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The promoter and the 5′-untranslated region of rice metallothionein OsMT2b gene are capable of directing high-level gene expression in germinated rice embryos

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Abstract

Key message

Critical regions within the rice metallothionein OsMT2b gene promoter are identified and the 5′-untranslated region (5′-UTR) is found essential for the high-level promoter activity in germinated transgenic rice embryos.

Abstract

Many metallothionein (MT) genes are highly expressed in plant tissues. A rice subfamily p2 (type 2) MT gene, OsMT2b, has been shown previously to exhibit the most abundant gene expression in young rice seedling. In the present study, transient expression assays and a transgenic approach were employed to characterize the expression of the OsMT2b gene in rice. We found that the OsMT2b gene is strongly and differentially expressed in germinated rice embryos during seed germination and seedling development. Histochemical staining analysis of transgenic rice carrying OsMT2b::GUS chimeric gene showed that high-level GUS activity was detected in germinated embryos and at the meristematic part of other tissues during germination. Deletion analysis of the OsMT2b promoter revealed that the 5′-flanking region of the OsMT2b between nucleotides −351 and −121 relative to the transcriptional initiation site is important for promoter activity in rice embryos, and this region contains the consensus sequences of G box and TA box. Our study demonstrates that the 5′-untranslated region (5′-UTR) of OsMT2b gene is not only necessary for the OsMT2b promoter activity, but also sufficient to augment the activity of a minimal promoter in both transformed cell cultures and germinated transgenic embryos in rice. We also found that addition of the maize Ubi intron 1 significantly enhanced the OsMT2b promoter activity in rice embryos. Our studies reveal that OsMT2b351-ubi(In) promoter can be applied in plant transformation and represents potential for driving high-level production of foreign proteins in transgenic rice.

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Acknowledgments

We thank Dr. Pei-Yin Wu for critical review of the manuscript. We also thank Dr. Maurice SB Ku for scientific discussions and valuable comments. This work was supported by grants from the National Sciences Council (NSC95-2311-B-415-004 and NSC96-2311-B-415 -003-MY3) of the Republic of China.

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  1. Department of Bioagricultural Science, National Chiayi University, Chiayi, 60004, Taiwan, ROC

    Chung-Shen Wu, Dai-Yin Chen, Chung-Fu Chang, Min-Jeng Li, Kuei-Yu Hung & Peng-Wen Chen

  2. Institute of Molecular Biology, National Chung Hsing University, Taichung, 40227, Taiwan, ROC

    Liang-Jwu Chen

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Supplementary material 1 (DOC 88 kb)

299_2013_1555_MOESM2_ESM.jpg

Supplementary material 2 (JPEG 1177 kb) Histochemical staining analysis of OsMT2b::GUS fusion gene in independent transgenic rice lines during seed germination and later stage of seedling growth and development. a. T2 seeds of homozygous transgenic lines MT-2-1, MT-5-1, and MT-7-1 carrying the OsMT2b::GUS gene were germinated and grown for 6 days and stained for GUS activity. b. T2 seeds of transgenic line MT-2-1 were germinated and grown for 10, 15, and 20 days and stained for GUS activity

299_2013_1555_MOESM3_ESM.jpg

Supplementary material 3 (JPEG 743 kb) Identification of the 5′-end of OsMT2b mRNA by primer extension analysis. Rice Seeds were germinated and grown in a 14-hour light/10-hour dark cycle at 28℃ for 6 days. Total RNA was purified from the embryo and subjected to primer extension analysis. A 25-nt MT-PE primer was 5′-end labeled with γ-32P ATP and used to synthesize cDNA fragment from total RNA isolated from germinated rice embryo. The extended products were electrophoresed and the resulting autoradiograph was shown. Lanes C, T, A, G provide the sequence ladders of the 5′-flanking region of OsMT2b cDNA using the MT-PE primer as a sequencing primer. The transcription start sites are indicated by the bold letters

299_2013_1555_MOESM4_ESM.jpg

Supplementary material 4 (JPEG 780 kb) The activity of αAmy8-ubi(In) promoter is much higher than the αAmy8-mt(In) promoter in rice embryo. a. Schematic representation of constructs for αAmy8-mt(In) and αAmy8-ubi(In) promoter analysis. b. Transient expression assay of luciferase activity in rice embryos. Rice embryos were transfetced with plasmids pαAmy8(+mt intron1)-Luc and pαAmy8(+ubi intron1)-Luc by particle bombardment. Transfected embryos were incubated at 28℃ for 24 h in the MS liquid medium lacking sucrose, and then pooled for luciferase activity assays. The luciferase activity reading obtained from αAmy8-ubi(In) were normalized to the reading obtained from αAmy8-mt(In), which was set to 1X. Error bars indicate the standard deviation of three replicate experiments for each construct

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Wu, CS., Chen, DY., Chang, CF. et al. The promoter and the 5′-untranslated region of rice metallothionein OsMT2b gene are capable of directing high-level gene expression in germinated rice embryos. Plant Cell Rep 33, 793–806 (2014). https://doi.org/10.1007/s00299-013-1555-6

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