烟草线粒体基因atp6的SNP及其与CMS的相关性

doi:10.3724/SP.J.1006.2009.01655

摘要/Abstract

摘要：

为更好地在烟草育种中利用雄性不育，对烟草胞质雄性不育(CMS)的分子机理进行了研究。atp6基因是影响植物CMS的一个重要候选基因。本研究以7个烟草CMS系及其相应的保持系为材料，利用PCR特异引物扩增其线粒体基因atp6，通过直接测序和比对，发现atp6中A-59C、T-92C、T-185G、T-253C、T-418C、T-768C 6个核苷酸位点存在碱基变异，其中第59位、92位、185位和418位碱基的变化导致了相应位点编码氨基酸残基的改变，另两个位点(第253位和768位)为同义突变。对atp6基因中第59位A→C的突变进行了大量个体(共222个烟草植株个体)的PCR-RFLP检测及分析，结果表明，所有保持系单株的线粒体atp6基因片段都可以被Bst1107 I酶切，酶切后出现两种条带；而96%以上的雄性不育系单株的线粒体atp6基因片段部分能被Bst1107 I酶切，部分由于第59位A→C突变不能被Bst1107 I酶切，酶切后出现3种条带。说明atp6基因第59位的SNP位点与烟草CMS特性存在显著的相关性。

关键词: 烟草, CMS, atp6基因, 单核苷酸多态性

Abstract:

The cytoplasmic male sterility (CMS) of higher plants has great significance for the use of heterosis in crop production and the researches of cytoplasmic heredity and the interaction between cytoplasm and nucleolus. The mitochondrial genome is considered as the carrier of sterility gene of cytoplasm, its mutation and recombination are closely related to CMS. ATP synthase participates in the reactions of oxidative phosphorylation and photophosphorylation and provides energy for the plant growth and development. The atp6 gene is one of the subunits of ATP synthase in mitochondria. The base mutation of DNA sequence of atp6 gene results in the abnormal synthesis of amino acids, causing the dysfunction of ATP synthase and the shortage of energy in plant cells. So the normal physiological function of cells is difficulty to be maintained, which results in the male sterility in higher plant.Single nucleotide polymorphism (SNP) is a frequent sort of genetic variation in organism genome DNA and this genetic variation is generally the direct reason of the changes of biologic traits. To better use the male-sterile lines to breed tobacco varieties, we investigated the molecule mechanism of tobacco CMS. The atp6 is a significant candidate gene carried by chondriosome related to CMS in higher plants. In this study, using specific primers designed, the mitochondrial atp6 gene was distinctively amplified by PCR from seven tobacco (Nicotiana tabacum) CMS lines (named as MS Yunyan 85, MS Zhongyan 90, MS K346, MS K326, MS G28, MS Nordel, and MS Jingyehuang) and their corresponding maintainer lines. Six nucleotide variations at A-59C, T-92C, T-185G, T-253C, T-418C, and T-768C in mitochondrial atp6 gene of CMS were detected by sequencing directly and comparing the sequences of atp6 gene. Four nucleotide variations at 59, 92, 185 and 418 resulted in the changes of amino acids coded by the changed nucleotide.The rest two nucleotide variations at 253 and 768 didn’t cause the changes of amino acids, which was a synonymy mutation. The 59 A→C mutation of atp6 gene was detected and analysed by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) with a total of 222 individual tobacco plants, indicating that all the mitochondrial atp6 gene fragments of maintainer lines plants could be entirely digested by Bst1107 I and showed 2 bands in the electrophoresis pattern of atp6 gene, while these of 96% CMS plants displayed 3 bands in the electrophoresis pattern of atp6 gene could be partly digested by Bst1107 I due to the mutation of A→C at 59. So the A-59C SNP site in atp6 gene exhibited a significant correlation with the tobacco CMS.

Key words: Tobacco, Cytoplasmic male sterility, atp6 gene, Single nucleotide polymorphism

赵婷,朱滕义,刘齐元,张美良,蒋海燕. 烟草线粒体基因atp6的SNP及其与CMS的相关性[J]. 作物学报, 2009, 35(9): 1655-1661.

ZHAO Ting,ZHU Teng-Yi,LIU Qi-Yuan,ZHANG Mei-Liang,JIANG Hai-Yan. SNP in Tobacco Mitochondrial Gene atp6 and Its Correlation with CMS[J]. Acta Agron Sin, 2009, 35(9): 1655-1661.

