Methyl-CpG binding domain protein acts to regulate the repair of cyclobutane pyrimidine dimers on rice DNA

UVB radiation causes cyclobutane pyrimidine dimers (CPDs) to form on the DNA of living organisms. This study found that overexpression of the silicon absorbance gene Lsi1 reduced the accumulation of CPDs in rice, which profited from the reactivation by photolyase. The transcript abundance of deoxyribodipyrimidine photolyase (Os10g0167600) was generally correlated with the silicon content of the rice, and the up-regulation of Os10g0167600 was found to be highest in the UVB-treated Lsi1-overexpressed (Lsi1-OX) rice. A trans-acting factor, methyl-CpG binding domain protein (OsMeCP), was found to interact with the cis-element of Os10g0167600. The nucleic location of OsMeCP effectively enabled the transcriptional regulation. Compared with the WT, the level of OsMeCP was lower in the Lsi1-OX rice but higher in the Lsi1-RNAi line. Rice cultured in a high silicate-concentration solution also exhibited less OsMeCP abundance. Overexpression of OsMeCP led to lower Os10g0167600 transcript levels and a higher CPD content than in the WT, but the reverse was true in the OsMeCP-RNAi line. These findings indicate that OsMeCP acts as a negative regulator of silicon, and can mediate the repression of the transcription from Os10g0167600, which inhibits the photoreactivation of the photolyase involved in the repair of CPDs.


Silicon, CPD, and 6-4 PPs contents in the Lemont rice accession with Lsi1-OX, Lsi1-RNAi, and WT.
Overexpression of Lsi1 in the Lemont rice accession (Lsi1-OX) resulted in increased silicon content in the leaves, and the silicon content in the Lsi1-RNAi transformed Lemont was notably lower than that in the WT (Fig. 1A). Exposing the rice to UVB radiation led to increased CPD and 6-4 PP contents in the leaf DNA. There was no obvious difference in the 6-4 PPs contents between the transformed rice and the WT under the same light conditions (Fig. 1B). In contrast, the CPD content was significantly lower in the leaves of the Lsi1-OX line than in the WT after exposure to UVB radiation. A 35.17% decrease was found in the Lsi1-OX line compared with the WT. However, a notable increase of 33.16% was found in the Lsi1-RNAi line compared with the WT (Fig. 1C). These results indicate that the increased Lsi1 expression in the rice enhanced the silicon content in the leaves and reduced the CPD content in the DNA of the leaves under UVB irradiation, while inhibition of Lsi1 expression in the rice samples led to the opposite result.
Photolyase gene expression and the photoreceptor in the Lemont rice accession with Lsi1-OX, Lsi1-RNAi, and WT. Based on our transcriptome sequencing data from the Lsi1-OX and Lsi1-RNAi transformed Lemont rice lines and the WT rice, which were irradiated with UVB for 5h, and the control group (full data not shown here), we found seven genes that encoded photolyase or the photoreceptor, and which were differentially expressed in the three UVB-treated rice lines and the control condition. Among these genes, the transcription of Os10g0167600 was up-regulated when the rice samples were exposed to UVB. Changes of 1.51, 2.59, and 1.57 times were detected in the WT, Lsi1-OX, and Lsi1-RNAi lines, respectively, when comparing the UVB group to the control group. The gene encodes deoxyribodipyrimidine photolyase to repair the CPDs on the UVB-damaged DNA. Similarly, up-regulation of the Os03g0343400 gene expression increased by 1.57, 1.12, and 1.31 times in the three rice lines, and the gene encoded a putative photolyase/blue-light receptor PHR2. In contrast, the transcript abundance of Os09g0532700, which is encoded as the deoxyribodipyrimidine photolyase protein-like family, was down-regulated in all three UVB-treated rice lines. Os02g0204400 gene expression was slightly down-regulated in the UVB-treated WT and Lsi1-RNAi transformed lines, but it was obviously up-regulated in the Lsi1-OX transformed line. Os02g0204400 encodes the (6-4) DNA photolyase that catalyzes the photoreactivation of the 6-4 PPs.
