利用酵母双杂交筛选菠萝AcSWEET11的互作蛋白

Screening of AcSWEET11 Interacting Proteins Using Yeast Twohybrid System in Pineapple(Ananas comosus L.)

SWEET(sugars will eventually be exported transporter)基因在植物开花过程中具有重要的作用,但AcSWEET11在菠萝成花中的作用机制尚不清楚。通过鉴定成花过程中与AcSWEET11的互作蛋白,为菠萝成花机制的解析奠定基础。本研究利用共转化的方法在菠萝成花过程的cDNA膜文库中筛选AcSWEET11的互作蛋白,分析候选蛋白的表达量。结果表明,pBT3-STE-AcSWEET11+pPR3-N对NMY51酵母细胞无毒性,但有自激活活性。进一步研究结果显示,在TDO/3?AT培养基和QDO培养基上自激活受到抑制。利用该系统筛选到了81个阳性克隆,经测序鉴定出48个与AcSWEET11互作的候选蛋白,包括E3 ubiquitin-protein ligase RING1-like、Trehalose-phosphate synthase 7、Cytochrome P450、TranscriptionfactorLUX等。GO和KEGG分析结果显示,48个蛋白主要分布在细胞进程、代谢过程、刺激反应和催化活性等生物过程,参与脂类代谢、氨基酸代谢和碳水化合物代谢、信号转导和运输与分解代谢等新陈代谢途径。Trehalose-phosphate synthase 7(XP_020105459.1)、Protein TIFY 3-like(XP_020082835.1)、40S ribosomal protein S27(XP_020092770.1)、Heterogeneous nuclear ribonucleoprotein 1-like(XP_020112516.1)等4个基因与AcSWEET11表达趋势一致,在菠萝成花过程中下调表达;Dihydrolipoyl dehydrogenase 2(XP_020113798.1)、Putative lipid-transfer protein DIR1(XP_020086640.1)、clathrin assembly protein At4g32285(XP_020108161.1)等3个基因在菠萝成花过程中上调表达。这些结果表明,AcSWEET11可能通过与Trehalose-phosphatesynthase7等蛋白发生互作,参与菠萝成花过程。本研究进一步丰富了AcSWEET11的蛋白互作网络,为AcSWEET11在菠萝成花中的调控机制的解析奠定基础。

SWEET(sugars will eventually be exported transporter)plays an important role in pant flowering.However,the mechanism of flower was unclear in pineapple.In this study,the interacting proteins of AcSWEET11 were identified,in order to lay a foundation for analysis of the flowering mechanism of pineapple.In this study,the interacting proteins of AcSWEET11 were screened from the pineapple flowering cDNA membrane library by co-transformed,and then the expression levels of the candidate proteins during flowering were analyzed.pBT3-STE-AcSWEET11+pPR3-N was nontoxicity for NMY51 yeast cells but had autonomous activation.Further study showed that autonomous activation was restrained after adding 3ʹAT in TDO and QDO.A total of 81 positive clones were screened by yeast two-hybrid,and 48 proteins were identified by sequencing which were probably interactive with AcSWEET11.The proteins were contained E3 ubiquitin-protein ligase RING1-like,Trehalose-phosphate synthase 7,Cytochrome P45090A1-like and Transcription factor LUX.GO and KEGG revealed that 48 proteins may participate in lipid metabolism,amino acid metabolism,carbohydrate metabolism,signal transduction and transport and catabolism.And the expression of Trehalose-phosphate synthase 7(XP_020105459.1),Protein TIFY 3-like(XP_020082835.1),40S ribosomal protein S27(XP_020092770.1)and Heterogeneous nuclear ribonucleoprotein 1-like(XP_020112516.1)were down regulated during flowering induction that consistent with AcSWEET1 while the expression of Dihydrolipoyl dehydrogenase 2(XP_020113798.1),Putative lipid-transfer protein DIR1(XP_020086640.1),Clathrin assembly protein At4g32285(XP_020108161.1)were up regulated.AcSWEET11 might interact with the proteins such as Trehalose-phosphate synthase 7 to participate in pineapple flowering.The protein interaction network of AcSWEET11 was plentiful to provide a foundation for analysis the regulatory mechanism of AcSWEET11 in pineapple flowering.