of soybeans. The first green revolution in the early 60s of the last century is characterized in that the high stalks of rice and wheat are changed into short stalks, and the problem of grain yield reduction caused by lodging is solved due to the introduction of the short stalk genes, so that the key function of plant height in the yield determination is realized. The pod of the soybean is planted in the node, and the number of the node determines the number of the pod of each plant, thereby determining the yield of the soybean. The molecular breeding is used for controlling the plant height of the soybean, and breeding the soybean variety with lodging resistance, more nodes and dense pods is an effective means for improving the yield of the soybean.

Disclosure of Invention

The invention aims to provide a soybean GmSPLE gene, and a protein coded by the same and application thereof.

The nucleotide sequence of the soybean GmSPLE gene is shown as SEQ ID NO: 1 is shown.

The amino acid sequence of the coded protein of the soybean GmSPLE gene is shown as a sequence table SEQ ID NO: 2, respectively.

The invention provides application of a soybean GmSPLE gene in regulating and controlling soybean plant height.

The invention has the beneficial effects that:

the invention discloses a soybean GmSPLE gene and a coded protein and application thereof. According to the invention, the soybean GmSPLE gene is overexpressed in soybeans, so that the plant height of the soybean GmSPLE gene is regulated and controlled, the number of main stem nodes of the soybeans is reduced, and the soybean internode distance is promoted to be reduced, thereby dwarfing the soybeans. Therefore, the soybean GmSPLE gene has important application prospect in lodging resistance or increasing soybean yield by regulating plant height.

Drawings

FIG. 1 is a semi-quantitative RT-PCR analysis of GmSPLE gene in over-expressed transgenic and non-transgenic soybeans;

FIG. 2 is Western blot analysis of GmSPLE gene in over-expressed transgenic soybean and non-transgenic soybean;

FIG. 3 shows the plant height phenotype of GmSPLE over-expressed transgenic soybean and non-transgenic soybean;

FIG. 4 shows the flowering phenotype of GmSPLE overexpressing transgenic and non-transgenic soybeans;

FIG. 5 shows statistics of the number of major stem nodes of GmSPLE over-expressed transgenic soybean and non-transgenic soybean;

FIG. 6 is a GmSPLE overexpressing transgenic soybean and non-transgenic soybean internodal phenotype;

FIG. 7 is a statistics of GmSPLE overexpression transgenic soybean and non-transgenic soybean internodal distance data.

Detailed Description

The following examples are given to illustrate the present invention, and the following examples are carried out on the premise of the technical solution of the present invention, and give detailed embodiments and specific procedures, but the scope of the present invention is not limited to the following examples.

Example 1: construction of GmSPLE gene overexpression vector

Firstly, using cDNA of soybean variety Karyutaka as template, using GmSPLEmyc-F1 and GmSPLEmyc-R1 as primers, adopting high fidelity enzyme Fastpfu Fly DNA Polymeras purchased from Beijing all-purpose gold Biotechnology Limited to make PCR amplification, and making PCR reaction condition of pre-denaturation at 94 deg.C for 5min, (denaturation at 94 deg.C for 30s, annealing at 58 deg.C for 30s, and extension at 72 deg.C for 60s) × 6 circulation, denaturation at 94 deg.C for 30s, annealing at 68 deg.C for 30s, extension at 72 deg.C for 60s, 26 circulation, and extension at 72 deg.C for 5min, and adopting cDNA purchased from Promega

The method comprises the steps of carrying out Gel recovery on SV Gel and PCR Clean Up System, obtaining an amplification band of a GmSPLE gene through PCR amplification, introducing a BamH I restriction site at the upstream of the GmSPLE gene through the PCR amplification, carrying out double restriction on a BamH I restriction site and a Sac I restriction site of a downstream primer, carrying out T4 DNA ligase overnight connection on a PCR product obtained through amplification and a vector pBA-myc simultaneously, transforming escherichia coli competent cells, identifying positive clones through colony PCR, delivering the positive clones to a sequencing company for sequencing, wherein the sequencing result is shown in a sequence table SEQ ID NO. 1, the gene fragment is the soybean GmSPLE gene and consists of 417bp bases, the protein is coded and has an amino acid sequence of SEQ ID NO. 2 in the sequence table, the sequencing result shows that a vector genetically transformed in soybean by the GmSPLE gene is successfully constructed, the pBSPA-myc: GmLE vector contains a Bar screening marker gene (glufosinate), 35S drives a promoter, the resistance gene is added with a protein level tag 6 × on the GmSPLE gene, and the protein level is convenient to detect.

Primer GmSPLEmyc-F1: 5'-CGGGATCCTGCATATGGACGAAAG-3'

Primer GmSPLEmyc-R1: 5'-CGCGAGCTCTCAAAGTTCGTGGTAT-3'

The vector pBA-myc has been disclosed in an article (Wenfeng Ning, et al 2017).

The constructed vector pBA-myc, GmSPLE, is transferred into the genome of Dongnong variety 50 of soybean by the method of mediating soybean cotyledonary node with agrobacterium tumefaciens to obtain the GmSPLE gene over-expression transgenic soybean strain.

Example 2: molecular identification of GmSPLE gene overexpression transgenic soybean

1. By genetic transformation of soybean, at T₀A total of 28 independent transformation events were obtained, and were found at T by smearing with 160mg/L glufosinate-ammonium₁Four transformation events (35S: GmSPLE # L3, 35S: GmSPLE # L9, 35S: GmSPLE # L17, 35S: GmSPLE # L22) were still exhibited for the generation of glufosinate-ammonium resistance, and RNA was extracted from the mature leaves of soybeans of these four transformation events, according to the RNA extraction method described in the handbook of TRIzol kit of Invitrogen.