The other three genes also exhibited changes in their levels of expression. Os06g0661800 was notably up-regulated in all three rice lines, as it is encoded as cryptochrome with blue-light photoreceptor activity but lacks photolyase activity. In contrast, the transcription levels of Os02g0625000 and Os02g0573200 were down-regulated in the UVB-treated WT and Lsi1-OX lines, but were slightly up-regulated in the UVB-treated Lsi1-RNAi rice (Fig. 2). Comparison of the changes in the transcript levels of the genes suggests that Os10g0167600 is the most correlative to silicon in rice and plays a role in the photoactivation of CPDs on damaged DNA. The transcriptional abundance of the seven genes was calculated as the reads per kilobase per million reads (RPKM), which is a method of quantifying gene expression from RNA sequencing data by normalizing the total read length and the number of sequencing reads. The RNA sequencing data were generated from the mRNA of the UVB-treated Lsi1-OX, -RNAi, and WT lines, respectively, and the control groups. The full RNA sequencing data are not shown here.
Scientific RepoRts | 6:34569 | DOI: 10.1038/srep34569 Gene dynamic expression of Os10g0167600 in the Lsi1-OX, -RNAi transformed Lemont, and WT lines. qPCR was used to detect the gene dynamic expression of Os10g0167600 in the leaves of rice exposed to UVB irradiation for 0.5, 1, 2, 3, and 5 hours. Compared with the expression of Os10g0167600 in the rice samples before UVB treatment, UVB irradiation resulted in the up-regulation of Os10g0167600 gene expression in the Lsi1-OX transformed and WT lines, and the level of up-regulation increased with time. The increase in the transcription level was higher in the Lsi1-OX transformed line than in the WT and peaked at 5 h of irradiation, at which point there was a 2.02 times increase in the UVB-irradiated Lsi1-OX transformed line compared to its control before treatment. However, no obvious change in Os10g0167600 gene expression was found in the Lsi1-RNAi transformed line after UVB treatment (Fig. 3A). Compared with the control group under the same exposure time, the gene expression was also found to be up-regulated in the UVB-treated group, with a 2.95 times increase in up-regulation in the UVB-treated Lsi1-OX transformed line (Fig. 3B). The dynamic change in Os10g0167600 gene expression detected by qPCR confirmed the results of the RNA-seq. Based on these results, Os10g0167600 was selected for further study.
Promoter of Os10g0167600 and the interacted trans-acting factor. A 2154-bp fragment from the upstream region of the 5′ UTR of Os10g0167600 was amplified from Lemont ( Fig. 4A) and then incubated with the natural protein ( Fig. 4B) to reveal the interacting trans-acting factor according to the DNA pull-down method (Fig. 4C). Based on the results, an obvious protein band was obtained with SDS-PAGE separation. Nine proteins (see Table S2) were identified by LC-MS. Methyl-CpG binding domain containing protein (OsMeCP) was regarded as the putative interacting trans-acting factor (Table 1).

Subcellular localization of OsMeCP in rice.
It is well known that gene regulation usually occurs in the nucleus and that the trans-acting factor acts in the nucleus. As indicated by the nuclear marker tag (Fig. 5A), the The gene transcription levels of OsMeCP were then detected in the three rice samples. OsMeCP was down-regulated in the Lsi1-OX rice compared with the WT, but was up-regulated in the Lsi1-RNAi line (Fig. 6A), suggesting that the silicon in the rice negatively regulated OsMeCP gene expression. Similarly, Os10g0167600 gene expression was down-regulated in the Lsi1-OX rice and slightly up-regulated in the Lsi1-RNAi line, compared with the WT (Fig. 6B). This can be attributed to the low CPD content in the rice DNA under normal light condition (Fig. 1C). When the rice samples were exposed to UVB radiation, the Lsi1-OX rice still showed down-regulation of OsMeCP gene expression compared with the WT, and the reverse was true in the Lsi1-RNAi line (Fig. 6C). However, in contrast to OsMeCP gene expression in the rice samples, Os10g0167600 gene expression was found to be up-regulated in the Lsi1-OX line but down-regulated in the Lsi1-RNAi line compared with the WT (Fig. 6D). Thus, it can be deduced that OsMeCP may have transcriptionally repressed Os10g0167600 gene expression in the rice under UVB radiation.

