Research Report

Expression of a Seed Storage Protein AtSSA3 of Arabidopsis thaliana in Escherichia coli  

Le Chang1,2 , Shenkui Liu3 , Yuanyuan Bu1,2
1 Key Laboratory of Saline-Alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin 150040, China
2 College of Life Science, Northeast Forestry University, Harbin 150040, China
3 The State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Lin’An, 311300, China
Author    Correspondence author
Molecular Soil Biology, 2021, Vol. 12, No. 1   doi: 10.5376/msb.2021.12.0001
Received: 02 Mar., 2021    Accepted: 03 Mar., 2021    Published: 10 Mar., 2021
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This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Chang L., Liu S.K., and Bu Y.Y., 2021, Expression of a seed storage protein AtSSA3 of Arabidopsis thaliana in Escherichia coli, Molecular Soil Biology, 12(1): 1-6 (doi:10.5376/msb.2021.12.0001)

Abstract

Mature seeds are rich in storage proteins, which not only affect the quality of seeds, but also the energy source of seed germination and seedling growth. In this study, a gene encoding a seed storage protein SSA3 from Arabidopsis thaliana was isolated. The AtSSA3 gene was expressed in Escherichia coli BL21 (DE3). The fusion protein pET-32a(+)-AtSSA3 was induced under the optimized conditions of 1mM IPTG and 25°C for 4h. Western blot analysis the optimal conditions of protein expression. This study provides a theoretical and practical basis for the further study of seed storage protein genes in the future.

Keywords
AtSSA3; Protein induction; Prokaryotic expression

Introduction

The content of protein in seeds is relatively high, among which the content of seed storage protein (seed storage proteins, SSP) is the most abundant, accounting for about 60% of the total protein content of seeds, which is an important energy source for seed germination, growth and so on (Zhou, 2015). The stored proteins in dry seeds can not only provide a buffer for seeds, help seeds from oxidative stress, and protect important proteins needed for seed germination and seedling formation. and affect the life span of seeds by changing the activities of some enzymes in the metabolic cycle (Nguyen et al., 2015). The expression of seed storage protein is mainly controlled by the transcriptional regulation at the filling stage, and the protein accumulation is closely related to the corresponding mRNA. As a member of the TF family containing B3 domain, LEC2 can and activate SSP gene (Braybrook et al., 2006). Comparing SSP promoter with DNA-protein binding sequence, two main conserved factor binding sites, RY/G motif and Bmurbox, were found, and they play a synergistic role (Vicente-Carbajosa and Carbonero, 2005). Seed storage proteins combine to form larger precursors on rough endoplasmic reticulum (ER) and are classified as protein storage vacuoles, where they are transformed into mature forms (Jolliffe et al., 2005). It has been found that AtVSR1 is responsible for accumulating appropriate mature storage proteins in seeds and acting as sorting receptors for storage proteins in mature seed cells (Shimada et al., 2003). Arabidopsis thaliana MAG1/AtVPS29 protein effectively divides seed storage proteins by participating in the recovery of plant receptors (Shimada et al., 2006).

 

In this study, we isolated an Arabidopsis cDNA that encodes AtSSA3 gene, and cloned into the protein expression vector pET-32a(+) to form AtSSA3 plasmid. The AtSSA3 gene was expressed better in pET-32a(+) under 1 mM IPTG and 25°C for 4h conditions.

 

1 Materials and Methods

1.1 Strains, carriers and main reagents needed

The competent state of Esc-herichia coli DH5α, Pure Plasmid Mini kit and Gel Extraction kit were purchased from CWBIO. Esc-herichia coli BL21(DE3) competent cells and PBS buffer were prepared and preserved in our laboratory. Low molecular weight protein Marker was purchased from TransGen Biotech. The high-fidelity ExTaq enzyme series, pMD18-T Vector, dNTP Mix and the reverse transcription kit were obtained from TaKaRa. The T4 DNA ligase was obtained from ThermoFisher Scientific. The sheep anti-rabbit IgG (peroxidase covalent binding) was purchased from Beyotime. The eECL Western Blot kit were also purchased from CWBIO. The primers used were synthesized by GENEWIZ.

 

1.2 Extraction of total RNA and total protein

Fresh leaves of Arabidopsis thaliana were extracted, and the total RNA of Arabidopsis thaliana was extracted by TRIzol method and referring to the instructions of reagents. The total protein of Arabidopsis thaliana leaves was extracted by PBS. The extracted total RNA and total protein were stored in the -80°C refrigerator in our laboratory.

