Screening and Analysis of Proteins Interacting with TaPDK from Physiological Male Sterility Induced by CHA in Wheat
Screening and Analysis of Proteins Interacting with TaPDK from Physiological Male Sterility Induced by CHA in Wheat
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DOI：
10.1016/s2095-3119(13)60471-2
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发表时间：
2013-06
影响因子：
4.8
通讯作者：
Longyu Zhang;Gaisheng Zhang;Xin-liang Zhao;Shu-Ling Yang
中科院分区：
文献类型：
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作者：
Longyu Zhang;Gaisheng Zhang;Xin-liang Zhao;Shu-Ling Yang
为了进一步研究丙酮酸脱氢酶激酶（pyruvate dehydrogenase kinase，简称TaPDK）在化学杂交剂（CHA） SQ-1诱导小麦生理雄性不育（PHYMS）中的调控网络，构建了另一个花药cDNA文库，并利用酵母双杂交技术筛选了与TaPDK相互作用的蛋白。随后，通过实时定量PCR检测核苷酸表达水平的候选蛋白。将含有BD-TaPDK诱饵质粒的酵母菌Y2HGold与含有花药cDNA文库质粒的酵母菌Y187进行杂交筛选。将二倍体酵母细胞置于合成dropout营养培养基（SD/-Ade/-His/-Leu/-Trp） （QDO）上，再置于含有AbA和X-α-Gal的QDO培养基上孵育。通过共转化验证进一步证实了TaPDK与阳性菌落蛋白之间的相互作用。从蓝色菌落中提取质粒DNA并测序后，用生物信息学方法对测序结果进行分析。最终获得24个菌落，包括非特异性脂质转移蛋白前体（TanLTP）、多泛素（TaPUbi）、甘油醛-3-磷酸脱氢酶、增殖细胞核抗原（TaPCNA）、CBS结构域蛋白（TaCBS）、肌动蛋白、鸟嘌呤核苷酸结合蛋白β亚基、查尔酮合成酶等8个基因，以及3个功能未知的新基因。定量RT-PCR结果显示，与可育系相比，PHYMS花药中TanLTP、TaPUbi和TaPCNA的表达水平明显上调，而TaCBS的表达仅在三细胞期升高。而在PHYMS细胞系中，TaPDK的表达明显下调。综上所述，这些数据表明，大部分候选蛋白可能与PHYMS系花粉败育有关，这进一步表明TaPDK在花粉发育中除了参与调节丙酮酸脱氢酶复合物活性外，还发挥着多种作用。
To further research the regulatory network of pyruvate dehydrogenase kinase (designated as TaPDK) in physiological male-sterility (PHYMS) of wheat induced by chemical hybridizing agent (CHA) SQ-1, an anther cDNA library was constructed, and the proteins interacting with TaPDK were screened via yeast two-hybrid technique. Subsequently, a few candidate proteins in nucleotide expression levels were detected by real-time quantitative PCR. Yeast-two hybrid screening was performed by mating yeast strain Y2HGold containing BD-TaPDK bait plasmid with yeast strain Y187 including anther cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Ade/-His/-Leu/-Trp) (QDO), and further were incubated on QDO medium containing AbA and X-α-Gal. The interactions between TaPDK and the proteins obtained from positive colonies were further confirmed by co-transformation validation. After plasmids DNA were extracted from blue colonies and sequenced, the sequences results were analyzed by bioinformatic methods. Finally, 24 colonies were obtained, including eight genes, namely non-specific lipid-transfer protein precursor (TanLTP), polyubiquitin (TaPUbi), glyceraldehyde-3-phosphate dehydrogenase, proliferating cell nuclear antigen (TaPCNA), CBS domain containing protein (TaCBS), actin, guanine nucleotide-binding protein beta subunit, chalcone synthase, and three new genes with unknown function. The results of quantitative RT-PCR showed that the expression levels of TanLTP, TaPUbi, and TaPCNA were obviously up-regulated in PHYMS anther, and TaCBS expression was only increased at the tricellular stage in PHYMS anther compared with in fertile lines. Whereas, the expression of TaPDK was obviously down-regulated in PHYMS lines. Collectively, these datas indicated that the majority of candidate proteins might be related to pollen abortion in PHYMS lines, which further suggested that TaPDK plays multiple roles in pollen development, besides participating in regulating pyruvate dehydrogenase complex activity.