Protein expression of OsMeCP in the Lsi1-OX, -RNAi transformed Lemont, and WT lines.
The protein expression levels of OsMeCP were also detected in the transformed and WT rice lines under UVB radiation. It was found that the protein expression obviously increased in the rice exposed to UVB irradiation. The overexpression of Lsi1 inhibited the expression of OsMeCP, and the reverse was true in the Lsi1-RNAi line from the control group and UVB-treated group conditions (Fig. 7). These results further confirmed that the silicon in the rice played a negative role in the regulation of OsMeCP protein expression.

OsMeCP expression and CPD content in rice cultured with different silicon concentrations.
Rice cultured in different silicate concentrations of the hydroponic solution showed different levels of OsMeCP. Increasing the silicate concentration in the solution resulted in reduced protein expression levels of OsMeCP in the rice. The protein expression level of OsMeCP was increased in the UVB-treated rice compared with the control group, where the two rice samples were under the same silicate concentration. Moreover, the protein level of OsMeCP was still the highest in the rice cultured in the medium containing 0.5 mM silicate, and lowest in that containing 2.5 mM of silicate (Fig. 8A). The gene expression level of Os10g0167600 decreased slightly less in the UVB normal condition than in the rice cultured in the increasing silicate concentration. This may have been because the rice under the normal growth conditions had little DNA damage, as indicated by the lower CPD content on the rice leaves. When the three different cultured strains of rice were exposed to UVB radiation, the gene transcription of Os10g0167600 increased in all cases and was highest in the 2.5 mM silicate cultured rice (Fig. 8B). Moreover, the CPD content was highest in the 0.5 mM silicate cultured rice, significantly decreased in  the rice cultured with the hydroponic nutrient solution that contained 1.5 mM silicate, and was notably lowest in the 2.5 mM condition. Detection of the CPD content in the leaves of rice cultured in the different silicate concentrations showed that increasing the silicate concentration in the solution obviously repressed OsMeCP protein expression, and resulted in enhanced Os10g0167600 gene expression in the UV-B treated rice, which strengthened the photoreactivation and significantly reduced the CPD content in the cultured rice leaves (Fig. 8C).

OsMeCP-OX and -RNAi transformed rice and DNA damage. The respective OsMeCP-OX and -RNAi
transformed Lemont rice lines were generated. The protein expression level of OsMeCP in the overexpression transformed line was found to be greater than in the WT, and it was obviously reduced in the OsMeCP-RNAi line (Fig. 9A). Comparison of the changes in Os10g0167600 gene expression in the two transformed rice lines and the WT line under normal growth conditions showed that Os10g0167600 gene expression was up-regulated in the OsMeCP-RNAi line but down-regulated in the OsMeCP-OX line compared with the WT (Fig. 9B). When the three lines were exposed to UVB radiation, both OsMeCP-RNAi and the WT showed up-regulation of Os10g0167600 gene expression compared with the control group, and the increase in the up-regulation was higher in the OsMeCP-RNAi transformed line than in the WT. In contrast, a slight down-regulation of Os10g0167600 gene expression was found in the OsMeCP-OX transformed line (Fig. 9C). The highest up-regulation of Os10g0167600 gene transcription in the OsMeCP-RNAi transformed line led to the lowest CPD content in the UVB-treated leaves, compared with that of the WT and the OsMeCP-OX transformed line. Moreover, the OsMeCP-OX transformed line had the highest CPD content among the three rice lines (Fig. 9D), owing to the slightly higher down-regulation of Os10g0167600 than in the WT. These results further confirmed the transcriptional repression of OsMeCP on Os10g0167600 gene expression under UVB radiation, which resulted in reduced photolyase activity in the UVB-exposed rice. Discussion UVB radiation causes the formation of covalent links by reactions localized on the C= C double bonds, which results in the accumulation of CPDs and 6-4PPs in DNA 19 . To reduce the damage, the photoreactivation of the CPD photolyase repairs the CPD in the plant chloroplasts, mitochondria, and nuclei 20 , which is essential for plant survival after exposure to UVB-containing sunlight.