 

1.3 Cloning of AtSSA3 and expression vector construction

The specific primers of seed storage protein gene were designed by Primer Premiers 5.0. The forward primers wereAtSSA3-F (5’-GGTACCATGGCTAACAAGCTCTTCCT-3’, containing KpnI restriction sites), and the reverse primers were AtSSA3-R (5’-GAGCTCCTAGTAGTAAGGAGGGAAGA-3’, containing SacI restriction sites). The total RNA extracted from Arabidopsis thalianawas used as a template to obtain cDNA according to the reverse transcription kit instructions of TaKaRa. Using cDNA as template, 1 μL of upstream primers and 10 μM of downstream primers were added, and a series of high fidelity ExTaq enzymes were added to make the total volume up to 20 μL. The PCR reaction was carried out under the conditions of 94°C 30 s, 55°C 40 s and 72°C 90 s. The reaction lasted for 35 cycles. After 1.5% agarose gel electrophoresis and purification with Gel Extraction kit, the amplified product was connected to pMD18-T intermediate vector and transformed into DH5α. The intermediate vector plasmid containing the target gene was obtained by colony PCR, plasmid PCR and double restriction endonuclease digestion. The plasmid was digested by KpnI and SacI, and the target fragment was purified and ligated with the prokaryotic expression vector pET-32a(+). The plasmid was transformed into Esc-herichia coli DH5α, identified and sequenced by PCR and double restriction endonuclease digestion, and the recombinant plasmid containing the target gene, pET-32a(+)-AtSSA3, was obtained.

 

1.4 Induced expression of pET-32a(+)-AtSSA3 fusion protein 

The expression vector pET-32a(+)-AtSSA3 was transformed into Esc-herichia coli BL21(DE3) plysS, 37°C incubator overnight. The next day, the clones were picked into LB culture medium (Amp resistance) and cultured. The correct strains identified by double enzyme digestion were stored in a refrigerator at -80°C for prokaryotic induced expression test.

 

The preserved bacterial liquid 10 μL was cultured overnight in the liquid medium of LB (100 μg/ mL Amp) at 37°C. 200 μL overnight bacteria solution was added to 800 μL LB liquid medium containing 100 μg/mL Amp, shaken for about 2 hours in 37°C, 160 rpm/min shaker. When the OD600 of the bacteria solution was about 0.6, the induction test was carried out. In order to explore the effect of temperature on the expression of pET-32a(+)-AtSSA3 fusion protein, the strain was cultured at 37°C, 30°C and 25°C (rotational speed 160 rpm/min) when the concentration of IPTG was 1 mM. The bacterial liquid was collected and the protein was extracted by PBS. The protein expression was detected by SDS-PAGE electrophoresis at different temperatures. According to the study of time on the expression of pET-32a(+)-AtSSA3 fusion protein, the bacterial solution of 1mM with the final concentration of OD600=0.6, IPTG was cultured at 25°C and 160 rpm/min shaker for 0 h, 1 h, 3 h and 4 h, respectively. The protein was extracted and the expression of fusion protein was detected by SDS-PAGE. In order to explore the effect of different concentrations of inducer on the expression of pET-32a(+)-AtSSA3 fusion protein, 200 μL of bacterial solution after the same treatment was added to the experimental group with the final concentration of IPTG of 0.2 mM, 0.5 mM, 1 mM and 2 mM, and 0 mM IPTG as the control group. 25°C, 160 rpm/min shaker culture for 4 h. The expression of recombinant protein induced by different concentrations of IPTG was also detected by SDS-PAGE.

 

According to the expression of pET-32a(+)-AtSSA3 fusion protein at different temperature, time and IPTG concentration, the optimal conditions for protein expression were determined. The optimal conditions induced by pET-32a(+)-AtSSA3 fusion protein were verified by Western Blot.

 

Required reagents: 12% Separation Gel (15 mL): 6 mL 30% Acrylamide, 3.8 mL 1.5 M Tris-HCl (pH8.8), 0.15 mL 10% SDS, 0.15 mL 10% Ammonium Persulphate, 0.006 mL TEMED, 4.9 mL Deionized Water; 5% Concentrated Gel (8 mL): 1.3 mL 30% Acrylamide, 1 mL 1M Tris-HCl (pH6.8), 0.08 mL 10% SDS, 0.08 mL 10% Ammonium Persulfate, 0.008 mL TEMED, 5.5 mL Deionized water; 20×BufferIII (pH10.0): 121 g/L Tris-Base, 112 g/L NaCl; Electrophoresis Buffer (1 L): 3 g Tris-Base, 18.8 g Glycine, 1 g SDS; Staining Solution (1 L): 1 g Coomassie brilliant blue R250, 250 mL Isoamyl Alcohol, 100 mL Aceticacid, 650 mL Deionized Water; Decolorizing Solution (1 L): 50 mL Ethanol, 100 mL Aceticacid, 850 mL Deionized Water; Transfer Buffer (1 L): 3 g Tris-Base, 14.4 g Glycine, 300 mL Methanol; 1% Blocking Reagent: 10% Blocking Reagent was diluted with Maleic Acid.

 

First of all, the induced protein was electrophoretic by SDS-PAGE. After that, the protein glue was cut off and soaked in the transfer buffer with the reference of the two-color pre-staining protein Marker. The vesicles were activated in methanol for about 15 seconds, then soaked in the transfer buffer and separated from the protein glue. Put the membrane and protein glue together in the film transfer instrument, from negative plate to positive plate are sponge, filter paper, protein glue, membrane soaked in transfer buffer, thickened filter paper, sponge. The voltage is 90 V and the time is about 1 h. After the membrane was transferred, it was immersed in 1.5% Blocking Reagent for about 2 hours, and the first antibody was added according to the ratio of 1: 1000, and spent the night at 4°C. Secondly, take out the membrane the next day, add appropriate amount of 1 × PBS buffer (covering the membrane surface), wash 3 times, each time 10 min. The second antibody was added according to the ratio of 1: 1000, and shaken at room temperature for 2 hours. The membrane was washed twice and 5 min each time. Finally, the film was exposed and observed in LAS-4000 by eECL Western Blot kit.

 

2 Results and Discussion

2.1 Construction of the expression vector pET-32a-AtSSA3

In this study, we cloned the AtSSA3 gene using the total cDNA of Arabidopsis thaliana as a template (Figure 1A). After the intermediate vector pMD18-T, was identified correctly (Figure 1B), it was cloned into the plant protein expression vector pET-32a(+) between KpnI and SacI. The transformation of Esc-herichia coli DH5α strain showed that the pET-32a(+)-AtSSA3 vector was successfully constructed (Figure 1C). Seed storage proteins, which have the characteristics of large accumulation and special aggregation, are water-soluble and highly rich proteins. They are decomposed after seed germination seedlings begin to grow, thus providing nitrogen and sulfur for developing seedlings (Shutov et al., 2003; Galili, 2004; Mylne et al., 2014). The proteolytic enzymes that degrade storage proteins are mainly cysteine proteases, but also serine, aspartic acid and metalloproteinases (Tan-Wilson and Wilson, 2012).

 

Figure 1 Construction of prokaryotic protein expression vector of AtSSA3

Note: A: cDNA template cloning of AtSSA3; M: DS2000 Marker; 1: PCR product electrophoresis results, fragment size 495 bp; B: Identification by double restriction endonuclease digestion of AtSSA3 ligated pMD18-T vector; M: DS2000 Marker; 1: the results of double restriction endonuclease digestion electrophoresis showed that there were two bands with the size of 2 600 bp and 495 bp; C: Double restriction endonuclease digestion of AtSSA3 ligated pET-32a(+) terminal vector; M: DS5000 Marker; 1: end carrier double enzyme digestion electrophoresis results: there were two bands, the size of which were 5 900 bp and 495 bp

 

2.2 Prokaryotic induced expression Expression of pET-32a(+)-AtSSA3 fusion protein in E.coli

The protein size affects its expression in E.coli, and the protein with more than 100 kDa can not be expressed correctly in bacteria (Palacios et al., 2001). The size of the protein encoded by AtSSA3 is 19 kDa, while the size of His-tag and other tags in pET-32a(+) vector is about 15 kDa. Therefore, the total relative molecular weight of the fusion protein pET-32a(+)-AtSSA3 is about 34kDa, which is basically consistent with the results of SDS-PAGE.

 

The Fusion protein induced by 37°C, 30°C and 25°C was collected and extracted. When the recombinant protein was induced at 30°C and 37°C for 4 hours, the color of the bands in each lane did not change significantly (Figure 2A; Figure 2B). Temperature is an important factor affecting the growth of recombinant bacteria, the stability of plasmid and the formation of recombinant products (Ye et al., 2002).

