Design of Construct Carrying GmDREB 6 to Enhance Soybean Gene Expression Related to Abiotic Stress Response

Glycine max (L.) Merrill is a crop that brings a lot of economic and nutritive values, however soybean is quite sensitive to stress. The applications of gene technology can improve resistance of soybean plants against external stress factors. The aim of this study was to conduct a transgenic vector containing GmDREB6 gene and determine the expression of gene encoding GmDREB6 in Nicotiana tabacum before transforming into soybean plants. The GmDREB6 artificial gene was synthesized containing nucleotide fragment encoding 230 amino acids, nucleotide fragment encoding cmyc antigen and nucleotide fragments with cut-off points of XbaI/SacI enzyme pair. The results indicated that the 35S-GmDREB6-cmyc construct was designed and transferred into tobacco plants. The GmDREB6 was incorporated in the genome and was expressed in transgenic tobacco plants at the transcriptional level. The transgenic vector pBI121_GmDREB6 well worked on the model tobacco plants. Therefore, it can be used for transferring into soybean plants to enhance soybean tolerance to abiotic stress.


I. INTRODUCTION
Soybean (Glycine max (L.) Merrill) not only brings economic, nutritive value but also can improve soil fertility.Soybean is considered as a crop that is quite sensitive to adverse impacts from externalities.Biotic (such as virus, bacteria and fungi) and abiotic (like drought, salinity, heat shock) stress have seriously affected the growth, development, productivity and quality of soybean.Some studies on improvement biotic and abiotic stress tolerance of Published on June 29, 2019.Thi Ngoc Lan Nguyen is with the Thainguyen University of Education, Thainguyen University, Thai Nguyen, Vietnam (e-mail: nguyenngoclan@dhsptn.edu.vn)Phutthakone Vaciaxa is with the Khangkhay Teacher Training College, Xiangkhoang, Laos (e-mail: phutthakone.kkct@gmail.com).
Van Son Le is with the Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi, Vietnam (e-mail: levanson@ibt.ac.vn).
Hoang Mau Chu is with the Thainguyen University of Education, Thainguyen University, Thai Nguyen, Vietnam (e-mail: chuhoangmau@tnu.edu.vn) ( * Corresponding Author) crops and soybeans using gene transfer have been reported [1]- [5].In 2015, Lo et al. generated a GmEXP1 construct to express recombinant expansion protein in tobacco plants.It was observed that overexpression of the GmEXP1 gene in transgenic tobacco plants.The transgenic tobacco plants have improved drought tolerance as a result of an increase of the length and volume of transgenic plant roots in comparison to non-transgenic plants [1].The 35S-GmDREB2-cmyc construct carrying GmDREB2 gene was designed and transformed into Nicotiana tabacum [2].According to these authors, the proline accumulation of the transgenic tobacco lines was higher than on non-transgenic plants during drought conditions.DREB (dehydrationresponsive element binding) transcription factor has been identified and its function is to activate genes involved in abiotic stress response.The cis-element of DREB gene and the trans-factor of DREB protein play an important role in gene expression to react to abiotic stress.Trans-factors bind to cis-elements to activate expression of functional genes in plants under stressed conditions.In soybean, it was detected that GmDREB6 gene has locus LOC100101914 on chromosome 5, the CDS region is 693 bp in length which encodes 230 amino acids.DNA-binding domain in plant proteins includes AP2 and EREBP.AP2 region of DREB6 protein is 59 amino acids in length.DNA binding site with 11 amino acids, which have positions at 60, 61, 63, 65, 67, 69, 73, 75, 82, 84, 87 respectively [6].The promoter region of GmDREB6 contains the cis-elements of GT-1 and DRE, namely DRE (-1113), GT-1 (-133, -1398, -1488, -1560, -1993).Based on the analysis results of OsDREB2A expression in transgenic soybean plants, Zhang et al. (2013) suggested that salt tolerance of the transgenic plants was improved because of the overexpression of OsDREB2A.The expression of 10 abiotic stress-response genes was identified to illustrate molecular mechanisms of OsDREB2A under salt stress conditions.There were four genes in OsDREB2A transgenic soybeans, including GmDREB6, GmP5CS, GmERF3, and GmERF7 that had a considerable rise in expression in comparison to none-transgenic plants [7].However, the relationship between the activity of GmDREB6 transcription factor and the expression level of GmP5CS gene in transcription and translation stages of soybean is an issue to be proved by experiments.In this study, we present design results of the construct carrying GmDREB6 genes and initially evaluate the activities of this structure on the model of tobacco plants.The results of this study will be fundamental for overexpression of the

