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Molecular Breeding

, Volume 30, Issue 1, pp 23–32 | Cite as

Mitochondrial genes atp6 and atp9 cloned and characterized from ramie (Boehmeria nivea (L.) Gaud.) and their relationship with cytoplasmic male sterility

  • Xiao-Li Liu
  • Shou-Wen Zhang
  • Ji-Qiang Duan
  • Guang-Hui Du
  • Fei-Hu Liu
Article

Abstract

Cytoplasmic male sterility (CMS) in plants is known to be associated with structural change and the presence of new chimeric genes in mitochondrial DNA (mtDNA). In this study, the atp6 and apt9 gene fragments were cloned from mtDNA of a CMS line and its maintainer and restorer lines of ramie (Boehmeria nivea (L.) Gaud.) using PCR and degenerate primers that were designed according to the conserved sequences within the coding region of chondriogenes atp6 and atp9 of some dicotyledons recorded in GenBank. In spite of incompleteness of the coding region, the cloned fragments showed a homology of over 94% and over 85% with atp6 and atp9 genes, respectively, from the dicotyledons in GenBank. The whole sequences of atp6 and atp9 genes including the complete open reading frames were cloned by amplifying the 3′ and 5′ end unknown sequences of these gene fragments using a DNA Walking strategy. The atp6 gene showed no difference among the CMS line, maintainer and restorer lines of ramie in DNA sequence, transcription and translation, and in the levels of protein. However, compared to the atp9 gene from the maintainer and restorer lines, within the coding region, the atp9 gene from the CMS line had a number of different nucleotides and a sequence deficiency of as many as 21 nucleotides at the 3′ end. An unusual high expression of the atp9 gene in the CMS line at the budding and full-bloom stages was revealed by RT-PCR analysis. The results indicated that the variation in DNA sequence and its encoding product, and/or the abnormal expression of the atp9 gene in the CMS line, may be closely related to male sterility in ramie. This work provides basic knowledge for understanding the function of the atp9 gene causing CMS in ramie and subsequently the molecular genetic mechanisms.

Keywords

Ramie (Boehmeria nivea (L.) Gaud.) Cytoplasmic male sterility (CMS) atp6 gene atp9 gene DNA Walking RT-PCR 

Notes

Acknowledgments

The authors are grateful to the financial support provided by the National Natural Science Foundation of China (Project No: 30360058 and Project No 30971825).

Supplementary material

11032_2011_9595_MOESM1_ESM.docx (15 kb)
Supplementary material 1 (DOCX 15 kb)
11032_2011_9595_MOESM2_ESM.doc (2 mb)
Supplementary material 2 (DOC 2054 kb)

