Introduction

The expression of plant gene is closely related to the biological clock which is involved in the growth regulation of plant. Plant biological clock system is mainly composed of input part, central oscillator, output part and the valve effect device (Li, 2015). Previous studies showed that LHY and CCA1 were a type of class R1 and class Myb DNA binding transcription factors, LHY/CCA1 and TOC1 forms the central oscillator in biological clock central control loop of Arabidopsis thaliana, TOC1 positively regulates the expression of LHY/CCA1. When the expression of LHY/CCA1 reached a certain value, CCA1 and LHY will be combined with the night element of TOC1 promoter to negatively inhibite the expression of TOC1 (Harmer et al., 2000; Alabadí et al., 2001). The mRNA expression in TOC1 can be increased because of the decrease of CCA1/LHY expression level could gradually release its inhibitory effect on TOC1 during the day. When the expression of TOC1 reaching a certain amount of time, it will positively regulate the expression of CCA1 and LHY,, which can increase the  expression of LHY/CCA1, thus forming a physiological cycle of oscillation.

We used the method of bioinformatics to identify 4 LHY orthologous candidate genes in soybean. In order to further validate the reliability and validity of our bioinformatics analysis, We designed primers according to homologous genes of LHY in soybean, amplified a full-length cDNA sequence by RT-PCR , compared the similarity with the 4 GmLHY we got according to bioinformatics analysis, then named GmLHY2. Later on, we built GmLHY2 into PGEX expression vector, expressed the GST-GmLHY2 fusion protein in the host (BL21), and explored the expression conditions of GST - GmLHY2 preliminary.

1 Results and Analysis

1.1 PCR Amplification of LHY in Soybean

We used two pairs of specific primers, which were designed to make PCR amplification for Glycine Max. and got a specific amplification from the second pairs of primers, the molecular weight is about 2300 bp (Figure 1; Figure 2).

1.2 Cloning of PCR products

1.2.1 Recycle of amplified fragment

pMD18-T vector was used to connect and transform the human Enterobacter Gm109 strain, we selected the positive clones and verified the insertion fragment size by double enzyme.

1.2.2 Sequencing and in silico analysis of positive cloning

We picked positive clone GmMYBLHY1 sequencing to abtain cloned fragment whose size was about 2252 bp, in which has an ORF with a total length of 2250 bp, that can be translated into protein of 749 amino acid sequences. Compare the amino acid sequence with 4 LHY orthologous candidate genes from soybean, we found that the similarity with GmLHY1、GmLHY2、GmLHY3 and GmLHY4 is 90.1%、98.1%、71.3% and 71.4% respectively. Thus, we concluded that the gene we cloned is GmLHY2.

1.3 Prokaryotic Expression of GmLHY2

1.3.1 Construction of expression vector and transformation of BL21 expressing strain

We used the primers which was designed according to the expression vector PGEX for PCR, and connected to the cloning vector PMD18-T after recycling PCR gel, then turned into clone strains Gm109. After extraction of plasmid and double enzyme identification, it was connected with expression vector PGEX and turned into clone strains Gm109. We used kit to extracte plasmid, made double enzyme identification on EcoRI and SaII (Figure 3), and then turned into expression strain BL21 and PCR identification (Figure 4).

1.3.2 Time gradient induced expression for target protein of GST - GmLHY2

The host BL21 strain, which was identified as a recombinant vector containing PGEX-GmLHY11, was placed at 28℃ and 37℃, It was induced for 6 hours under the conditon of IPTG concerntration were 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 μM respectively. The results showed that the expression of fusion protein of engineering bacteria which contains PGEX-GmLHY11 recombinant vector, under 37ºC was better than 28ºC, and have better induction effect in the IPTG concentration of 0.4 μM and 0.6 μM conditions. We selected an engineering bacteria with better express, under the condition of 37ºC, IPTG concentration was 0.4 μM, to do the time gradient induction (Figure 5). From the diagram, we can see the molecular weight of fusion protein was greater than 97 KDa. The protein encoded by gene GmLHY2 contains 749 amino acids, analyzed by related online software, the molecular weight of the protein was 82 KDa, plus the 23 KDa protein coding by GST, its expected size was 105 KDa. But as shown in the figure, the molecular weight of fusion protein was greater than predicted; it may be the following reasons: first, the software forecast of protein size was not accurate; second, after protein GST - GmLHY2 expressed in BL21 host bacteria, protein modification affected, such as glycosylation, forming dimers with itself or other proteins, etc. If it was the second case, that will increase the difficulty of the subsequent purification work.

