Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (16): 3264-3276.doi: 10.3864/j.issn.0578-1752.2014.16.012

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Expression Analysis of Fertility-Related Genes for the Genic Male Sterile Line in Pepper (Capsicum annuum L.)

1、College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193; #br# 2、Guangzhou Academy of Agricultural Sciences, Guangzhou 510308   

  1. 1、College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193;
    2、Guangzhou Academy of Agricultural Sciences, Guangzhou 510308
  • Received:2013-12-30 Online:2014-08-18 Published:2014-06-15

Abstract: 【Objective】To investigate the correlativity between the four screened ESTs and the genic male sterility, full length cDNA of the four genes were obtained, and the expression patterns of different materials and different organs were analyzed.【Method】The expression patterns of the four genes were detected by RT-PCR. Full length cDNA were cloned using the rapid-amplification of cDNA ends technique (RACE). Physical and chemical properties of proteins were analyzed with the bioinformatics softwares. RT-PCR was employed to detect expression patterns of the genes in different organs (anther, ovary, petal, sepal and leaf) and anthers from different developmental stages (tetrad stage, early- or mid-uninucleate stage, late-uninucleate stage and binucleate stage).【Result】According to the results of Blastx, the four genes were named as CaSEP1, CaPROF, CaOle e 6 and CaPCP, separately. Full length of CaSEP1 is 1 108bp, which codes 221 amino acids and contains a MADS domain and a K domain. The full length CaPROF includes 767 base pairs and 131 amino acids, which has a PROF domain. CaOle e 6 is 523 bp in full length, codes 85 amino acids with an Ole e 6 domain. Full length of CaPCP includes 563 base pairs and 66 amino acids. Amino acid sequence alignments and phylogeny reconstructions indicate that CaSEP1 is most similar to the tomato SEP1 protein and they have the closest relationship. CaPROF is more similar to profilins from tobacco and tomato than other species and shows the closest genetic distance with tomato profiling, CaOle e 6 is similar to the tomato Ole e 6 protein and they are classified into the same cluster, CaPCP shows the most similarity with tea pollen like protein and they are closest in genetic distance. RT-PCR showed that CaSEP1 expressed only in the reproductive organs of the fertile line with higher expression in anther, ovary and petal than sepal and leaf. CaPROF expressed significantly higher in anther than leaf of the fertile line, while no expression in other organs. CaOle e 6 expressed significantly higher in anther than other organs of the fertile line. CaPCP showed specific expression in anther of the fertile line. The expression of CaSEP1 increased gradually before decreasing in the binucleate stage of fertile anther, while it increased gradually in sterile anther during microspore development. The gene shows a similar expression between fertile line and sterile line at the tetrad stage of microspore development. At early- or mid-uninucleate stage and late-uninucleate stage, the gene expresses higher in fertile line. While the expression in fertile line is lower than sterile line during binucleate stage of microspore development. CaPROF, CaOle e 6 and CaPCP only express at the late stages (late-uninucleate stage and binucleate stage).【Conclusion】Analysis of sequences and expression profilings of the four genes indicates close relationships to male fertility. This study provides important information for revealing the mechanism of male sterility and regulating male fertility in pepper.

Key words: pepper , genic male sterility , expression , gene clone , sequence analysis

[1]Liu C, Ma N, Wang P Y, Fu N, Shen H L. Transcriptome sequencing and de novo analysis of a cytoplasmic male sterile line and its near-isogenic restorer line in chili pepper (Capsicum annuum L.). PLoS ONE, 2013, 8(6): e65209.

[2]Hong S T, Chung J E, An G, Kim S R. Analysis of 176 expressed sequence tags generated from cDNA clones of hot pepper by single-pass sequencing. Journal of Plant Biology, 1998, 41(2): 116-124.

[3]Chen C M, Hao X F, Chen G J, Cao B H, Chen Q H, Liu S Q, Lei J J. Characterization of a new male sterility-related gene Camf1 in Capsicum annum L. Molecular Biology Reports, 2011, 39(1): 737-744.

[4]Shifriss C, Frankel R. A new male sterility gene in Capsicum annuum L. Journal of the American Society for Horticultural Science, 1969, 94: 385-387.

[5]Wang D Y, Bosland P W. The genes of Capsicum. HortScience, 2006, 41(5): 1169-1187.

[6]王得元, 杨凤梅, 李颖, 王恒明. 辣椒核雄性不育基因研究进展. 中国蔬菜, 2008, 9: 40-43.

Wang D Y, Yang F M, Li Y, Wang H M. Progress in hot pepper genic male sterility genes research. China Vegetables, 2008, 9: 40-43. (in Chinese)

[7]Lee J, Han J H, An C G, Lee W P, Yoon J B. A CAPS marker linked to a genic male-sterile gene in the colored sweet pepper ‘Paprika’ (Capsicum annuum L.). Breeding Science, 2010, 60(1): 93-98.