Design of Construct Carrying GmDREB6 to Enhance Soybean Gene Expression Related to Abiotic Stress Response
Thi Ngoc Lan Nguyen, Phutthakone Vaciaxa, Thi Mai Thu Lo, Thi Hai Yen Nguyen, Thi Thanh Nhan Pham, Van Son Le, Hoang Mau Chu transcription factor GmDREB6 in the transgenic soybean plants.

A. Materials
The tobacco plants, K326, were used as the model plant for evaluation of the construct carrying GmDREB6 gene.pBI121 transgenic vector; the enzymes XbaI, SacI; Agrobacterium tumefaciens CV58.pBI121 transgenic vector contains neomycin-phosphotransferase II gene (nptII) and cauliflower mosaic virus 35S promoter (35S promoter).

B. Generating GmDREB6 transgenic construct and recombinant A. tumefaciens
Transgenic vector carrying GmDREB6 gene was designed following two basic steps: (1) design of independent structure including GmDREB6 gene, cmyc segment and the cutting positions of XbaI/SacI enzyme pair (GmDREB6cmyc); (2) insert the structure into plant transgenic vector, pBI121 to form recombinant vector pBI121_GmDREB6.

C. Agrobacterium-mediated transformation
Agrobacterium-mediated transformation via leaf infection and regeneration of tobacco plants was done as previously described by Topping (1998) [8].Tobacco leaves were cut into 1×1 cm pieces, soaked in cell suspension of recombinant A.tumefaciens in 10 mins, and then regenerated multi-shoots on MS medium with added BAP and kanamycin.The shoots transferred into RM medium with added kanamycin for root generation.The plantlets were grown in greenhouses.

D. Analysis of transgenic tobacco plants
Total DNA from tobacco leaves was extracted according to method of Saghai-Maroof et al. (1984) [9] using the Trizol Reagents Kit (Invitrogen).cDNA was synthesized from RNA using Maxima® First Strand cDNA Synthesis Kit (Fermantas).
GmDREB6 transgene was amplified from transgenic tobacco plants by PCR and transcription of GmDREB6 transgene was confirmed by RT-PCR.The primer pair, XbaI-DREB6-F/ DREB6-SacI-R is used for PCR: XbaI-DREB6-F: 5'-ATGAAGTTCAACCAACCACTTCAT-3' DREB6-SacI-R: 5'-ATTCAGATCCTCTTCTGAGATGAGT-3'.Incorporation of GmDREB6 transgene in the genome of tobacco plants was confirmed by Southern blot analysis (Southern 1975) [10].The GmDREB6 gene was amplified using PCR with the primer pair XbaI-DREB6-F/ DREB6-SacI-R.The probe DNA was marked with a Biotin Labeling Kit DNA DecaLabel using biotin-11-dUTP.Genomic DNA samples from the PCR-positive transgenic plants in T0 generation were cut by restriction enzyme SacI at 37°C.DNA fragments cut by SacI enzyme were evaluated by agarose gel electrophoresis and then DNA fragments were transferred from electrophoresis gel to the hybrid membrane and implement hybrid with probe DNA.The membrane was hybridized overnight at 44°C.The hybridization result was displayed on X-ray film.
Expression of GmDREB6 gene at the transcriptional level was analysed by RT-PCR with primer pair XbaI-DREB6-F/ DREB6-SacI-R.