参考文献

[1] Belliard G, Vedel F, Pelletier G. Mitochondral recombination in cytoplasmic hybrids of Nicotiana tabacum by protoplast fusion. Nature, 1979, 281: 401-403
[2] Hanson M R. Plant mitochondrial mutations and male sterility. Annu Rev Genet, 1991, 25: 461-486
[3] Dewey R E, Levings C S III, Timothy D H. Novel recombinations in the maize mitochondrial genome produce aunique transcriptional unit in the Texas male-sterile cytoplasm. Cell, 1986, 44: 439-449
[4] Levings C S III, Pring D R. Restriction endonuclease analysis of mitochondria DNA from normal, Texas cytoplasmic male-sterile maize. Science, 1976, 193: 158-169
[5] Pring D R, Levings C S III. Heteroheneity of maize cytoplasmic henomes among male sterile cytoplasm. Genetics, 1978, 89: 121-136
[6] Nair C K K. Mitochondrial genome organization and cytoplasmic male sterility in plants. J Biosci, 1993, 18: 407-422
[7] Kohler R H, Horn P, Lossl A, Zetsche K. Cytoplasmic male sterility in sunflower is correlated with the co-transcription of a new open reading frame with the atpA gene. Mol Gen Genet, 1991, 227: 369-376
[8] Dieterich J H, Braun H P, Schmitz U K. Alloplasmic male sterility in Brassica napus (CMS ‘Tournefortii-Stiewe’) is associated with a special gene arrangement around a novel atp9 gene. Mol Genet Genom, 2003, 269: 723-731
[9] Kadowaki K, Harada K. Differential organization of mitochondrial genes in rice with normal and male sterile cytoplasm. Jpn J Breed, 1989, 39: 179-186

[10] Yang J-S(杨金水), Ge K-L(葛扣麟), Walbot V. Studies of restriction patterns of mitochondrial DNA from male sterile and maintainer lines of rice BT CMS. Acta Agric Shanghai (上海农业学报), 1992, 8(1): 1-8 (in Chinese with English abstract)

[11] Yang J-S(杨金水), Walbot V. Restriction patterns analysis of mitochondrial DNA between rice cytoplasmic male sterile line of wild abortion and maintainer line of indica. Acta Agron Sin (作物学报), 1995, 21(2): 181-187 (in Chinese with English abstract)
[12] Makaroff C A, Apel I J, Palmer J D. The atp6 coding region has been disrupted and novel reading frame generated in the mitochondrial genome of cytoplasmic male-sterile radish. J Biol Chem, 1989, 264: 11706-11713
[13] Landgren M, Zetterstrand M, Sundberg E, Glimelius K. Alloplasmic male-sterile Brassica lines containing B. tournefortii mitochondria express an ORF 3’ of the atp6 gene and a 32 kDa protein. Plant Mol Biol, 1996, 32: 879-890

[14] Zhang B(张冰), Sun C(孙超), Huang M-R(黄敏瑞), Sun J-L(孙俊丽). SNP detection in Luchuan pig leptin gene and its associations with partial production trait. Acta Agric Boreali-Occident Sin (西北农业学报), 2007, 16(5): 26-29 (in Chinese with English abstract)

