Yeast one-hybrid system (Y1H) not only can be used to discover new transcription factors, but also to analyze the structure of transcription factors (Fields and Song, 1989; Kim et al., 1997). Many transcription factors found with Y1H are not from yeast, but from plants or animals. Generally, transcription factors from non-yeast need to be further verified in the cells of their source species. In order to verify the plant transcription factors found with Y1H, a set of plant or protoplast transient expression analysis system (Figure 1) matched with Y1H vector (pGBKT7) was constructed. The vector system consists of 4 vectors: pHQEff-6, pHQRep(3x), pHQRep(6x), and negative control vector pHQEff-1.

The effect vector pHQEff-6 (NCBI No. KJ184338.1) contains 35S promoter, DNA binding region of yeast transcription factor GAL4 (Ma et al., 1988), transcriptional activation region CRII of Arabidopsis leucine zipper transcription factor AtDPBF4 (Ma et al., 2019, Chinese patent, ZL 2015 1 0376978.2), and NOS terminator. The control vector phqeff-1 was the same as the effect vector. The control vector pHQEff-1 (NCBI No.KM985459) did not contain CRII, and others was the same as the effect vector. Report vector pHQRep(3x) (NCBI No.KJ184340.1) and pHQRep(6x) (NCBI No. KJ184341.1) contain mini35S promoter, DNA binding region recognition element UAS (upstream activating sequence), reporter gene GUS, and NOS terminator.

In the system constructed in this study, because the reporter vector used the UAS sequence recognized by GAL4, not only the reporter gene was not started by endogenous transcription factors, but also the transcription factors to be verified in the effect vector could be subcloned directly from Y1H vector. In addition, the two report vectors pHQRep(3x) and pHQRep(6x) constructed in this study contain different UAS copy numbers. There are three UAS in pHQRep(3x) and six UAS in pHQRep(6x). One of the reasons for this construction is that the expression intensity of reporter genes can be selected according to the needs of research work, and the other is to understand whether doubling the number of UAS will double the expression intensity of reporter genes. The results showed that the expression of report genes was significantly increased by doubling the number of UAS, but there was no quantitative relationship between the activity of report genes and the number of UAS. 

1 Results and Analysis

1.1 Construction of plant transient expression analysis vector

pUC19 plasmid was used as the starting vector, four recombinant fragments were inserted into its polyclonal sites, and four recombinant vectors were obtained for plant transient expression analysis: 1 control vector pHQEff-1 (NCBI No.KM985459), 1 effect vector pHQEff-6 (NCBI No.KJ184338.1), and 2 report vectors pHQRep(3x) (NCBI No.KJ184340.1), and pHQRep(6x) (NCBI No.KJ184341.1) (Figure 1; Figure 2).

1.2 Transient expression analysis of GUS gene of leaf protoplasts in Arabidopsis thaliana

Because of its low endogenous activity and wide dynamic range, GUS is widely used in plant transient expression system. However, with the extension of culture time, GUS enzyme activity increased steadily, and GUS enzyme had endogenous activity of protoplasts inArabidopsis thaliana (Figure 3).

We analyzed the GUS activity of mesophyll protoplasts in Arabidopsis thaliana cultured for 12 h after transformation. The relationship between the reaction time and the concentration of 4-MU and the analysis of CRII transcriptional activity of AtDPBF4 (Figure 3) showed that 4-MU concentration curve and the effect of total protein content on CRII transcription activation activity in experimental group 1 (pHQEff-6+pHQRep(3×)) was 0.73 nmol 4-MU·mg^-1·min^-1, and the GUS enzyme activity of experimental group 2 (pHQEff-6+pHQRep(6×)) was 0.96 nmol 4-MU·mg^-1·min^-1. The GUS enzyme activity of control group 1 (pHQEff-1+pHQRep(3×)) was 0.47 nmol 4-MU·mg^-1·min^-1, while the GUS enzyme activity of control group 2 (pHQEff-1+pHQRep(6×)) was 0.52 nmol 4-MU·mg^-1·min^-1. And the enzyme activity of mesophyll protoplasts in Arabidopsis thaliana without any vector treatment was 0.45 nmol 4-MU·mg^-1·min^-1. From the above results, we can see that the GUS activity of the experimental group is significantly higher than that of the control group, indicating that CRII of AtDPBF4 also has transcriptional activation activity in plant cells. Meanwhile, the combination of the reporter gene vector with 6 repeats of GAL4 binding site UAS_Gal1 and the effect vector with AtDPBF4 CRII had higher GUS activity than the combination of the reporter gene vector with 3 repeats of UAS_Gal1 and the effect vector with AtDPBF4 CRII, but the increasing trend of GUS activity was not linear with the number of repeats of UAS_Gal1.

