CN111333706B

CN111333706B - Soybean GmSPLE gene and encoding protein and application thereof

Info

Publication number: CN111333706B
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: 2022-04-15
Anticipated expiration: 2040-03-11
Also published as: CN111333706A

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, cDNA of a soybean variety Karyutaka is taken as a template, GmSPLEmyc-F1 and GmSPLEmyc-R1 are taken as primers, and high fidelity enzyme Fastpfu Fly DNA Polymeras purchased from Beijing all-purpose gold Biotechnology Limited is adopted for PCR amplification. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5min, (denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 60s) x 6 cycles, denaturation at 94 ℃ for 30s, annealing at 68 ℃ for 30s, extension at 72 ℃ for 60s, 26 cycles, and extension at 72 ℃ for 5min, using a pre-denaturation strain from Promega

The SV Gel and PCR Clean Up System was used for Gel recovery. An amplification band of the GmSPLE gene is obtained through PCR amplification, a BamH I restriction enzyme site is introduced into the upstream of the GmSPLE gene through the PCR amplification, and a Sac I restriction enzyme site is introduced into the downstream of the GmSPLE gene. After the PCR product obtained by amplification and the vector pBA-myc are subjected to double enzyme digestion by BamH I and Sac I, T4 DNA ligase is connected overnight to transform the competent cells of escherichia coli. Identifying positive clones by colony PCR, and sequencing the positive clones by a sequencing company, wherein the sequencing result is shown in a sequence table SEQ ID NO: 1, the gene fragment is soybean GmSPLE gene and consists of 417bp basic groupsAnd (4) forming. The code has the sequence shown in SEQ ID NO: 2 in the presence of a protease. The sequencing result shows that a vector for genetic transformation of the GmSPLE gene in soybean is successfully constructed, the pBA-myc vector contains a Bar screening marker gene (glufosinate resistance), the GmSPLE gene is driven by a 35S promoter, and the vector adds a 6 Xcmyc label at the N end of the GmSPLE gene, so that the detection on the protein level is facilitated.

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 as qTUA5-F1 and qTUA5-R1, and primer sequences of the internal reference gene TUA5 as published sequences (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, and compared with non-transgenic soybean Dongnong 50, the expression level of the GmSPLE gene is obviously higher than that of 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 proteins of the mature leaves of the soybeans from three of the transformation events were extracted, the fully developed soybean leaves were taken, ground in liquid nitrogen with a mortar, and the powder was quickly 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 XRoche protease inhibitor) was added, shaken on a shaker at 4 ℃ for 20min, centrifuged at 16,100 Xg for 10min, and the supernatant was transferred to a clean centrifuge tube. A proper amount of protein is added into a5 × loading buffer, water bath is carried out at 95 ℃ for 5min, the mixture is cooled to room temperature and then is centrifuged briefly, and the mixture is separated by SDS-PAGE, and Western Blot hybridization is carried out by using anti-cmyc (Thermo Fisher, Cat. No. MA1980) antibody. Western blot results are shown in FIG. 2, the non-transgenic soybean Dongnong 50 has no target band, and 3 independent transformation events can generate the target band due to the fact that the GmSPLE gene with the cmyc label is transferred, and therefore the GmSPLE gene can successfully achieve over-expression in soybeans.

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 sowed in a pot, 40-50 plants of each plant line are planted under natural light (long-day 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

<110> institute of geography and agroecology of northeast China academy of sciences

<120> soybean GmSPLE gene and coding protein and application thereof

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<212> DNA

<213> Glycine genus (Glycine max L. Merr.)

<400> 1

atggacgaaa gttggagtga gggaaaaagg agcatgagtt acaaggagga ggatgagtac 60

gaagaagagg aagaggagga ggtgagtgag tatagagatg atggtaggaa aaagaaggtg 120

gtgagtagta agagagggtc caaagctgga ggctcagtgc caccttcatg tcaagttgat 180

ggttgtagcg ctgatctaag tgaagctaag ccctaccata ggcgtcacaa ggtttgtgag 240

taccatgcca aggctcctgc cgtactcatt ggagaccagc accaacggtt ttgccaacaa 300

tgtagtaggt ttcatgagct atcagaattc gatgactcaa aaaggagttg cagaagacgt 360

ttggctggac ataatgagag gcgtcgcaaa aatgcatctg aataccacga actttga 417

<210> 2

<211> 138

<212> PRT

<213> Glycine genus (Glycine max L. Merr.)

<400> 2

Met Asp Glu Ser Trp Ser Glu Gly Lys Arg Ser Met Ser Tyr Lys Glu

1 5 10 15

Glu Asp Glu Tyr Glu Glu Glu Glu Glu Glu Glu Val Ser Glu Tyr Arg

20 25 30

Asp Asp Gly Arg Lys Lys Lys Val Val Ser Ser Lys Arg Gly Ser Lys

35 40 45

Ala Gly Gly Ser Val Pro Pro Ser Cys Gln Val Asp Gly Cys Ser Ala

50 55 60

Asp Leu Ser Glu Ala Lys Pro Tyr His Arg Arg His Lys Val Cys Glu

65 70 75 80

Tyr His Ala Lys Ala Pro Ala Val Leu Ile Gly Asp Gln His Gln Arg

85 90 95

Phe Cys Gln Gln Cys Ser Arg Phe His Glu Leu Ser Glu Phe Asp Asp

100 105 110

Ser Lys Arg Ser Cys Arg Arg Arg Leu Ala Gly His Asn Glu Arg Arg

115 120 125

Arg Lys Asn Ala Ser Glu Tyr His Glu Leu

130 135

<210> 3

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<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgggatcctg catatggacg aaag 24

<210> 4

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<213> Artificial Sequence (Artificial Sequence)

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cgcgagctct caaagttcgt ggtat 25

<210> 5

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cttcatgtca agttgatggt tgtag 25

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtgctggtct ccaatgagta cg 22

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tgccaccatc aagactaaga gg 22

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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accaccagga acaacagaag g 21

Claims

1. The application of the soybean GmSPLE gene in regulating the height of soybean plants; the nucleotide sequence of the soybean GmSPLE gene is shown as SEQ ID NO: 1 is shown.

2. The use according to claim 1, 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.

3. The use according to claim 1, 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; the plant is soybean.