羽衣甘蓝不同组织及柱头发育实时荧光定量PCR内参基因的筛选

doi:10.7525/j.issn.1673-5102.2016.04.012

植物研究 ›› 2016, Vol. 36 ›› Issue (4): 565-572.doi: 10.7525/j.issn.1673-5102.2016.04.012

羽衣甘蓝不同组织及柱头发育实时荧光定量PCR内参基因的筛选

李晗, 李治龙, 李晓屿, 李玉花, 蓝兴国

东北林业大学生命科学学院, 哈尔滨 150040

收稿日期:2015-12-17 出版日期:2016-07-15 发布日期:2016-06-15
通讯作者: 蓝兴国,E-mail:lanxingguo@126.com E-mail:lanxingguo@126.com
作者简介:李晗(1991-),女,硕士研究生,主要从事植物发育生物学研究。
基金资助:
中央高校基本科研业务费专项基金项目(DL13CA13);国家自然科学基金项目(31070275)

Selection of Reference Genes for Real-time Fluorescence Quantitative PCR in Different Tissues and Stigma Development from Ornamental Kale

LI Han, LI Zhi-Long, LI Xiao-Yu, LI Yu-Hua, LAN Xing-Guo

College of Life Sciences, Northeast Forestry University, Harbin 150040

Received:2015-12-17 Online:2016-07-15 Published:2016-06-15
Supported by:
Central Universities Fundamental Research Special Fund Project(DL13CA13);National Natural Science Foundation of China(31070275)

摘要/Abstract

摘要： 以羽衣甘蓝(Brassica oleracea var. acephala)S_13-bS_13-b自交不亲和系为试材,利用实时荧光定量PCR(qRT-PCR)技术检测Actin、Cpi6、EF-1β、GAPDH、Tub-α3、Tub-α6、Ubc7候选内参基因在不同组织和5个不同发育时期柱头的表达情况,并运用geNorm和BestKeeper软件统计分析候选内参基因的表达稳定性。在不同组织中,geNorm软件统计分析的结果表明Tub-α6和EF-1β的表达较稳定;BestKeeper软件统计分析的结果表明Ubc7和Tub-α6的表达较稳定。在不同发育柱头中,geNorm软件统计分析的结果表明Actin和Ubc7的表达较稳定;BestKeeper软件统计分析的结果表明EF-1β和Tub-α6的表达较稳定。以筛选到的内参基因Tub-α6和Actin,分别分析羽衣甘蓝柱头S-位点糖蛋白基因(SLG)在不同组织和不同发育时期柱头的表达水平,结果显示出SLG主要在柱头中表达,而且SLG在柱头发育成熟时期表达量达到最高。

关键词: 羽衣甘蓝, 实时荧光定量PCR, 内参基因, 柱头发育, SLG

Abstract: We used real-time quantitative polymerase chain reaction to test the expression of Actin, cysteine proteinase inhibitor 6(Cpi6), elongation factor 1-beta(EF-1β), glyceraldehyde-3-phosphate dehydrogenase(GAPDH), tubulin alpha-3(Tub-α3), tubulin alpha-6(Tub-α6) and ubiquitin conjugating enzyme7(Ubc7), and then identified the most suitable reference genes in a given set of tissues and different developmental stigmas of ornamental kale(Brassica oleracea var. acephala) S_13-bS_13-b homozygotes by using the geNorm and BestKeeper software programs. By a geNorm analysis, Tub-α6 and EF-1β were the most suitable reference genes among the given set of tissues, and Actin and Ubc7 were the most stable genes during stigma development. By a BestKeeper analysis, Ubc7 and Tub-α6 were the most suitable reference genes among the given set of tissues, and EF-1β and Tub-α6 were the most stable genes during stigma development. Tub-α6 and Actin were the most suitable reference genes among the given set of tissues and during stigma development, respectively. Furthermore, the expression of SLG normalized with Tub-α6 or Actin showed that SLG was predominantly expressed in stigma among the given set of tissues and reached its highest level at approaching anthesis during stigma development.