2 Discussion

In this research, we designed primers according to homologous genes of LHY in soybean, and successfully amplified the fragment whose ORF span was 2250 bp, encoding 749 amino acids. Compared the amino acid sequence with 4 LHY orthologous candidate genes from soybean, we found that the similarity of GmLHY1, GmLHY2, GmLHY3 and GmLHY4 was 90.1%, 98.1%, 71.3% and 71.4%, so we can draw a conclusion that the gene we cloned was GmLHY2. However, the similarity of GmLHY2 gene we got according to bioinformatics analysis was not 100%, this may due to the difference of soybean materials we used.

The molecular weight of fusion protein in prokaryotic expression was greater than predicted, it may be the following reasons: first, the software forecast of protein size was not accurate; second, after protein GST - GmLHY2 expressed in BL21 host bacteria, protein modification affected, such as glycosylation, forming dimers with itself or other proteins, etc. If in second case that will increase the difficulty of subsequent purification work.

3.1 Materials3 Materials and Methods

Soybean material was high resistance SCN varieties “grey cloth” (Glycine Max, from Shanxi Province Xing County, native variety, ZDD2315). The colony strain we used was E.coli Gm109, without resistant; Its carrier was pMD18 - T and the resistance was Amp50 (50 μg/mL); Prokaryotic expression host strains was E.coli, without resistance, its expression vector was PGEX and the resistance was Amp100. The culture medium we used was LB liquid medium (Yeast extract 5 g/L, Tryptone 10 g/L, NaCl 10 g/L, PH: 7.2) and LB solid medium (LB liquid medium＋1.5% (w/v) agar) for the liquid and solid culture of E.coli respectively. The Inoue transformation buffer for competent cell of E.coli (10.88 g MnCl2, 2.20 g CaCl2•H2O, 18.65 g KCl soluble in water, add 20 mL 0.5M PIPES (PH 6.7), then add ddH2O to 1 L, they were divided into small pieces in -20℃ after filter and degerming by Millipore filters membrane (0.22 μm aperture), which was processed in advance. The solution for extract of plasmid DNA was prepared by boiling method. (Sam brook, 2002). The solution for prokaryotic expression: we dissolved 8 g IPTG (Sigma products) into 10 mL dual steaming water, filtered and degermed by 0.22 μm filters membrane, subpackaged, and saved in - 20℃, then we got IPTG storage solution (Sam brook, 2002). According to the GST fusion system (Pharmacia biotech, Inc, 29) and on the preparation of E.coli BL21 competent cells required TSS gene buffer.

3.2 Methods

3.2.1 Primer design

One pair of primers were designed according to 4 homologous genes of GmLHY, they were composed by Shanghai (Sangon) biological process company: upstream primer P1: 5---TAATGGACGCCGACTCCTCT---3, downstream primer P2: 5---TCAAGTTGAAGCCTCCCCAT---3.

3.2.2 Soybean plant sampling

We took the whole plant in the growing period for rapid cleaning,, wrapped in tinfoil, quickly frooze it in liquid nitrogen for 2 ~ 5 min, then reserved it in -70°C.

3.2.3 Extraction of total RNA from soybean genome

The extraction of total RNA from soybean was carried out in accordance with Biozol total RNA extraction kit specification of BioFlux Company. RNase existed in a wide range, and it was difficult to inactivate. In order to prevent the pollution of RNase, all the instrument related to RNA operation was dealed with 0.1% hydrogen peroxide (H2O2) soak overnight, 70ºC bake for 2 h, sterilized 30 mins under 120ºC high pressure. We added deionized water into the glass bottle without RNA enzyme treatment, then added DEPC to 0.1% (DEPC/ - v/v deionized water), mixed and sterilized for 30 mins under 120ºC high pressure, to get DEPC water.

3.2.4 Synthesis of the first cDNA chain

Prepared reagent: “RNA kit (AMV) Ver3.0” Reverse transcription kit from TakaRa Company, Oligo dt-Adaptor primer, Mg cl2, 5 X AMV Reverse Tra, RNase inhibitor, dNTP mixture, and RNase Free dH2O. The step of reverse transcription was reference to the instructions of “RNA kit (AMV) Ver3.0” reverse transcription kit from TakaRa Company.