2 Discussion

In the analysis of transcriptional activation activity, several transcription factors from plant cells can show transcriptional activation activity in yeast cells. For example, Wang et al. (2016) screened out proteins interacting with R2R3-MYB plant transcription factor SmPAP1 in Salvia miltiorrhiza with yeast two-hybrid system. While Han et al. (2016) constructed the pGBKT7 fusion expression vector containing the NAC plant transcription factor family genes of H. ammodendron and transferred it into yeast AH109 to analyze the transcriptional activation function. However, not every transcription factor can activate transcription in yeast and plant cells. For example, the full-length yeast transcription factor GAL4, mediated by GAL4 binding site, cannot activate the transcription of reporter gene in potato cells (Ma et al., 1988). On the contrary, the transcriptional activation domain of wheat HALF-1 transcription factor has transcriptional activation activity in potato BY2 cells but cannot activate the expression of downstream reporter genes in yeast cells (Okanami et al., 1996). In this study, a plant transient expression analysis system matched with Y1H system was constructed to determine whether CRII of AtDPBF4 can activate the transcription of reporter gene in plant cells. Experimental data show that CRII of AtDPBF4 has transcriptional activation activity in plant cells, which is consistent with previous results obtained by Y1H in yeast (Ma et al., 2019, Chinese patent, ZL 2015 1 0376978.2). In addition, the GUS activity of the combination of the reporter gene vector with 6 repeats of GAL4 binding site UAS_Gal1 and the effect vector with CRII containing AtDPBF4 increased by 31.5% compared with the combination of the reporter gene vector with 3 repeats of UAS_Gal1 and the effect vector with CRII containing AtDPBF4. As we expected, increasing the repeat sequence of GAL4 binding site (UAS_Gal1) in the reporter gene can improve the expression of downstream reporter genes, but this increase in activity does not have a linear relationship with the number of repeats of UAS_Gal1. In this study, the CRII of AtDPBF4 of mesophyll protoplasts in Arabidopsis thaliana not only laid the foundation for the study of transcription activation and regulation mechanism of transcription factor AtDPBF4, but also provided reference information for the selection of repeat sequence of GAL4 binding site UAS_Gal1 in the construction of transient expression vector and reporter gene vector.

3 Materials and Methods

3.1 Experimental materials

Escherichia coli DH5α, Saccharomyces cerevisiae AH109, Columbia-0 Arabidopsis thaliana, initiator plasmid pUC19, genetic element donor plasmid pCAMBIA1301, pGBKT7, and pUC19-AtDPBF4 were all preserved in the laboratory of Jianzhong Ma.

3.2 Construction of plant transient expression analysis vector

pCAMBIA1301 was used as template, three primers (Table 1) were for amplification of the CaMV35S-NosTer fragment. EcoRⅠ and HindⅢ were digested and ligated into pUC19 plasmid digested by the same restriction enzyme, and to obtain recombinant plasmid pUC19-CaMV35S-NosTer. New restriction sites XbaⅠ and BamHⅠ were introduced between 35S promoter and NosTer terminator. Meanwhile, pGBKT7 and pGBKT7-CRII were digested by XbaⅠ and BamHⅠ respectively to obtain Gal4DBD and Gal4DBD-CRII fragments, and then connected with pUC19-35Spro-Noster at XbaⅠ and BamHⅠ sites to obtain the control vector pHQEff-1. Besides, an amplified fragment Gal4DBD-CRII was inserted between XbaⅠ and BamHⅠ sites. And the recombinant plasmid named pHQEff-6 was used as the effect vector. Conserved region II is a piece of 22 amino acids, from a basic leucine zipper AtDPBF4 transcription factor, which plays a role of transcriptional activation in yeast and plants.

The primers (Table 2) from pCAMBIA1301 were for amplification of the Mini35S-GUS-NosTer fragment. This primer introduced XbaⅠ and Hind Ⅲ two restriction enzyme sites. The amplified fragment was subcloned into pUC19 between XbaⅠ and HindⅢ sites, and to prepare recombinant plasmid pHQMini35S-GUS-NosTer.

3.3 Construction of report gene

After chemical synthesis, the UAS bound to GAL1 and GAL4 was repeated three times in two Oligo-DNA fragments Gal4BS-F and Gal4BS-R (Table 3). The sticky ends of the two fragments were annealed, digested with EcoRⅠ and BamHⅠ, and then inserted into the mini-35S promoter (Table 3). The obtained plasmid was named as report vector pHQRep(3×) (Figure 1). The undigested annealed fragment was cloned into XbaⅠ site of pHQRep(3x) to make a report vector pHQRep(6×) (Figure 1).