 

Figure 2  SDS-PAGE analysis of the effect of different temperature on the expression of His-AtSSA3 fusion protein

Note: D: Expression of 37°C fusion protein; M: Low molecular weight standard protein; 0, 1, 2, 3, 4: Results of protein electrophoresis induced by 0, 0.2 mM IPTG, 0.5 mM IPTG, 1 mM IPTG, 2 mM IPTG at 37°C for 4 hours; E: Expression of 30°C fusion protein; M: Low molecular weight standard protein; 0, 1, 2, 3, 4: Results of protein electrophoresis induced by 0, 0.2 mM IPTG, 0.5 mM IPTG, 1 mM IPTG, 2 mM IPTG at 30°C for 4 hours; F: Expression of 25°C fusion protein; M: Low molecular weight standard protein; 0, 1, 2, 3, 4: Results of protein electrophoresis induced by 0, 0.2 mM IPTG, 0.5 mM IPTG, 1 mM IPTG, 2 mM IPTG at 25°C for 4 hours

 

However, when the temperature is 25°C, the color of the band near 35 kDa deepens (Figure 2C), indicating that the protein expression is increased at this time. It is speculated that the fusion protein may be more easily induced at 25°C.

 

The recombinant protein treated with 25°C and 1 mM IPTG for 0 h, 1 h, 3 h and 4 h was collected, and extracted by PBS. SDS-PAGE analysis showed that there was a band near lane 1, 2 and 3 35 kDa compared with lane 0, and the color of band 3 was darker and more obvious (Figure 3). The expression of the recombinant protein did increase with the increase of induction time, and the expression of the fusion protein was the best at 4 hours.

 

Figure 3 SDS-PAGE analysis of the effect of different induction time on the expression of His-AtSSA3 fusion protein

Note: M: Low molecular weight standard protein; 0: Control group; 1: 25°C, 1 mM IPTG induced the expression of His-AtSSA3 fusion protein in a small amount for 1 hour; 2: 25°C, 1 mM IPTG induced the expression of His-AtSSA3 fusion protein in a small amount for 3 hours; 3: 25°C, 1 mM IPTG induced the expression of His- AtSSA3 fusion protein in a small amount for 4 hours

 

After adding different concentrations of IPTG to pET-32a(+)-AtSSA3 recombinant protein, it was found that compared with the control group, the experimental group had a band near 35 kDa after adding IPTG. When the concentration of IPTG increases, the color of the bands becomes darker. It means that the expression of recombinant protein pET-32a(+)-AtSSA3 increased. Compared with the protein induced by 1 mM at the final concentration of IPTG, the expression of the fusion protein did not increase significantly when the final concentration of IPTG was increased to 2 mM (Figure 4). It is speculated that the toxicity of IPTG may affect the expression of protein (Xiang et al., 2011). Therefore, it can be concluded that the final concentration of IPTG is 1 mM, which is the best concentration for the expression of recombinant protein pET-32a(+)-AtSSA3.

 

Figure 4 SDS-PAGE analysis of the effect of different concentrations of inducer on the expression of His-AtSSA3 fusion protein

Note: M: Low molecular weight standard protein;0:Expression of recombinant Hismura AtSSA3 fusion protein induced in a small amount for 4 hours without adding IPTG; 1: 25°C, 0.2 mM IPTG induced the expression of the fusion protein His-AtSSA3 in a small amount for 4 hours; 2: 25°C, 0.5 mM IPTG induced the expression of His-AtSSA3 fusion protein in a small amount for 4 hours; 3: 25°C, 1 mM IPTG induced the expression of His-AtSSA3 fusion protein in a small amount for 4 hours; 4: 25°C, 2 mM IPTG induced the expression of His-AtSSA3 fusion protein in a small amount for 4 hours

 

The expression of plant protein is affected by many factors, not only including temperature, time, IPTG concentration, etc., but also related to the properties of genes and protein expression vectors (Kaur et al., 2018). Thus, Western blotting was used to detect the results of protein induction by specific antibody against AtSSA3 gene. Compared with the control group 1, the target band of lane 2 was darker (Figure 5), the protein expression was increased. It is suggested that the condition of 25°C and adding 1 mM IPTG for 4 h is more favorable for the expression of pET-32a(+)-AtSSA3 fusion protein.

 

Figure 5 Detection of small amount of fusion protein induction by Western-Blot

Note: 1 Protein induced by 25°C, 0 mM IPTG for 4 hours; 2: 25°C, protein induced by 1mM IPTG for 4 hours

 

In summary, the study of seed storage protein gene is of great significance in bioscience, agricultural production and other fields. The research work related to Arabidopsis thaliana seed storage proteins need to be carried out in depth.

 

Acknoledgements

This work was supported by Heilongjiang Province Government Postdoctoral Science Foundation (LBH-Q18008) awarded to Yuanyuan Bu. Further supported by the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT17R99) awarded to Shenkui Liu. The funders had no role in study design.

 

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