[8]Lee J, Yoon J B, Hart J H, Lee W P, Kim S H, Park H G. Three AFLP markers tightly linked to the genic male sterility ms3 gene in chili pepper (Capsicum annuum L.) and conversion to a CAPS marker. Euphytica, 2010, 173(1): 55-61.

[9]Lee J, Yoon J B, Han J H, Lee W P, Do J W, Ryu H, Kim S H, Park H G. A codominant SCAR marker linked to the genic male sterility gene (ms1) in chili pepper (Capsicum annuum L.). Plant Breeding, 2010 129(1): 35-38.

[10]Bartoszewski G, Waszczak C, Gawronski P,  Stepien I, Bolibok- Bragoszewska H, Palloix A, Lefebvre V, Korzeniewska A, Niemirowicz-Szczytt K. Mapping of the ms8 male sterility gene in sweet pepper (Capsicum annuum L.) on the chromosome P4 using PCR-based markers useful for breeding programmes. Euphytica, 2012, 186(2): 453-461.

[11]韩清, 冯淼, 刘辰, 马宁, 沈火林. 利用cDNA-AFLP技术研究辣椒核雄性不育两用系的基因差异表达. 农业生物技术学报, 2012, 20(10): 1117-1125.

Han Q, Feng M, Liu C, Ma N, Shen H L. Differential expression analysis of genie male sterile-fertile line in pepper (Capsicum annuum L.) by cDNA-AFLP. Journal of Agricultural Biotechnology, 2012, 20(10): 1117-1125. (in Chinese)

[12]杨高强, 霍秀文. 利用cDNA-AFLP研究辣椒细胞核雄性不育差异表达基因. 北方园艺, 2012, 13: 132-136.

Yang G Q, Huo X W. Differentially distinguished expressed genes in chili pepper genic male sterility (GMS) using cDNA-AFLP. Northern Horticulture, 2012, 13: 132-136. (in Chinese)

[13]黄炜. 辣椒核雄性不育两用系创制及其不育机理研究[D]. 杨凌: 西北农林科技大学, 2012.

Huang W. Breeding of genic male sterile line and studies on sterile mechanism in pepper. Yangling: Northwest Agriculture & Forestry University, 2012. (in Chinese)

[14]Chen C M, Chen G J, Hao X F, Cao B H, Chen Q H, Liu S Q, Lei J J. CaMF2, an anther-specific lipid transfer protein (LTP) gene, affects pollen development in Capsicum annuum L. Plant Science, 2011, 181(4): 439-448.

[15]Chen C M, Liu S Q, Hao X F , Chen G J, Cao BH, Chen Q H, Lei J J. Characterization of a pectin methylesterase gene homolog, CaPME1, expressed in anther tissues of Capsicum annuum L.. Plant Molecular Biology Reporter, 2011, 30(2): 402-413.

[16]李国琴, 吉姣姣, 巩振辉, 黄炜, 李大伟. 辣椒CaCP26基因的cDNA克隆和分析. 西北农业学报, 2011, 20(11): 111-116.

Li G Q, Ji J J, Gong Z H, Huang W, Li D W. Cloning and analysis of CaCP26 gene cDNA of pepper. Acta Agriculturae Boreali- occidentalis Sinica, 2011, 20(11): 111-116. (in Chinese)

[17]张菊平, 巩振辉, 刘珂珂, 黄炜, 李大伟. 辣椒小孢子发育时期与花器形态的相关性. 西北农林科技大学学报(自然科学版), 2007, 35(3): 153-158.

Zhang J P, Gong Z H, Liu K K, Huang W, Li D W. Interrelation of cytological development period of pepper’s microscope and the morphology of flower organ. Journal of Northwest A & F University (National Science Edition), 2007, 35(3): 153-158. (in Chinese)

[18]Tamura K, Peterson D, Peterson N, Stecher G, Nei M, and Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 2011, 28: 2731-2739.

[19]丛楠, 程治军, 万建民. 控制花器官发育的ABCDE模型. 中国农学通报, 2007, 23(7): 124-128.

Cong N, Cheng Z J, Wan J M. The ABCDE model of floral organ development. Chinese Agricultural Science Bulletin, 2007, 23(7): 124-128. (in Chinese)

[20]Theissen G. Development of floral organ identity: stories from the MADS house. Current Opinion in Plant Biology, 2001, 4(1): 75-85.

[21]Ferrario S, Immink R G, Shchennikova A, Busscher-Lange J, Angenent G C. The MADS-box gene FBP2 is required for SEPALLATA function in petunia. Plant Cell, 2003, 15(4): 914-925.