3.4 Protoplast preparation and transformation in Arabidopsis thaliana and determination of report enzyme GUS activity

In this study, rosette leaves were collected from 3-4-week-old Arabidopsis thaliana seedlings for protoplast preparation and purification (Yoo et al., 2007), and GUS activity was determined by fluorescence spectrophotometer (F97Pro13007, Shanghai Lengguang Technology Co., Ltd.) (Gong et al., 2007; Sahoo et al., 2014). All plasmids were extracted with UNIQ-500 column plasmid DNA Extraction Kit (Sangon Biotech, China). In this study, 5 groups of transformation vector combinations were designed to analyze the transient expression of GUS gene of protoplasts in Arabidopsis thaliana. The experimental group included the combination of pHQEff-6+pHQRep(3x) and the combination of pHQEff-6+pHQRep(6x). The control group included the combination of pHQEff-1+pHQRep(3x) and the combination of pHQEff-1+pHQRep(6x), and the negative control group without any vector. 15 μg of each plasmid was used for transformation of protoplasts in Arabidopsis thaliana (Yoo et al., 2007). Protein content and GUS activity were determined after incubation in 37℃ water baths (Bradford et al., 1976; Jefferson et al., 1987). All the experimental groups and control groups were set up three parallel experiments, and each group was repeated three times. All the data were analyzed by SPSS20.0.

Authors’ contributions

ZY, HF, MYL designed and carried out the study. ZY, and MYL completed the data analysis and drafted the manuscript. ZY and MYL participated in the design of the study and results analysis. MJZ conceived of the project, directed the design of the study, data analysis, draft and revision. All authors read and approved the final manuscript. 

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 31560073, and No. 31860063).

Reference

Bradford M.M., 1976, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72: 248-254

https://doi.org/10.1016/0003-2697(76)90527-3

Fields S., and Song O.K., 1989, A novel genetic system to detect protein-protein interactions, Nature, 340(6230): 245-246

https://doi.org/10.1038/340245a0

PMid:2547163

Gong Q.H., Yu W.G., Dai J.X., Liu H.Q., Xu R.F., Guan H.S., and Pan K.H., 2007, Efficient gusA transient expression in Porphyra yezoensis protoplasts mediated by endogenous beta-tubulin flanking sequences, Journal of Ocean University of China, 6(1): 21-25

https://doi.org/10.1007/s11802-007-0021-x

Han J.D., Zhang H., Ni Z.Y., Yao Z.P., Wang Z., Ren C., Chen Q.J., and Ma H., 2016, Cloning and transcriptional activation analysis of NAC gene family from Haloxylon ammodendron, Jiyinzuxue Yu Yingyong Xueshengwu (Genomics and Applied Biology), 35(11):3163-3171

Jefferson R.A., Kavanagh T.A., and Bevan M.W., 1987, GUS fusions beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants, EMBO J., 6(13): 3901-3907

https://doi.org/10.1002/j.1460-2075.1987.tb02730.x

PMid:3327686 PMCid:PMC553867

Kim S.Y., Chung H.J., and Thomas T.L., 1997, Isolation of a novel class of bZIP transcription factors that interact with ABA-responsive and embryo-specification elements in the Dc3 promoter using a modified yeast one-hybrid system, Plant J., 11(6): 1237-1251

https://doi.org/10.1046/j.1365-313X.1997.11061237.x

PMid:9225465

Ma J., Przibilla E., Hu J., Bogorad L., and Ptashne M., 1988, Yeast activators stimulate plant gene expression, Nature, 344(6183): 631-633

https://doi.org/10.1038/334631a0

PMid:3165494

Okanami M., Meshi T., Tamai H., and Iwabuchi M., 1996, HALF-1, a bZIP-type protein, interacting with the wheat transcription factor HBP-1a contains a novel transcriptional activation domain, Genes Cells, 1(1): 87-99

https://doi.org/10.1046/j.1365-2443.1996.06006.x

PMid:9078369

Sahoo D.K., Sarkar S., Raha S., Das N.C., Jee J.B., Dey N., and Maiti I.B., 2014, Analysis of Dahlia Mosaic Virus full-length transcript promoter-driven gene expression in transgenic plants, Plant Molecular Biology Reporter, 33: 1572-9818

https://doi.org/10.1007/s11105-014-0738-9

Wang H.Q., Guo X.R., Yang X.B., Su J., and Cao X.Y., 2016, Screening of the proteins interacting with SmPAP1 of R2R3-MYB transcription factor from Salvia miltiorrhiza Bunge by Yeast two-hybrid system, Jiyinzuxue Yu Yingyong Xueshengwu (Genomics and Applied Biology), 35(10): 2819-2826 

Yoo S.D., Cho Y.H., AND Sheen J., 2007, Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis, Nat. Protoc., 2(7): 1565-1572

https://doi.org/10.1038/nprot.2007.199

PMid:17585298