3.2.5 Cloning of target gene

PCR amplification: we diluted 5 multiple of the reverse transcription product, gradient PCR reaction system and process, agarose gel electrophoresis was reference to Guo (2008). Recovery of PCR products: PCR products were separated by 1% agarose gel electrophoresis, the objective DNA fragment was cut off under the ultraviolet lamp, recovered and purificated it by the DNA Extraction Kit from Fermentas life science company (If not reclaim immediately, we can put the plastic strip on the 1.5 mL centrifuge tube, stored at -20℃). We can search the concrete steps refer to the instructions of DNA Extraction Kit from KitFermentas life science company. We connected the purified PCR product to Vector pMD18-T (TAKARA company), and operated according to instructions of pMD18-T Vector reagent kit (TAKARA company). As for transformation process, we took 50 μL new prepared Gm109 competent cell and 5 μL connected product mixed, stewing on ice for 30 mins, made heat shock under 42ºC for 1 min (precise timing), ice bath for 2~3 mins, added 300 μL LB liquid medium which was preheated under 37ºC, preculture with violent shock for 1 h. 200 μL bacterial fluid was scrawled on the LB solid medium which obtains 50 mg/L ampicillin (Amp), then inverted culture under 37ºC overnight. We took out the flat plate and preserved it under 4ºC. We used SacⅠ and SmaⅠ to extract plasmid DNA. The recombinant plasmid was identified by double enzyme digestion. The enzyme digestion system: Buffer 1X Tango™: 1.5 μL, SacⅠ: 0.5 μL, SmaⅠ: 0.5 μL, DNA: 3 μL, ddH2O: 9.5 μL, Total Volume: 15 μL. 30ºC water bath for 90 mins and 37ºC bath for 90 mins after mixed. Strains were identified as positive clones, then sent to the company (Shanghai) for sequencing, the clones were named GmLHY, and preserved in -70ºC. Positive clones were sequenced by Shanghai Technology Company, and the sequencing results was spliced by BioEdit, they translated it into amino acid sequence. Nucleotide sequence and amino acid sequence results in the NCBI database to carry on the similarity comparison as well as other bioinformatics analysis.

3.2.6 Prokaryotic expression of target gene

The construction of pGEX-GmR4g31 recombinant plasmid was designed according to the sequence of GmLHY2: Upstream primer (EcoRⅠ): 5---GAATTCAATGGACGCATACTCCTC---3, Downstream primer (SalⅠ): 5---GTCGACTCAAGTCGAAGTCTCC--- 3. PCR reaction was based on the use of pMD18-T-GmR4g31 as template, and the annealing temperature was 58ºC, annealing time was 1 min, reaction system was 50 μL. We dealed the PCR product with glue recovery, connected it to pMD18-T carrier, then transferred into Gm109 strain. After identified by bacterial fluid PCR, we extracted pMD18-T-GmR4g31 (Plus enzyme cutting site) by kit. pGEX and pMD18-T-GmR4g31 plasmid were used for enzyme digestion, collection and connection of pGEX and pMD18-T-GmR4g31,, which were transformed into E.coli JM109 and identificated the recombinant plasmid pGEX-GmR4g31 with enzyme digestion. Preparation of competent cell of E.coli BL21: transferred recombinant plasmid pGEX-GmR4g31 into E.coli BL21 (Liu et al., 2006). Small inducible expression of fusion protein GST-GmR4g31: we vaccinated the identified monoclonal E.coli BL21 which obtained plasmid pGEX and pGEX-GmR4g31 to LB medium contained 100 mg L/Amp, then shaking culture overnight under 37ºC; 200 L overnight cultures were added to the medium containing 100 mg L/Amp in a 2.8 mL LB medium, oscillating culture under 37ºC for 1 h. When the bacteria liquid reached the logarithmic growth phase (OD6oo=0.8-1.0), we induced culture under 37ºC and 28ºC, the concentrations of inducer IPTG were 1, 0.5, 0.2, and 0.1 mM. When the bacteria were induced to 0 min, 60 min, 120 min, 240 min, 480 min, 960 min, 13000 rpm /1sec collected the bacteria liquid, then added suspended sediment PBST and the same amount of sample buffer 2×SDS, 13000 rpm /10 min centrifuge after having boiling water bath for 5 mins. We took 20 μL samples performed SDS-PAGE electrophoresis, then we made gel dyeing by Coomassie brilliant blue solution after electrophoresis.

Authors' contributions

Li Zongfei was the executor of this experiment, responsible for the experimental design, implementation, data analysis and draft writing; Wu Zhongqi, Wei Fang, Zhang Jie and Cai Mengdie were participated in the data analysis, the formation and amendment of the draft; Liu Zhenpeng was participated in the revision of the manuscript, translation and proofreading; Fang Xuanjun determined the research project conception, guided writing and revising paper. All authors have read and approved the final manuscript.

Acknowledgements

This research was supported by Cuixi Academy of Biotechnology Zhuji (Life science and Biotechnology Innovation Fund, No 201601201). Guo Tianxia made English review for the whole manuscript, express our thanks here.

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