In rice plants, Os10g0167600 encodes the deoxyribodipyrimidine photolyase, which is involved in the repair of UV radiation-induced DNA damage and catalyzes the light-dependent monomerization (300-600 nm) of CPDs, which is required for plant survival in the presence of UVB light. However, photolyase is not involved in the repair of 6-4 PPs 21,22 . In contrast to Os10g0167600, Os02g0204400 encodes the (6-4) DNA photolyase that catalyzes the photoreactivation of 6-4 PPs. The Os03g0343400 protein is a putative photolyase/blue-light receptor PHR2, and Os09g0532700 is deoxyribodipyrimidine photolyase family protein-like, and both have hydrolase activity. Unlike the photolyase, Os02g0625000, Os02g0573200, and Os06g0661800 encode cryptochromes with blue-light photoreceptor activity, but there is a lack of photolyase activity.
Although the transcript levels of these seven genes were detectable, we observed different expression levels in the three rice lines with different levels of Lsi1 abundance. Among these genes, the transcription of Os10g0167600 was correlated with the silicon content in the rice, and the highest increase was found in the Lsi1-OX transformed rice under UVB radiation. The resulting CPD content was negatively related to the gene abundance, and were lowest in the Lsi1-OX transformed rice. Such a reduction in the DNA lesion would maintain the UVB resistance of rice. Recently, Teranishi et al. 13 found that CPD photolyase was overexpressed in both UVB-sensitive O. sativa Norin 1 (japonica), and UVB-hypersensitive O. sativa Surjamkhi (indica) when determining its function in relation to UVB resistance in rice. Their results showed that CPD photolyase is a vital factor for evaluating the UVB sensitivity of O. sativa 13,14 . Similar results were found in a study on Arabidopsis thaliana 23,24 .
Gene expression can be regulated by trans-acting factors, which are known as transcription factors. These regulators interact with the cis-regulatory elements to activate gene transcription. In our study, the Os10g0167600 promoter was associated with the CpG islands, and OsMeCP was found to interact with the promoter to regulate the gene transcription. Protein subcellular locations are closely linked to their biological functions, and, in this study, the subcellular location of OsMeCP was found to be in the nucleus, which confirms its role in the regulation of transcription. A decrease in the protein abundance of OsMeCP in the rice cultured under the higher silicon condition indicates that silicon acts as a negative factor in the regulation of OsMeCP expression. The methyl-CpG-binding domain, which consists of about 70 residues, possesses a unique α /β -sandwich structure with characteristic loops, and is able to bind single methylated CpG pairs as a monomer, which is involved in recruiting histone deacetylases to methyl CpG-enriched regions in the genome to repress transcription 25 . We found that rice with a higher silicon content exhibited lower protein abundance of OsMeCP in both the Lsi1-OX transformed line and the WT cultured in high silicate-concentration solutions. This suggests that OsMeCP is a negative feedback of silicon. However, the changes in the gene transcription levels of Os10g0167600 in the Lsi1-OX and Lsi1-RNAi transformed lines compared with the WT, and in the rice cultured in different silicon-concentration solutions, were opposite to the OsMeCP expression level in the same rice. This finding indicates the repression of OsMeCP in the transcription of Os10g0167600 in the UVB-treated rice. Nan et al. 26 documented that an abundant nuclear protein, the methyl-CpG-binding protein MeCP2, interacts specifically with methylated DNA and mediates the transcriptional repression. MeCP2 binds tightly to the chromosomes in a methylation-dependent manner and contains a transcriptional repression domain that can function at a distance in vitro and in vivo.
