Screening and Analysis of Proteins Interacting with TaPDK from Physiological Male Sterility Induced by CHA in Wheat

ZHANG Long-yu; ZHANG Gai-sheng; ZHAO Xin-liang; YANG Shu-ling

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Screening and Analysis of Proteins Interacting with TaPDK from Physiological Male Sterility Induced by CHA in Wheat

机译：小麦CHA诱导的生理雄性不育与TaPDK相互作用蛋白的筛选与分析。

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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.

机译：为了进一步研究由化学杂交剂（CHA）SQ-1诱导的小麦的生理性雄性无菌（PHYMS）的丙酮酸脱氢酶激酶（指定为TAPDK）的调节网络，构建了一种花药cDNA文库，蛋白质与烟雾器相互作用通过酵母双混合技术筛选。随后，通过实时定量PCR检测核苷酸表达水平的几种候选蛋白质。通过使用酵母菌菌株Y187的含有Bd-Tapdk诱饵质粒的致酵母菌菌株Y2HGold进行酵母菌菌株Y2HGold进行酵母 - 两个杂化筛选。二倍体酵母细胞在合成的辍学营养培养基（SD / -RE / -HIS / -LEU / -TRP）（QDO）上涂覆，并进一步在含有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和从阳性菌落获得的蛋白质之间的相互作用。从蓝色菌落中提取质粒DNA后并测序，通过生物信息化方法分析序列结果。最后，获得了24种菌落，包括八个基因，即非特异性脂转移蛋白前体（TanlTP），络合剂（Tapubi），甘油醛-3-磷酸脱氢酶，增殖细胞核抗原（Tapcna），CBS结构域蛋白质（塔累），肌动蛋白，鸟嘌呤核苷酸结合蛋白β亚基，Chalcone合酶和三种具有未知功能的新基因。定量RT-PCR的结果表明，TanlTP，Tapubi和TapCNA的表达水平明显在Phyms花药中明显上调，并且在与肥沃线相比，TACBS表达仅在细胞间阶段增加。虽然，Tapdk的表达明显以Phyms线条下调。总的来说，这些数据表明大多数候选蛋白质可能与Plyms系列的花粉流产有关，这进一步表明，除了参与调节丙酮酸脱氢酶复合活性之外，Tapdk在花粉发育之外发挥着多种作用。

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《农业科学学报（英文版）》 |2013年第6期|941-950|共10页
作者
ZHANG Long-yu; ZHANG Gai-sheng; ZHAO Xin-liang; YANG Shu-ling;
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College of Agronomy, Northwest A&F University/Wheat Breeding Engineering Research Center, Ministry of Education/Key Laboratory of Crop Heterosis of Shaanxi Province, Yangling 712100, P.R.China;

College of Agronomy, Northwest A&F University/Wheat Breeding Engineering Research Center, Ministry of Education/Key Laboratory of Crop Heterosis of Shaanxi Province, Yangling 712100, P.R.China;

College of Agronomy, Northwest A&F University/Wheat Breeding Engineering Research Center, Ministry of Education/Key Laboratory of Crop Heterosis of Shaanxi Province, Yangling 712100, P.R.China;

College of Agronomy, Northwest A&F University/Wheat Breeding Engineering Research Center, Ministry of Education/Key Laboratory of Crop Heterosis of Shaanxi Province, Yangling 712100, P.R.China;

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Screening and Analysis of Proteins Interacting with TaPDK from Physiological Male Sterility Induced by CHA in Wheat

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