[22]Immink R G, Ferrario S, Busscher-Lange J, Kooiker M, Busscher M, Angenent G C. Analysis of the petunia MADS-box transcription factor family. Molecular Genetics and Genomics, 2003, 268(5): 598-606.

[23]曾维英, 杨守萍, 盖钧镒, 喻德跃. 大豆质核互作雄性不育系NJCMS1A及其保持系的花药差异蛋白质组学研究. 中国农业科学, 2007, 40(12): 2679-2687.

Zeng W Y, Yang S P, Gai J Y, Yu D Y. Proteomic study of anther differentiation between cytoplasmic-nuclear male-sterile line NJCMS1A and its maintainer in soybean [Glycine max (L)Merr.]. Scientia Agricultura Sinica, 2007, 40(12): 2679-2687. (in Chinese)

[24]Sharma B, Kramer E. Sub- and neo-functionalization of APETALA3 paralogs have contributed to the evolution of novel floral organ identity in Aquilegia (columbine, Ranunculaceae). New Phytologist, 2013, 197(3): 949-957.

[25]Arce Johnson J P, Poupin Swinburn M J, Medina Arevalo M C, Cadavid Labrada A, Federici Noe F. Method to produce sterile male flowers and partenocarpic fruits by genetic silencing, associated sequences and vectors containing said sequences: US, US 07994397. August 09, 2011.

[26]Whipple C J, Ciceri P, Padilla C M, Ambrose B A, Bandong S L, Schmidt R J. Conservation of B-class floral homeotic gene function between maize and Arabidopsis. Development, 2004, 131(24): 6083-6091.

[27]Ito T, Ng K H, Lim T S, Yu H, Meyerowitz E M. The homeotic protein AGAMOUS controls late stamen development by regulating a jasmonate biosynthetic gene in Arabidopsis. Plant Cell, 2007, 19(11): 3516-3529.

[28]Pelaz S, Ditta G S, Baumann E, Wisman E, Yanofsky M F. B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature, 2000, 405(6783): 200-203.

[29]Ditta G, Pinyopich A, Robles P, Pelaz S, Yanofsky M F. The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity. Current Biology, 2004, 14(21): 1935-1940.

[30]郭爽, 马宁, 杨文才, 沈火林. 辣椒花器官发育MADS-box基因的克隆与表达分析. 园艺学报, 2010, 37(10): 1591-1597.

Guo S, Ma N, Yang W C, Shen H L. Cloning and expression analysis of a flowering-related MADS-box gene in pepper. Acta Horticulturae Sinica, 2010, 37(10): 1591-1597. (in Chinese)

[31]Li X, Liu C, Da F J, Ma N, Shen H L. Expression pattern of class B gene PAP3 in flower development of pepper. International Journal of Molecular Sciences, 2013, 14: 24643-24655.

[32]Witke W. The role of profilin complexes in cell motility and other cellular processes. Trends in Cell Biology, 2004, 14(8): 461-469.

[33]Yu L X, Nasrallah J, Valenta R, Parthasarathy M V. Molecular cloning and mRNA localization of tomato pollen profiling. Plant Molecular Biology, 1998, 36(5): 699-707.

[34]Ribeiro H, Morales S, Salmeron C, Cruz A, Calado L, Rodriguez- Garcia M I, Alche J D, Abreu I. Analysis of the pollen allergen content of twelve olive cultivars grown in Portugal. Aerobiologia, 2013, 24(9): 513-521.

[35]Heslop-Harrison Y. Control gates and micro-ecology: The pollen- stigma interaction in perspective. Annals of Botany, 2000, 85(Supplement A): 5-13.