References

  1. Akagi H, Sakamoo M, Shinjyo C (1994) A unique sequence located downstream from the rice mitochondrial atp6 may cause male sterility. Curr Genet 25:52–58PubMedCrossRefGoogle Scholar
  2. Dewey RE, Timothy DH, Levings CS (1991) Chimeric mitochondrial genes expressed in the C male-sterile cytoplasm of maize. Curr Genet 20:475–482PubMedCrossRefGoogle Scholar
  3. Dieterich JH, Braun HP, Schmitz UK (2003) Alloplasmic male sterility in Brassica napus (CMS ‘Tournefortii-Stiewe’) is associated with a special gene arrangement around a novel atp9 gene. Mol Genet Genomics 269:723–731PubMedCrossRefGoogle Scholar
  4. Edqbist J, Bergman P (2002) Nuclear identity specifies transcriptional initiation in plant mitochondria. Plant Mol Biol Rep 49:59–68CrossRefGoogle Scholar
  5. Hanson MR, Bentolia S (2004) Interactions of mitochondrial and nuclear genes that affect male gametophyte development. Plant Cell 16:S154–S169PubMedCrossRefGoogle Scholar
  6. Hong D-F, Wang L–L, Yang G-S (2006) Estimation of techniques about cloning the flanking DNA sequence. Mol Plant Breed 4:280–288Google Scholar
  7. Horn R, Kohler RH, Zetsche K (1991) A mitochondrial 16 kDa protein is associated with cytoplasmic male sterility in sunflower. Plant Mol Biol Rep 17:29–36CrossRefGoogle Scholar
  8. Iwahashi M, Kyozuka J, Shimamoto K (1993) Processing followed by complete editing of an altered mitochondrial atp6 RNA restores fertility of cytoplasmic male sterile rice. EMBO J 12:1437–1446Google Scholar
  9. Kadowaki K, Harada K (1989) Differential organization of mitochondrial genes in rice with normal and male sterile cytoplasm. Jpn J Breed 39:179–186Google Scholar
  10. Kadowaki K, Suzuki T, Kazama S (1990) A chimeric gene containing the 5’ portion of atp6 is associated with cytoplasmic male-sterility of rice. Mol Gen Genet 224:106–116CrossRefGoogle Scholar
  11. Kaul MLH (1988) Male sterility in higher plants. Springer, BerlinCrossRefGoogle Scholar
  12. Kim DH, Kim BD (2006) The organization of mitochondrial atp6 gene region in male fertile and CMS lines of pepper (Capsicum annuum L.). Curr Genet 49:59–67PubMedCrossRefGoogle Scholar
  13. Levings CS III, Pring DR (1976) Restriction endonuclease analysis of mtDNA from normal and Texas male sterile maize. Science 193:158–160PubMedCrossRefGoogle Scholar
  14. Li Z-D (1989) Physiol-biochemistry and genetic breeding of ramie. China Agriculture Press, BeijingGoogle Scholar
  15. Liu F-H, Liang X-N, Huang H-Q (1998) Identification of fertility in ramie male sterile line. Acta Agri Univ Jiangxiensis 20:197–198Google Scholar
  16. Liu F-H, Liang X-N, Zhang S-W, Huang H-Q (2000) Identification and heredity analysis of sterility for ramie male sterile lines. China’s Fiber Crops 22:6–9Google Scholar
  17. Liu F-H, Liang X-N, Zhang S-W, Huang H-Q (2001) Analyses for ecology, biochemistry and genetics of ramie (Boehmeria nivea) male sterile lines. J Plant Genet Resour 2:1–7Google Scholar
  18. Mart CJ, Moneger F, Leaver CJ (1994) Cell-specific regulation of gene expression in mitochondria during anther development in sunflower. Plant Cell 6:811–825Google Scholar
  19. Mohr S, Schulte-Kappert E, Odenbach W, Oettler G, Kückl U (1993) Mitochondria DNA of cytoplasmic male sterile Triticum timopheevi: rearrangement of upstream sequence of the atp6 and orf25 genes. Theor Appl Genet 86:259–268CrossRefGoogle Scholar
  20. Sambrook J, Russel DW (2002) Molecular cloning: a laboratory manual. Science Press, BeijingGoogle Scholar
  21. Scotti N, Card T, Marechal-Drouard L (2001) Mitochondrial DNA and RNA isolation from small amounts of potato tissue. Plant Mol Biol Rep 19:67a–67hCrossRefGoogle Scholar
  22. Song J, Zhang Z-H, Pan G-T (2007) Physiological and biochemical characteristics in ramie male sterile lines. J Trop Subtrop Bot 15:423–428Google Scholar
  23. Spassova M, Moneger F, Leaver CJ, Petrov P, Atanassov A, Nijkamp HJJ, Hille J (1994) Characterisation and expression of the mitochondrial genome of a new type of cytoplasmic male-sterile sunflower. Plant Mol Biol 26:1819–1831PubMedCrossRefGoogle Scholar
  24. Wang G-L, Fang H-J (2002) Plant Gene Engineering. Science Press, BeijingGoogle Scholar
  25. Xue Y, Collin S, Davies DR, Thomas CM (1994) Differential screening of mitochondrial cDNA libraries from male-fertile and cytoplasmic male-sterile sugar-beet reveals genome rearrangements at atp6 and atpA loci. Plant Mol Biol 25:91–103PubMedCrossRefGoogle Scholar
  26. Zabaleta E, Mouras A, Hernould M, Suharsono Araya A (1996) Transgenetic male-sterile plant induced by an unedited atp9 gene is restored to fertility by inhibiting its expression with antisense RNA. Proc Natl Acad Sci USA 93:11259–11263PubMedCrossRefGoogle Scholar
  27. Zhang Z-H, Wei G, Yang Y, Shu Z-X (2005) Breeding and utilization of ramie male sterility line “C26”. China’s Fiber Prod 27:109–112Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Xiao-Li Liu
    • 1
  • Shou-Wen Zhang
    • 2
  • Ji-Qiang Duan
    • 1
    • 3
  • Guang-Hui Du
    • 1
  • Fei-Hu Liu
    • 1
  1. 1.Yunnan UniversityKunmingChina
  2. 2.Jiangxi College of Traditional Chinese MedicineNanchangChina
  3. 3.Nujiang Food and Drug AdministrationNujiangChina

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