2. Treating the total RNA extracted in the first step by DNase, and detecting the DNA content and quality of the treated total RNA by using NanoDrop purchased from Toyobo company;

3. taking 2 mu g of total RNA after reaching the standard through the detection of the NanoDrop in the step 2, and adopting the total RNA purchased from Beijing Quanzijin Biotechnology Co., Ltd

One-Step gDNAremoval and cDNASynthesis SuperMix kit, and dT attached to the kit₁₈Synthesizing cDNA by using the primers according to the operation manual of the kit;

4. mu.l of the cDNA diluted 5 times in step 3 was used as RT-PCR template, qGmSPLE-F1 and qGmSPLE-R1 were used as primers, and EasyTaq purchased from Beijing Quanyujin Biotechnology Ltd was used for PCR amplification. PCR conditions: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, 527 cycles of the internal reference gene TUA, 29 cycles of the GmSPLE gene, final extension for 72, 5min, primer sequences of the soybean internal reference gene TUA5 are qTUA5-F1 and qTUA5-R1, and a primer sequence of the internal reference gene TUA5 is a published sequence in the published papers (Ruibo Hu, et al.2019). And 5 mul of PCR product is taken for agarose gel electrophoresis, and the result is shown in figure 1, compared with non-transgenic soybean Dongnong 50, the expression level of the GmSPLE gene is obviously higher than that of the non-transgenic soybean in 4 independent transformation events. Indicating that the GmSPLE gene successfully realizes the overexpression in soybean.

Primer qGmSPLE-F1: 5'-CTTCATGTCAAGTTGATGGTTGTAG-3'

Primer qGmSPLE-R1: 5'-GTGCTGGTCTCCAATGAGTACG-3'

Primer qTUA 5-F1: 5'-TGCCACCATCAAGACTAAGAGG-3'

Primer qTUA 5-R1: 5'-ACCACCAGGAACAACAGAAGG-3'

5. The protein of the mature leaf of soybean of three transformation events was extracted, the fully developed leaf of soybean was taken, after grinding in liquid nitrogen with a mortar, the powder was rapidly transferred to a 1.5mL centrifuge tube, 1mL of a protein lysis extract (50mM/L Tris-HCl, ph8.0,120mm/L NaCl, 10% glycerol, 0.2% Triton X-100, 1.5g/L DTT, 0.1% SDS, 1mM/L PMSF, 1 × Roche protease inhibitor) was added, 20min, 16,100 16,100 × g was shaken in a 4 ℃ shaker for 10min, the supernatant was removed to a clean centrifuge tube, the protein was taken to add 5 × loadbuffer, water bath was carried out at 95 ℃ for 5min, centrifugation was carried out after cooling to room temperature, SDS-PAGE was used for separation, Western Blot was carried out using anti-cmyc antibody (Western Blot, cat # fisch, cat # 2) as a Western Blot, the Western Blot results showed that no single transgenic soybean with a transient gene expression signature of 50, no soybean gene was expressed in the soybean with a transient target gene.

Example 3: phenotypic analysis of GmSPLE gene overexpression transgenic soybean

Will T₂The transgenic soybean with the overexpression of the GmSPLE gene and a non-transgenic control variety Dongnong 50 are sown in pots, 40-50 plants of each plant line are planted under natural light (long sunlight), and the phenotype of the plants is investigated in detail. As a result, the pod bearing habit of the transgenic soybean was changed (FIGS. 3 and 4), the transgenic soybean bloomed earlier at the apical end (FIG. 4), the apical growth was terminated, and the pod bearing habit was changed to a less-limited pod bearing habit, while the pod bearing habit of the non-transgenic soybean was a sub-limited pod bearing habit. The change in pod bearing habit led to a decrease in the number of major stem nodes of transgenic soybean overexpressing the GmSPLE gene (FIG. 5), resulting in a significant shortening of the plants. In addition to pod bearing habit, internode spacing is also an important factor affecting soybean plant height, and we further investigated the internode spacing of transgenic and non-transgenic soybeans. Compared with non-transgenic plants, the pitch spacing of GmSPLE gene transgenic soybean is obviously shortened (FIG. 6 and FIG. 7).

Sequence listing

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ggttgtagcg ctgatctaag tgaagctaag ccctaccata ggcgtcacaa ggtttgtgag 240

taccatgcca aggctcctgc cgtactcatt ggagaccagc accaacggtt ttgccaacaa 300

tgtagtaggt ttcatgagct atcagaattc gatgactcaa aaaggagttg cagaagacgt 360

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Claims

1. The soybean GmSPLE gene is characterized in that the nucleotide sequence of the GmSPLE gene is shown as SEQ ID NO: 1 is shown.

2. The amino acid sequence of the protein encoded by the soybean GmSPLE gene of claim 1 is shown in a sequence table SEQ ID NO: 2, respectively.

3. The use of soybean GmSPLE gene as defined in claim 1 for regulating soybean plant height.

4. The use according to claim 3, wherein the regulation of the plant height of soybean is specifically to reduce the number of main stem nodes of soybean and reduce the distance between nodes of soybean, thereby promoting dwarfing of soybean.

5. The use according to claim 3, wherein the GmSPLE gene is introduced into plant cells, tissues or organs, and the transformed plant cells, tissues or organs are cultured into plants, so that the GmSPLE gene is expressed in the plants to obtain transgenic plants for regulating the plant height of soybean.

6. Use according to claim 5, characterized in that the plant is soybean.