[36]Yoo Y G, Lee S C, Kim S R. Identification of a flower-specific cDNA, RsPCP1, encoding putative pollen coat protein from radish. Journal of Plant Biology, 2003, 46(2): 130-133.
[1] SHEN LongXian, WANG LiTing, HE Ke, DU Xue, YAN FeiFei, CHEN WeiHu, LÜ YaoPing, WANG Han, ZHOU XiaoLong, ZHAO AYong. Effects of Melatonin and Nicotinamide Mononucleotides on Proliferation of Skeletal Muscle Satellite Cells in Goose [J]. Scientia Agricultura Sinica, 2023, 56(2): 391-404.
[2] GU LiDan,LIU Yang,LI FangXiang,CHENG WeiNing. Cloning of Small Heat Shock Protein Gene Hsp21.9 in Sitodiplosis mosellana and Its Expression Characteristics During Diapause and Under Temperature Stresses [J]. Scientia Agricultura Sinica, 2023, 56(1): 79-89.
[3] ZHANG KeKun,CHEN KeQin,LI WanPing,QIAO HaoRong,ZHANG JunXia,LIU FengZhi,FANG YuLin,WANG HaiBo. Effects of Irrigation Amount on Berry Development and Aroma Components Accumulation of Shine Muscat Grape in Root-Restricted Cultivation [J]. Scientia Agricultura Sinica, 2023, 56(1): 129-143.
[4] MO WenJing,ZHU JiaWei,HE XinHua,YU HaiXia,JIANG HaiLing,QIN LiuFei,ZHANG YiLi,LI YuZe,LUO Cong. Functional Analysis of MiZAT10A and MiZAT10B Genes in Mango [J]. Scientia Agricultura Sinica, 2023, 56(1): 193-202.
[5] DONG SangJie,JIANG XiaoChun,WANG LingYu,LIN Rui,QI ZhenYu,YU JingQuan,ZHOU YanHong. Effects of Supplemental Far-Red Light on Growth and Abiotic Stress Tolerance of Pepper Seedlings [J]. Scientia Agricultura Sinica, 2022, 55(6): 1189-1198.
[6] LI ShiJia,LÜ ZiJing,ZHAO Jin. Identification of R2R3-MYB Subfamily in Chinese Jujube and Their Expression Pattern During the Fruit Development [J]. Scientia Agricultura Sinica, 2022, 55(6): 1199-1212.
[7] LAI ChunWang, ZHOU XiaoJuan, CHEN Yan, LIU MengYu, XUE XiaoDong, XIAO XueChen, LIN WenZhong, LAI ZhongXiong, LIN YuLing. Identification of Ethylene Synthesis Pathway Genes in Longan and Its Response to ACC Treatment [J]. Scientia Agricultura Sinica, 2022, 55(3): 558-574.
[8] SHU JingTing,SHAN YanJu,JI GaiGe,ZHANG Ming,TU YunJie,LIU YiFan,JU XiaoJun,SHENG ZhongWei,TANG YanFei,LI Hua,ZOU JianMin. Relationship Between Expression Levels of Guangxi Partridge Chicken m6A Methyltransferase Genes, Myofiber Types and Myogenic Differentiation [J]. Scientia Agricultura Sinica, 2022, 55(3): 589-601.
[9] ZHAO HuiTing,PENG Zhu,JIANG YuSuo,ZHAO ShuGuo,HUANG Li,DU YaLi,GUO LiNa. Expression and Binding Properties of Odorant Binding Protein AcerOBP7 in Apis cerana cerana [J]. Scientia Agricultura Sinica, 2022, 55(3): 613-624.
[10] LI YuZe,ZHU JiaWei,LIN Wei,LAN MoYing,XIA LiMing,ZHANG YiLi,LUO Cong,HUANG Gui Xiang,HE XinHua. Cloning and Interaction Protein Screening of RHF2A Gene from Xiangshui Lemon [J]. Scientia Agricultura Sinica, 2022, 55(24): 4912-4926.
[11] GUO ShaoLei,XU JianLan,WANG XiaoJun,SU ZiWen,ZHANG BinBin,MA RuiJuan,YU MingLiang. Genome-Wide Identification and Expression Analysis of XTH Gene Family in Peach Fruit During Storage [J]. Scientia Agricultura Sinica, 2022, 55(23): 4702-4716.
[12] ZHANG Qi,DUAN Yu,SU Yue,JIANG QiQi,WANG ChunQing,BIN Yu,SONG Zhen. Construction and Application of Expression Vector Based on Citrus Leaf Blotch Virus [J]. Scientia Agricultura Sinica, 2022, 55(22): 4398-4407.
[13] HAO Yan,LI XiaoYing,YE Mao,LIU YaTing,WANG TianYu,WANG HaiJing,ZHANG LiBin,XIAO Xiao,WU JunKai. Characteristics of Volatile Components in Peach Fruits of 21shiji and Jiucui and Their Hybrid Progenies [J]. Scientia Agricultura Sinica, 2022, 55(22): 4487-4499.
[14] KANG Chen,ZHAO XueFang,LI YaDong,TIAN ZheJuan,WANG Peng,WU ZhiMing. Genome-Wide Identification and Analysis of CC-NBS-LRR Family in Response to Downy Mildew and Powdery Mildew in Cucumis sativus [J]. Scientia Agricultura Sinica, 2022, 55(19): 3751-3766.
[15] YuXia WEN,Jian ZHANG,Qin WANG,Jing WANG,YueHong PEI,ShaoRui TIAN,GuangJin FAN,XiaoZhou MA,XianChao SUN. Cloning, Expression and Anti-TMV Function Analysis of Nicotiana benthamiana NbMBF1c [J]. Scientia Agricultura Sinica, 2022, 55(18): 3543-3555.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!