[15] Yang G-L(杨广礼), Ren J(任军), Guo Y-M(郭源梅), Ma J-W(麻骏武), Lian Z-Q(连总强), Liu M-Z(刘孟洲), Huang L-S(黄路生). Polymorphism of porcine solute carrier family 6 member 14 (SLC6A14 )c. 1483G>A and its association with fatness and growth traits. Acta Agric Univ Jiangxiensis (江西农业大学学报), 2007, 29(6): 866-870(in Chinese with English abstract)
[16] Yang L(杨仑), Shen W-B(沈文飚), Chen H(陈虹), Jiang Y(姜洋), Zhang L(张琳), Xu L-L(徐朗莱). Developing putative rice SNPs through bioinformatics approaches. Chin J Rice Sci (中国水稻科学), 2004, 18(3): 185-191 (in Chinese with English abstract)
[17] Antoni R. Applications of single nucleotide polymorphisms in crop genetics. Curr Opin Plant Biol, 2002, 5: 94-100
[18] Zhang H-Y(张洪映), Mao X-G(毛新国), Jing R-L(景蕊莲), Xie H-M(谢惠民), Chang X-P(昌小平). Relationship between single nucleotide polymorphism of TaPK7 gene and drought tolerance in wheat. Acta Agron Sin (作物学报), 2008, 34(9): 1537-1543 (in Chinese with English abstract)
[19] Hang J-C(韩继成), Fang X-J(方宣钧). The SNP analysis of the conserved sequences of ethylene receptor gene in five tropical fruits. Mol Plant Breed (分子植物育种), 2003, 1(1): 72-81 (in Chinese with English abstract)
[20] Ding X-Y(丁小余), Wang Z-T(王峥涛), Xu L-S(徐珞珊), Xu H(徐红), Zhou K-Y(周开亚), Shi G-X(施国新). Study on sequence difference and SNP phenomenon of rDNA ITS region in F type and H type population of Dendrobium officinale. China J Chin Materia Med (中国中药杂志), 2002, 27(2): 85-89 (in Chinese with English abstract)
[21] Zhang C-F(张传福), Jing R-L(景蕊莲), Zhang G-S(张改生), Gao N(高宁). Application of single nucleotide polymorphism in plant research. J Plant Genet Resour (植物遗传资源学报), 2004, 5(3): 304-308 (in Chinese with English abstract)
[22] Liu Q-Y(刘齐元), Liu F-H(刘飞虎), Huang H-Q(黄海泉), He K-X(何宽信), Xiao F(肖伏). RAPD analysis of mitochondrial DNAs of cytoplasmic male sterile line and its maintainer in tobacco (Nicotiana tobacum). Acta Agric Univ Jiangxiensis (江西农业大学学报), 2005, 27(6): 826-830 (in Chinese with English abstract)
[23] Li D-D(李大东), Wang B(王斌). Rice atpA gene cloning and relationship with cytoplasmic male sterility. Hereditas (遗传), 1990, 12(4): 1-4 (in Chinese)
[24] Sabar M, Gagliardi D, Balk J, Leaver C J. ORFB is a subunit of F1F(O)-ATP synthase: Insight into the basis of cytoplasmic male sterility in sunflower. EMBO Rep, 2003, 4: 381-386
[25] Heazlewood J L, Whelan J, Millar A H. The products of the mitochondrial orf25 and orfB genes are FO components in the plant F1FO ATP synthase. FEBS Lett, 2003, 540: 201-205
[26] Mouras A, Suharsono, Hernould M, Zabaleta E, Araya A. Inheritance of an induced male-sterile trait in transgenic plants expressing an engineered unedited atp9 mitochondrial gene. Theor Appl Genet, 1999, 98: 614-621
[27] Zhou W(周玮), Liu Q-Y(刘齐元), Chen X-F(陈雪峰), Liu F-H(刘飞虎), Cao H(曹槐), Liu C-Q(刘次全). Studies on atp9 mRNA related to cytoplasmic male sterility in tobacco. Guihaia (广西植物), 2007, 27(1): 114-120 (in Chinese with English abstract)
[28] Wang J(汪静), Cao M-J(曹墨菊), Zhu Y-G(朱英国), Pan G-T(潘光堂), Rong T-Z(荣廷昭). The editing sites in the transcripts of mitochondrial atp6 gene of maize sterile lines and maintainer line. Hereditas (遗传), 2007, 29(6): 731-737 (in Chinese with English abstract)
[29] Zhu K-X(朱孔锡). Study of the development and relationship between mitochondria DNA ATPase 6, 8 gene mutation and alcoholic liver disease in rats. PhD Dissertation of Shandong University, 2006 (in Chinese with English abstract)
[30] Wang X-Z(王秀珍), Teng X-Y(滕晓月), Yan L-F(阎隆飞), Zhou R-G(周人纲). The relationship between the ATP content in anthers of maize and sorghum and cytoplasmic male-sterility. Acta Agron Sin (作物学报), 1986, 12: 177-181 (in Chinese with English abstract)
[31] Chen X-F(陈贤丰), Liang C-Y(梁承邺). Relation of energy metabolism in HPGMR abortive anthers and accumulation of H₂O₂ to male sterility. Plant Physiol Commun (植物生理学通讯), 1991, 27(1): 21-24 (in Chinese)
[32]Tang X-Y(唐雪元), Liu F(刘芬), Peng Y(彭娅), Xiang B(向波), Wang C-H(王成红), Yao C-J(姚晨姣), Shen S-R(沈守荣). Expression and SNP analysis of BRD7 gene in acute leukemia cells. J Cent South Univ (Med Sci)(中南大学学报·医学版), 2008, 33(8): 645-650 (in Chinese with English abstract)

[33] Lu F(陆峰), Wang L-S(王连生), Yang Z-J(杨志健), Zhu T-B(朱铁兵), Wang Z-G(王志广), Zhu H-J(朱怀军), Tang N-P(汤纳平), Cao K-J(曹克将), Huang J(黄峻), Gui M(桂鸣). Correlation between single nucleotide polymorphism (SNP+276 G/T) of adiponectin gene and coronary artery disease in non-diabetic population. Acta Univ Med Nanjing (Nat Sci) (南京医科大学学报·自然科学版),2007, 27(7): 736-739 (in Chinese with English abstract)
[34] Li C-X(李春笑), Jiang M-S(蒋美山), Chen S-Y(陈仕毅), Lai S-J(赖松家). Correlation analysis between single nucleotide polymorphism of FGF5 gene and wool yield in rabbits. Hereditas (遗传), 2008, 30(7): 893-899 (in Chinese with English abstract)

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed