读paper，做笔记

Arabidopsis thaliana Acyl-CoA-binding protein ACBP6 interacts with plasmodesmata-located protein PDLP8

Introduction

1. PDLP1-PDLP8 ,  predicted to range between 30 to 35 kD

2. PDLP5, as a regulator of plasmodesmal gating, was responsive to virulent Pseudomonas syringae 注射假单胞菌 and salicylic acid (SA)-dependent callose deposition at the plasmodesmata

3. Arabidopsis thaliana : acyl-CoAbinding protein (AtACBP) family share a conserved acyl-CoA binding domain 

(AtACBP) family 1-6的定位和功能：

Ankyrin repeat-containing AtACBPs, AtACBP1 and AtACBP2, a membrane-associated proteins that can facilitate protein-protein interactions as well as mediate heavy metal stress responses. 膜定位，重金属胁迫

AtACBP3 has been identified as an extracellularly-targeted protein which functions in pathogen defense and autophagy-mediated leaf senescence.  细胞外定位，叶片衰老

Both cytosolic AtACBP4 and AtACBP5 contain kelch-motifs that are potentially capable of protein-protein interactions and they play complementary roles in floral development.

The smallest(10-kDa) member, AtACBP6, is also a cytosolic protein and conferred freezing tolerance upon its ectopic expression in transgenic Arabidopsis rosettes and flowers.

More recently, immunoelectron microscopy using anti-AtACBP6 in 5-weekold Arabidopsis revealed that AtACBP6 was detected in the companion cells, sieve elements and the plasmodesmata.

本文使用等温滴定量热法（ITC）以及pull-down和双分子荧光互补（BiFC）测定法验证了PDLP8和AtACBP6的相互作用。利用qRT-PCR、对PDLP8突变体进行韧皮部渗出物分析，对PDLP8pro：：GUS和AtACBP6pro：：GUS转基因拟南芥系进行GUS测定，进一步探讨了PDLP8与AtACBP6之间的关系。

Results

Interaction of AtACBP6 and PDLP8 at the plasmamembrane

PDLP8结构： 两个a螺旋，四个b片和一个可见的环出现在PDLP8的3-D结构中

AtACBP6-PDLP8相互作用，与AtACBP6的a1和a2螺旋预测与PDLP8的b3和b4片相互作用

确定其相互作用后，提出问题：

由于PDLP8是一种跨膜蛋白，而AtACBP6蛋白位于细胞质基质中，因此对这两种蛋白在何处以及如何在细胞下相互作用进行了研究

(C-D) The interaction between (His)6-PDLP8 and GST-tagged AtACBP6 (with the GST tag cleaved) was visualized by 15% SDS-PAGE.

M, low-range rainbow marker; 

Lane 1, recombinant (His)6-PDLP8; 

Lane 2, flow-through after recombinant (His)6-PDLP8 is bound to the Ni-NTA magnetic agarose beads;

Lane 3, recombinant GST-tagged AtACBP6 (with the GST tag cleaved); 

Lane 4, flow-through after incubation of recombinant (His)6-PDLP8 and recombinant AtACBP6 on the Ni-NTA magnetic agarose beads following washing to remove excess recombinant AtACBP6; 

Lane 5, elution from Ni-NTA magnetic agarose beads using buffer B.

Arrows indicate the various recombinant proteins.

BiFC assays：

AtACBP6：：CYFP和PDLP8：：NYFP在边莲（烟草）叶细胞中的瞬时共表达在质膜上显示出YFP荧光信号（图1I），表明AtACBP6和PDLP8在质膜上的相互作用。

bZIP63：：NYFP和bZIP63：：CYFP组合作为我们的阳性对照，并在细胞核中检测到荧光信号（图1E）用同一个蛋白drive作为阳性control

阴性对照对（P35S：：NYFP  + P35S：：CYFP，AtACBP6：：CYFP + P35S：：NYFP和P35S：：CYFP C+PDLP8：：NYFP）未显示任何荧光信号

双分子荧光互补(bimolecular fluorescence complementation，BiFC)分析技术，是由Hu等在2002年最先报道的一种直观、快速地判断目标蛋白在活细胞中的定位和相互作用的新技术。

简介

将荧光蛋白在某些特定的位点切开，形成不发荧光的N和C端2个多肽，称为N片段(N-fragment)和C片段(C-fragment)。这2个片段在细胞内共表达或体外混合时，不能自发地组装成完整的荧光蛋白，在该荧光蛋白的激发光激发时不能产生荧光。

但是，当这2个荧光蛋白的片段分别连接到1组有相互作用的目标蛋白上，在细胞内共表达或体外混合这2个融合蛋白时，由于目标蛋白质的相互作用，荧光蛋白的2个片段在空间上互相靠近互补，重新构建成完整的具有活性的荧光蛋白分子，并在该荧光蛋白的激发光激发下，发射荧光。

简言之，如果目标蛋白质之间有相互作用，则在激发光的激发下，产生该荧光蛋白的荧光。反之，若蛋白质之间没有相互作用，则不能被激发产生荧光。

该技术巧妙地将荧光蛋白分子的两个互补片段分别与目标蛋白融合表达，如果荧光蛋白活性恢复则表明两目标蛋白发生了相互作用.其后发展出的多色荧光互补技术(multicolor BiFC)，不仅能同时检测到多种蛋白质复合体的形成，还能够对不同蛋白质间产生相互作用的强弱进行比较。目前，该技术已用于转录因子，G蛋白βγ亚基的二聚体形式，不同蛋白质间产生相互作用强弱的比较以及蛋白质泛素化等方面的研究工作上。

意义

该方法利用绿色荧光蛋白(greenfluorescent protein，GFP)及其突变体的特性作为报告基因，将荧光蛋白分割成两个不具有荧光活性的分子片段，再分别与目标蛋白连接。如果两个目标蛋白因为有相互作用而靠近，就使得荧光蛋白的两个分子片段在空间上相互靠近，重新形成活性的荧光基团而发出荧光。在荧光显微镜下，就能直接观察到两目标蛋白是否具有相互作用，并且在最接近活细胞生理状态的条件下观察到其相互作用发生的时间、位置、强弱、所形成蛋白质复合体的稳定性，以及细胞信号分子对其相互作用的影响等，这些信息对研究蛋白质相互作用有一定意义。

Comparison of PDLP8 and AtACBP6 expression in Arabidopsis tissues

Figure 2. Analysis in the expression pattern of AtACBP6 and PDLP8. Histochemical staining shows GUS expression of PDLP8pro::GUS (A-F) and AtACBP6pro::GUS (G-L),8-week-old inflorescence, 10-DAF mature flowers and 3-week-old rosette leaves. Different GUS staining times are shown (2 h in A-C and G-I; 24 h in D-F and J-L). GUSexpression was not detected in vector (pB101.3)-transformed control plants (M-R). (S-T) A comparison in expression of AtACBP6 and PDLP8 as carried out by analysis ofmicroarray data from Genevestigator (https://genevestigator.com/gv/). (S) Expression of AtACBP6 and PDLP8 in different developmental stages. The red line indicates theexpression of AtACBP6 and the blue represents the expression of PDLP8. (T) Heat map of the expression of AtACBP6 and PDLP8 in the phloem. CC, companion cells. (U)Quantitative real-time PCR analysis of PDLP8 and AtACBP6 expression in 4-week-old rosette leaves (RL), 4-week-old stems (ST), 4-week-old stems roots (RT), 1-week-oldseedlings (SD), buds (BD) and 8-week-old open flowers (OF). Results were normalized against the expression of ACTIN2. Each bar represents a mean value of six repeatsfrom two independent biological samples § SD.

GUS看时空表达，看在植物不同发育阶段的表达量，在不同组织中表达量，通过real-time PCR看mRNA的表达量

Reduced AtACBP6 accumulation in pdlp8 phloem exudates

1、首先确认pdlp8突变体可用，为salk line，T-DNA插入突变体，用三引物法；

2、qPCR检验pdlp8突变体的mRNA表达量

3、为了研究PDLP8是否会影响筛元件对AtACBP6的摄取，收集pdlp8突变植物的韧皮部渗出物，AtACBP6特异性抗体进行蛋白质印迹分析，Col-0和acbp6为对照，结果显示AtACBP6 accumulation was reduced in the absence of PDLP8 in the phloem

4、然而，叶子和花朵的AtaCBP6 mRNA表达水平在5周龄Col-0和pdlp8之间显示出无显着差异。因此we concluded that the absence of PDLP8 diminished AtACBP6

accumulation in the sieve elements.

Discussion

PDLP8不影响AtACBP6的表达，但是影响其积累