Overexpression of OsMeCP in rice also resulted in the down-regulation of Os10g0167600 gene expression, while the gene transcription level of Os10g0167600 was up-regulated in the OsMeCP-RNAi line. These results further clarify that OsMeCP acts as a negative regulatory factor in the transcription of Os10g0167600. The rice exposed to UVB radiation showed up-regulation of Os10g0167600 gene expression, but it was still highest in the OsMeCP-RNAi line, which led to the lowest amount of CPDs on the DNA. In contrast, the highest CPD content was found in the OsMeCP-OX transformed line, which was attributable to the slight down-regulation of Os10g0167600 gene expression under UVB treatment compared with the control group. Thus, the transcriptional repression on Os10g0167600 was obviously mediated by OsMeCP.
In conclusion, increasing the exogenous silicon concentration reduced the protein expression of OsMeCP in rice, and the silicon content in rice negatively regulated the protein expression of OsMeCP. The protein abundance of OsMeCP was also lower in the Lsi1-OX transformed line than in the WT rice, whereas the reverse was true for the Lsi1-RNAi line. OsMeCP repressed the transcription of Os10g0167600 gene expression, leading to down-regulation of Os10g0167600 in the rice samples. Inhibition of OsMeCP increased Os10g016760 gene expression, which reduced the CPD content in the rice under UVB radiation, while overexpression of OsMeCP led to the reverse results. Overall, our results indicate that OsMeCP is a negative regulator of silicon uptake in rice and plays a role in repressing Os10g0167600 gene transcription, which dominates the photorepair of damaged DNA in rice following UVB radiation (Fig. 10).

Materials and Methods
Plant materials. The Lemont rice accession (UVB-tolerant; introduced from the United States) was used in this study. The Lsi1-OX and Lsi1-RNAi transformed lines, which were generated in our previous study, had significantly higher or lower levels of silicon than the WT 18 .
The WT and transformed rice seeds were surface-sterilized with 25% NaClO for 30 min and then soaked in sterilized ddH 2 O overnight. The seeds were then placed in a temperature-controlled incubator to germinate at 30 °C, and the germinated seeds were sown in separate seedling plates. At the three-leaf stage, uniform seedlings from each plant were selected and transferred onto a Styrofoam plate (with holes spaced at 5 × 6 cm). The seedlings were affixed to the plate by inserting a cotton plug into each hole. The Styrofoam plate was allowed to float in a pot (45 × 35 × 15 cm) filled with 10 L of rice (Oryza sativa L.) culture solution (silicate-containing solution) with the following composition: 482 mg (NH 4  O. The solution was changed every week. The pot was continuously aerated, and its outer side was painted black to prevent algae growth. The pH value of the solution was maintained between 5.5 and 6.0 throughout the experiment. The method follows that of Fang et al. 18 , which is a minor optimization of the technique of Yoshida et al. 27 . At the five-leaf stage, the transformed rice line and WT rice seedlings were exposed to enhanced UVB radiation following the method described by Fang et al. 18,28 . The procedure was as follows. Fluorescent lamps (40 W, Beijing Electric Light Sources Research Institute, China) were used as the source of UVB radiation. The lamps were suspended above the rice plants, and UV radiation of wavelengths less than 280 nm (i.e., UVC) was eliminated by wrapping the lamps in a 0.1-mm film of cellulose diacetate (West Design Product Co., Ltd, United Kingdom). The distance between the rice canopy and the lamps was 30 cm, and four lamps were used. UVB radiation was applied at the top of the leaf canopy from 10 am to 3 pm at an intensity of 18.6 kJ • m −2 • d −1 . The rice seedlings used as a control were wrapped in a mylar film to filter UVB and UVC and placed under the lamps. The UVB-exposed and control rice leaves were sampled at 0.5, 1, 2, 3, and 5 h, and were immediately frozen in liquid nitrogen and stored at − 80 °C for isolation of the total RNA to detect Os10g0167600 gene expression. The sample of leaves treated for 5 h was also used to determine the silicon content and extract natural protein for the DNA-pull down.
Detection of the rice silicon content. The fresh rice leaves were ground into powder in liquid nitrogen and oven-dried at 70 °C. Each 0.1 g portion of the dry powder was digested in 3 ml of 50% NaOH (W/V) in an autoclave sterilizer at 121 °C for 30 min. The mixture was centrifuged, and the supernatant liquor was diluted with ddH 2 O to 50 mL. One milliliter of the solution was added to 30 ml of 20% acetic acid (V/V) and 10 ml of ammonium molybdate solution (54 g/L; pH 7.0). The mixture was blended and allowed to sit for 5 min; 5 ml of 25% tartaric acid solution (W/V) and 1 ml of reducing agent was then added, and the mixture was finally diluted with 20% acetic acid to 50 ml. To prepare the reducing agent, 25 ml of ddH 2 O was dissolved with 2 g of Na 2 SO 3 and 0.4 g of 1-amino-2-naphthol-4-sulfonic acid, and another 200 ml of ddH 2 O with 25 g NaHSO 3 was combined and diluted with ddH 2 O to 250 mL.
The absorbance of the rice leaf solution was detected with a spectrophotometer at a wavelength of 650 nm. For the standard curve of the silicon content, a gradient of Na 2 SiO 3 concentrations from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 mg/ml was taken to conduct the same reaction and then detected at OD 260 .  Damage ELISA Kit (CPD and 6-4 PP quantitation, respectively; Cell Biolabs, Inc., USA) following the manufacturers' instructions.

Detection of
Os10g0167600 gene expression in transformed and WT rice. The transcriptional abundance of the seven genes that encode photolyase or the photoreceptor was compared with our RNA-seq data to select the gene with the highest correlation with silicon. To detect the target photolyase gene expression in the UVB-irradiated Lsi1-OX and Lsi1-RNAi transformed lines and the WT, we designed gene-specific primers for Os10g0167600 (see Table S1), and used a real-time quantitative polymerase chain reaction (qPCR) to detect the relative mRNA expression levels of the genes in the UVB-treated group and the control group. The reaction was performed in an Eppendorf realplex 4 cycle, and the threshold cycle values (Ct) were recorded for each candidate mRNA in both the control and test samples. The 2 −△△Ct method was used to analyze the relative quantification of the gene expression 29 , and the gene expression level of β-actin was taken as a reference to normalize the candidate genes in the test sample.
Cloning of the specific photolyase gene promoter and pulling the interacted proteins. Based on the comparative study of photolyase gene expression in the UVB-treated Lsi1-OX and -RNAi lines and the WT, Os10g0167600 was selected as the available gene that was correlated with the silicon content of the rice strains and enhanced their capacity to repair the UVB damage on DNA. A 2134-bp upstream fragment of the 5′ end of the ORF of the gene was cloned and labeled with biotin (see Table S1). The natural proteins of the Lemont rice leaf were extracted with phosphate-buffered saline solution. The purified DNA fragment was mixed with the protein to reveal the interacted proteins. The DNA fragment was immobilized with streptavidin-coupled beads from the Dynabeads ® kilobaseBINDERTM Kit (Thermo Fisher Scientific Inc., USA), and the interacted proteins on the DNA fragment were then eluted with 1 M NaCl and forwarded to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Liquid chromatography-mass spectrometry (LC-MS; LTQ, Thermo Scientific) was performed to identify the interacted proteins.
Gene transcript levels of the DNA interacted proteins. A protein that contains the DNA binding domain, methyl-CpG binding domain protein (OsMeCP), was selected to detect the transcription levels in the Lsi1-OX, -RNAi transformed, and WT rice lines under UVB radiation for 5 h and the control. The specific primers for the encoding genes were as follows: OsMeCP-F: 5′ -CTC ATC TGG CAG AAA GGG-3′ and OsMeCP-R: 5′ -GGA AGG CTC AGT TGG GTT-3′ . qPCR was used to detect the gene expression levels in the test rice.

Protein expression detected by Western blot. To investigate the protein expression levels of OsMeCP
in the different UVB-treated rice lines and the control we used monoclonal antibodies of OsMeCP (Abmart Inc., USA). The leaf proteins of the three UVB-treated rice samples and the control were extracted and separated by SDS-PAGE, and the monoclonal antibodies were found to be immune to the total protein. A Clarity Western ECL Kit (Bio-Rad Laboratories, Inc., USA) was used for the staining, and the protein expression level was checked with the ChemiDoc ™ MP System (Bio-Rad Laboratories) under chemiluminescence.

OsMeCP protein expression levels in rice cultured in different silicon concentrations of hydroponic solution.
To detect the OsMeCP protein abundance, samples of the Lsi1-OX transformed Lemont rice were cultured in Yoshida culture solution with different silicate concentrations: 0.5 mM, 1.5 mM, and 2.5 mM. The rice under the three silicon concentrations was exposed to UVB radiation at the five-leaf stage, and rice wrapped in mylar film before being placed under the lamps was again used as the control. After 5 h, the respective rice leaves were sampled to detect OsMeCP protein abundance, Os10g0167600 gene expression, and the CPD content.
Protein subcellular localization of OsMeCP. The protoplast of rice was prepared to study the protein location. The full-length CDS of OsMeCP was amplified with gene-specific forward primer location CPG-F: 5′ -ATC GCT CTA GAA TGG CCA CGG CCG GCG ACG A-3′ and location CPG-R: 5′ -TAT AGG ACT AGT GGT GCA CTT CAC GGC AGA GG-3′ containing Xba I and Spe I sites (underlined). The gene was inserted into a plant binary expression vector, pCambia-eYFP-2300s, to create a recombinant plant transient expression vector and then transformed into the rice protoplast. The cell was cultured for two days to completely express the protein, and an Olympus Fluoview FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan) was used to observe the protein subcellular localization. The fluorescence images were acquired at 568-nm excitation and 592-nm emission. A monomeric far-red fluorescent protein TagFP635 (scientific name mKate) that tagged the nuclear localization signal protein localized to the nucleus was taken as the standard marker.
Overexpression and RNA interference of OsMeCP in rice. To further confirm the gene function of OsMeCP, the full-length CDS of the gene was again amplified with primers containing different recognition sites for Sac I and Bam H I, respectively, in forward primer OsMeCP-OX-F: 5′ -TAT ACG AGC TCA TCC CCA AAT CCC CAC ACG TC-3′ and reverse primer OsMeCP-OX-R: 5′ -TAG CGG GAT CCC CAG CAA CGT CAG TTC CTT GG-3′ . The gene was then inserted into another plant binary expression vector, pCambia-1300. A 347 base pair (bp) partial coding region downstream of the ATG of rice OsMeCP was cloned with a forward primer 5′ -CGGGATCCAAATGAAGAAGC GAAAGACG-3′ and a reverse primer 5′ -GGGGTACCAAGGCTCAGTTGGGTTGC-3′ containing BamH I and Kpn I sites (underlined), respectively. The same fragment was again amplified with the primers containing different recognition sites for Sac I and Spe I, respectively, in forward primer 5′ -CGAGCTCAAATGAAGAAGCGAAAGACG-3′ and reverse primer 5′ -GGACTAGTAAG GCTCAGTTGGGTTGC-3′ . Both fragments were inserted into pTCK303 to create an OsMeCP-RNAi stability vector. The two recombinant vectors were then transformed into the Agrobacterium strain EHA105, and the process of rice genetic transformation followed the methods described by Fang et al. 18 . After the positively transformed rice was confirmed and selected, the transformed rice and the WT were exposed to UVB radiation for 5 h, and the second top leaves of the rice plants were sampled to extract DNA and RNA, respectively, to detect the CPD contents and Os10g0167600 gene expression in the different types of rice under UVB irradiation.