分子鉴定拟南芥<em>myb26/mail sterile35</em>突变体中表达下调基因<em>At2g47500</em>

doi:10.7668/hbnxb.2014.05.013

华北农学报 ›› 2014, Vol. 29 ›› Issue (5): 71-79. doi: 10.7668/hbnxb.2014.05.013

所属专题： 生物技术；

分子鉴定拟南芥myb26/mail sterile35突变体中表达下调基因At2g47500

樊新萍^1,2, 乔永胜², 牛西午³, Zoe A Wilson⁴

1. 山西大学生命科学院, 山西太原 030006;
2. 山西省农业科学院果树研究所, 山西太谷 030800;
3. 山西省农业科学院, 山西太原 030006;
4. School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK

收稿日期:2014-02-17 出版日期:2014-10-28
作者简介:樊新萍(1973-), 女, 山西永济人, 副研究员, 硕士, 主要从事果树分子生物学研究。
基金资助:
山西省国际科技合作项目(2012081002)

Molecular Characterization of Down-regulated Gene At2g47500 Associated with myb26/mail sterile35 Mutant

FAN Xin-ping^1,2, QIAO Yong-sheng², NIU Xi-wu³, Zoe A Wilson⁴

1. College of Life Science, Shanxi University, Taiyuan 030006, China;
2. Pomology Institute, Shanxi Academy of Agricultural Sciences, Taigu 030800, China;
3. Shanxi Academy of Agricultural Sciences, Taiyuan 030006, China;
4. School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK

Received:2014-02-17 Published:2014-10-28

摘要/Abstract

摘要： 利用不可产生花粉的突变体作为研究花药和花粉发育的途径,以证实涉及花粉发育的基因及其在基因网络中的作用。研究结果表明,拟南芥中MYB26/MALE STERILE35(MS35)雄性不育基因会调控药室内壁次生加厚发育,影响其后的花药开裂,并可上调木质素生物合成途径。而At2g47500基因是在芯片分析ms35/myb26突变体中涉及花粉发育表达下调的一个未被鉴定的基因。通过生物信息学分析发现,At2g47500基因是一个与微管发育有关的基因,在整个植株都表达,包括花发育阶段。利用拟南芥基因库筛选该基因中的T-DNA插入突变体,并通过PCR鉴定,得到插入位点在启动子和外显子的纯合突变体。通过表型分析和表达筛选发现,At2g47500突变并未对表现型产生影响,表明T-DNA插入位点在外显子的突变体基因并未在花药中表达;但转录表达分析认为,该基因在花粉里表达,并通过启动子:GUS表达结构得到进一步证实。35S启动子超量表达At2g47500,并未导致其在野生型植物中异位的过量表达;将其转入突变体中,基因表达量恢复,也未有表现差异。说明其对花粉发育的影响不明显。定量PCR分析微管发育其他相关基因,以及花发育中次生壁发育有关基因在突变体中的表达,At2g47500对花粉发育影响不明显。在突变体中,大部分基因也未因其缺失、表达受到明显影响。综上所述,该基因在花药发育中不受ms35/myb26的调控,发挥次要作用,受到主效基因的调控。

关键词: At2g47500, ms35/myb26, 分子鉴定, GUS表达, 超量表达

Abstract: We have used mutants that are defective in the production of pollen as a means to study anther and pollen development.This has identified genes that are involved in pollen development and are likely to function in the same network.Previously,the Arabidopsis thaliana MYB26/MALE STERILE35(MS35)gene regulated endothecial cell development within the anther and acts upstream of the lignin biosynthesis pathway.Gene(At2g47500)related to microtubule identified from microarray analysis of ms35/myb26 mutant showed down-regulated gene expression in mutant were characterized by bioinformatics analysis.Insertional mutants was identified in knockout lines of Arabidopsis GenBank.Homozygous lines screened were then tested for altered expression of the genes of interest by reverse-transcriptase(RT)-PCR.Lines that show down-regulation were phenotypically characterized for changes in development.RT-PCR expression analysis suggested that theAt2g47500 was expressed in the anthers and confirmed using transformed promoter:GUS reporter constructs into wild type Arabidopsis plants by Agrobacterium transformation and analysis.Over-express At2g47500 in wild type plant and compensating in knock out lines,there was no much effect on general plant growth and specifically on pollen development and only recover expression in mutant.Quantity real time-PCR analysis some genes related to microtubule in mutant,expression as normal and similar to compare with some genes related to the secondary wall expression in myb26 buds(immature young and old stage).At2g47500 impact on pollen development is not obvious,it turns into mutant,the amount of gene expression,no performance difference.To sum up,the gene plays a secondary role in the anther development,and regulated by the main gene in anther.

Key words: At2g47500, ms35/myb26, Molecular identification, GUS reporter, Over-express of At2g47500

中图分类号:

樊新萍, 乔永胜, 牛西午, Zoe A Wilson. 分子鉴定拟南芥myb26/mail sterile35突变体中表达下调基因At2g47500[J]. 华北农学报, 2014, 29(5): 71-79. doi: 10.7668/hbnxb.2014.05.013.

FAN Xin-ping, QIAO Yong-sheng, NIU Xi-wu, Zoe A Wilson. Molecular Characterization of Down-regulated Gene At2g47500 Associated with myb26/mail sterile35 Mutant[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2014, 29(5): 71-79. doi: 10.7668/hbnxb.2014.05.013.

http://www.hbnxb.net/CN/Y2014/V29/I5/71

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分子鉴定拟南芥myb26/mail sterile35突变体中表达下调基因At2g47500

Molecular Characterization of Down-regulated Gene At2g47500 Associated with myb26/mail sterile35 Mutant

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