Key words: Brassica oleracea var. acephala, real-time quantitative PCR, reference genes, stigma development, SLG

中图分类号:

S635

李晗, 李治龙, 李晓屿, 李玉花, 蓝兴国. 羽衣甘蓝不同组织及柱头发育实时荧光定量PCR内参基因的筛选[J]. 植物研究, 2016, 36(4): 565-572.

LI Han, LI Zhi-Long, LI Xiao-Yu, LI Yu-Hua, LAN Xing-Guo. Selection of Reference Genes for Real-time Fluorescence Quantitative PCR in Different Tissues and Stigma Development from Ornamental Kale[J]. Bulletin of Botanical Research, 2016, 36(4): 565-572.

参考文献

1.Li Y H,Yu X Y.Pollination with laser-irradiated pollens breaks cross-incompatibility between zicaitai(Brassica campestris var.purpurea) and ornamental kale(Brassica oleracea var.acephala) to produce hybrids with the aid of ovule culture[J].Scientia Horticulturae,2006,108(4):397-402.
2.解莉楠,丁兵,李玉花.羽衣甘蓝新品种‘红罗裙’和‘白罗裙’[J].园艺学报,2007,34(4):1071.
Xie L N,Ding B,Li Y H.New ornamental kale cultivars of ‘Hongluoqun’ and ‘Bailuoqun’[J].Acta Horticulturae Sinica,2007,34(4):1071.
3.王江,丁兵,李玉花.观赏羽衣甘蓝新品种‘粉玉’[J].园艺学报,2014,41(11):2369-2370.
Wang J,Ding B,Li Y H.A new ornamental kale cultivar ‘Fenyu’[J].Acta Horticulturae Sinica,2014,41(11):2369-2370.
4.Lan X G,Yang J,Cao M M,et al.Isolation and characterization of a J domain protein that interacts with ARC1 from ornamental kale(Brassica oleracea var.acephala)[J].Plant Cell Reports,2015,34(5):817-829.
5.Horisaki A,Niikura S.Developmental and environmental factors affecting level of self-incompatibility response in Brassica rapa L.[J].Sexual Plant Reproduction,2008,21(2):123-132.
6.Nasrallah J B,Kao T H,Goldberg M L,et al.A cDNA clone encoding an S-locus-specific glycoprotein from Brassica oleracea[J].Nature,1985,318(6043):263-267.
7.Stein J C,Howlett B,Boyes D C,et al.Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea[J].Proceedings of the National Academy of Sciences of the United States of America,1991,88(19):8816-8820.
8.Goring D R,Rothstein S J.The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase[J].The Plant Cell,1992,4(10):1273-1281.
9.Gu T S,Mazzurco M,Sulaman W,et al.Binding of an arm repeat protein to the kinase domain of the S-locus receptor kinase[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(1):382-387.
10.Takasaki T,Hatakeyama K,Suzuki G,et al.The S receptor kinase determines self-incompatibility in Brassica stigma[J].Nature,2000,403(6772):913-916.
11.Murase K,Shiba H,Iwano M,et al.A membrane-anchored protein kinase involved in Brassica self-incompatibility signaling[J].Science,2004,303(5663):1516-1519.
12.蓝兴国,杨佳,赵昕,等.羽衣甘蓝ARC1的基因分离、表达及与SRK相互作用的分析[J].园艺学报,2011,38(12):2342-2348.
Lan X G,Yang J,Zhao X,et al.Isolation expression of ARC1 from ornamental kale and interaction analysis between ARC1 and SRK[J].Acta Horticulturae Sinica,2011,38(12):2342-2348.
13.Indriolo E,Safavian D,Goring D R.The ARC1 E3 ligase promotes two different self-pollen avoidance traits in Arabidopsis[J].The Plant Cell,2014,26(4):1525-1543.
14.Bustin S A.Quantification of mRNA using real-time reverse transcription PCR(RT-PCR):trends and problems[J].Journal of Molecular Endocrinology,2002,29(1):23-39.
15.Nolan T,Hands R E,Bustin S A.Quantification of mRNA using real-time RT-PCR[J].Nature Protocols,2006,1(3):1559-1582.
16.Vandesompele J,Depreter K,Pattyn F,et al.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J].Genome Biology,2002,3(7):research0034
17.Pfaffl M W.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Research,2001,29(9):e45.
18.Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT Method[J].Methods,2001,25(4):402-408.
19.Bustin S A,Beaulieu J F,Huggett J,et al.MIQE précis:practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments[J].BMC Molecular Biology,2010,11:74.
20.Magneschi L,Kudahettige R L,Alpi A,et al.Expansin gene expression and anoxic coleoptile elongation in rice cultivars[J].Journal of Plant Physiology,2009,166(14):1576-1580.
21.Bracha-Drori K,Shichrur K,Lubetzky T C,et al.Functional analysis of Arabidopsis postprenylation CaaX processing enzymes and their function in subcellular protein targeting[J].Plant Physiology,2008,148(1):119-131.
22.Brentner L B,Mukherji S T,Merchie K M,et al.Expression of glutathione S-transferases in poplar trees(Populus trichocarpa) exposed to 2,4,6-trinitrotoluene(TNT)[J].Chemosphere,2008,73(5):657-662.
23.Bomal C,Bedon F,Caron S,et al.Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis:a comparative in planta analysis[J].Journal of Experimental Botany,2008,59(14):3925-3939.
24.Aquea F,Gutierrez F,Medina C,et al.A novel Otubain-like cysteine protease gene is preferentially expressed during somatic embryogenesis in Pinus radiata[J].Molecular Biology Reports,2008,35(4):567-573.
25.Guénin S,Mauriat M,Pelloux J,et al.Normalization of qRT-PCR data:the necessity of adopting a systematic,experimental conditions-specific,validation of references[J].Journal of Experimental Botany,2009,60(2):487-493.
26.Kitashiba H,Liu P,Nishio T,et al.Functional test of Brassica self-incompatibility modifiers in Arabidopsis thaliana[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(44):18173-18178.
27.Indriolo E,Tharmapalan P,Wright S I,et al.The ARC1 E3 ligase gene is frequently deleted in self-compatible Brassicaceae species and has a conserved role in Arabidopsis lyrata self-pollen rejection[J].The Plant Cell,2012,24(11):4607-4620.
28.Xu X Y,Yang Z P,Sun X L,et al.Selection of reference genes for quantitative real-time PCR during flower bud development in CMS7311 of heading Chinese cabbage(Brassica rapa L.ssp.pekinensis)[J].Acta Physiologiae Plantarum,2014,36(3):809-814.
29.Qi J N,Yu S C,Zhang F L,et al.Reference gene selection for real-time quantitative polymerase chain reaction of mRNA transcript levels in Chinese cabbage(Brassica rapa L.ssp.pekinensis)[J].Plant Molecular Biology Reporter,2010,28(4):597-604.
30.Andersen C L,Jensen J L,Ørntoft T F.Normalization of real-time quantitative reverse transcription-PCR data:a model-based variance estimation approach to identify genes suited for normalization,applied to bladder and colon cancer data sets[J].Cancer Research,2004,64(15):5245-5250.

[1]	栗赫铭, 秦洪涛, 魏德强, 李旭, 蓝兴国. 羽衣甘蓝BoMAPK4原核表达、抗体制备及蛋白表达分析[J]. 植物研究, 2022, 42(4): 584-591.
[2]	李阳, 施亚坤, 高士科, 李晓屿, 蓝兴国. 羽衣甘蓝类蛋白质二硫键异构酶PDIL1-2的基因克隆及蛋白表达分析[J]. 植物研究, 2020, 40(2): 293-300.
[3]	高彩球;刘桂丰;王玉成;姜静;杨传平. 柽柳(Tamarix androssowii)Tadir*基因的克隆及分析[J]. 植物研究, 2010, 30(1): 81-86.

羽衣甘蓝不同组织及柱头发育实时荧光定量PCR内参基因的筛选

Selection of Reference Genes for Real-time Fluorescence Quantitative PCR in Different Tissues and Stigma Development from Ornamental Kale

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 3

编辑推荐

Metrics

本文评价