A. Construction of transgenic vector carrying GmDREB6 gene
Based on information of GmDREB6 (cDNA) isolated from DT2008 Vietnamese soybean cultivar and GmDREB6 with EF551166 code on GenBank (Liu et al., 2007) [6], artificial GmDREB6 gene was designed with 741 bp in size and nucleotide sequence of GmDREB6 gene as follows: In which 5'gctctaga is the sequence contains the cutting position of XbaI enzyme and gagctcg-3 is the sequence contains the cutting position of SacI enzyme.
Gene GmDREB6 was structured by 693 bp coding region, 8 bp (GCTCTAGA) at 5' end (the cutting position of XbaI) and 7 bp (GAGCTCG) enzymes in 3' end (the cutting position of the enzyme SacI).A segment of 33 bp which encodes the cmyc antigen at the 3' end was added.Compared with the sequence of GmDREB6 isolated from soybean DT2008 and sequence of GmDREB6 with the code EF551166 on GenBank, the artificial GmDREB6 sequence has G replaced by T at position 417.
GmDREB6 gene was inserted to pUC18 vector.Transgenic vector pBI121 contained GUS gene.XbaI/SacI enzyme pair was used to cut and remove GUS gene from pBI121_GUS, and then insert GmDREB6 transgene into pBI121 transgenic vector, creating construct carrying transgene pBI121_GmDREB6 (Fig 1).The results of cutting pUC18-GmDREB6 to obtain GmDREB6 gene and removing GUS gene from vector pBI121_GUS were shown in Figure 2A.Recombinant vector pBI121_GmDREB6 which was transferred into E.coli DH5α, was cloned and tested by colony-PCR (Fig. 2B).
Plasmid pBI121_GmDREB6 extracted from E.coli DH5α was transferred into A.tumefaciens.The recombinant A. tumefaciens carrying GmDREB6 gene was cloned and selected (Fig. 3).The positive colonies of A.tumefacines was used to infect into tobacco.

B. Transferring the pBI121_GmDREB6 construct into tobacco plants
Pieces of tobacco leaf that were about 1cm 2 in size were cultured on MS media for 48 hours; after that, the leaf pieces were immersed in the A.tumeffaciens suspension in 20 minutes.The bacterial infected leaf pieces were transferred to the co-cultivation medium, the multi-shoot regeneration medium, the rooting medium respectively, to create transgenic tobacco plants (Fig. 4).From 180 samples in three independent experiments, 539 shoots were induced on multi-shoot regeneration and shoot elongation media supplemented with kanamycin, in which 309 elongated shoots were rooted.And 101 rooted plantlets were planted on the pots, among which 48 plantlets were transferred to the greenhouse.Thirteen transgenic plants which have well grown and developed, were analyzed to determine the presence of the GmDREB6 transgene.The results of PCR analysis with XbaI-DREB6-F/ DREB6-SacI-R primer pair showed that nine positive plants with the emergence of a DNA band in the same size of GmDREB6 (741 bp) (Fig. 5A).The PCR-positive transgenic plants in the T0 generation were labeled as T0-4, T0-5, T0-6, T0-7, T0-8, T0-9, T0-11, T0-12, T0-13.

IV. CONCLUSION
The GmDREB6 gene was designed with the size of 741 bp encoded 230 amino acids and the nucleotide segment encodes the cmyc antigen used to test the presence of recombinant GmDREB6 protein in transgenic plants.The 35S-GmDREB6-cmyc construct in recombinant vector pBI121 was designed and transferred into tobacco plants by Agrobacterium-mediated transformation.The GmDREB6 transgene was incorporated in the genome and expressed in transgenic tobacco plants at the transcriptional level.The transgenic vector pBI121_GmDREB6 well worked on the model tobacco plants, so it can be used to transferre into target plants improves abiotic stress tolerance.

Fig. 4 .
Fig. 4. Results of creating transgenic tobacco plants by infecting combinant through leaf pieces.A: The leaf pieces in the A.tumefaciens suspension; B: The transformed samples were transferred to the co-cultivation medium; C, D: Samples on multi-shoot regeneration medium and the shoot elongation medium; E: The shoots on the rooting medium; (F): The transgenic tobacco plants grown on substrates in the green house.