苹果叶片叶绿体分离及其蛋白提取、双向电泳方法的优化

Optimization of isolation and extraction of chloroplast from apple leaves,2-DE of the chloroplast protein

【目的】筛选苹果叶片叶绿体及其蛋白的提取方法,建立叶绿体蛋白双向电泳分离技术体系。【方法】以苹果叶片为试材,采用4种Percoll密度梯度离心法提取苹果叶片叶绿体,检测提取叶绿体的完整度、纯度及提取率。采用酚抽提法、改良酚抽提法提取叶绿体蛋白,检测蛋白样品的质量。优化胶条长度、上样量、等电聚焦程序、分离胶浓度等双向电泳条件。【结果】Percoll密度梯度离心法D提取的叶绿体纯度高、提取率高,优于其他3种方法;改良酚抽提法提取的叶绿体蛋白,经2-DE分离所得蛋白点数多、分离效果好,优于酚抽提法。蛋白样品经优化后的双向电泳体系分离,获得了较为理想的2-DE图谱。【结论】Percoll密度梯度离心法D、改良酚提法适于苹果叶片叶绿体及其蛋白的提取。苹果叶片叶绿体蛋白使用优化后的双向电泳体系进行分离,可以获得比较理想的2-DE图谱。

[Objective]In order to study chloroplast proteomics in apple leaves,we designed our research to establish an efficient isolation of chloroplast from apple leaves,an optimal extraction of chloroplast protein and a suitable two-dimensional electrophoresis（2-DE） system for the protein.[Methods]The majority of chloroplast isolation of plant material,was comminuted and homogenized,and the crude extracts were obtained after centrifugation.Then,the chloroplast fraction was isolated from the crude extracts,through a density gradient liquid.In this experiment,the apple leaves were picked from the apple breeding nursery at the Institute of Pomology of the Chinese Academy of Agricultural Sciences（CAAS） in Xingcheng city,Liaoning province,China.There were four types of percoll density gradient centrifugation methods used to isolate the chloroplast from apple leaves.During the isolation experiments,three types of homogenate techniques（freezing grinding homogenate,grinding homogenate and machine homogenate） were used to comminute apple leaves and obtain the crude extracts.The chloroplast fraction was isolated from the crude extracts by using different types of percoll density gradient liquids.The purified chloroplast was observed under an optical microscope,allowing for counting the number of intact chloroplast,in order to test the integrity rate and the purity of the chloroplast sample.The chloroplast isolated by method D was observed using a TEM（transmission electron microscope）.The catalase enzyme assay of the crude extracts and the chloroplast（the two both extracted by method D） was performed.Phenol extraction is a classic method to extract plant protein.The theory of phenol extraction is as follows：after the vortex homogenate or grinding homogenate of the plant material,the sample extraction liquid is obtained from the homogenate liquid by TRIS,then it is mixed with the flocculants,and the protein is agglomerated.After high speed centrifugation,the protein is precipitated and extracted.In our paper,the modified phenol extraction was improved from the phenol extraction.Phenol extraction and modified phenol extraction were used to extract chloroplast protein in apple leaves during our experiment.The determination of the protein concentration,SDSPAGE and 2-DE were used to test the qualities of the chloroplast protein.The chloroplast protein was dissolved by sample lysis liquids,and the concentration of chloroplast protein was tested by using a UV Spectrophotometer（HITACHI U-3900）.The operational steps for SDS-PAGE were as follows：loaded protein samples 15 μg;50 V,4%gel concentration,30 minutes electrophoresis;100 V,15%gel concentration,2.5 hours electrophoresis.The map of SDS-PAGE was obtained from the gel dyed by using Coomassie brilliant blue R-350（CBB R-350）,with the help of the Image Scanner.The operating system of 2-DE was the improved method in our paper.The operating system of 2-DE includes many steps：the selection of IPG strips,the selection of loaded samples,the adjustment of the isoelectric focusing program,the selection of gel concentration and so on.These steps were improved during our research,in order to set up the2-DE system for the best applicable method to analyze chloroplast proteomics in apple leaves.[Results]The three types of percoll density gradient liquids were stratified,after the centrifugation,and during the methods A,B and D.The percoll density gradient liquid wasn＇ t stratified,after the centrifugation,during method C.The components（10%-30%-50%） of percoll density gradient liquids of methods A,B and D were best suited to isolate the chloroplast.The use level of the three types of percoll liquids（10%,30%,50%） was improved to save material and cost,during method D.The apple leaves were homogenized three times and the isolation rate of the chloroplast was improved,during method D.As observed under the optical microscope,there was less than other subcellular structures and broken chloroplast,except for the whole chloroplast,used during methods A,B and D.The intact rates of the chloroplast isolated by methods A,B and D,were（69.86±1.502）%,（80.80±0.384）%and（90.33±0.847）%,respectively.The isolation rates of the chloroplast samples extracted by methods A,B and D,were（0.24±0.002）%,（0.28±0.009）%and（1.01 ±0.018）%,respectively.The integrity of the isolated chloroplast by method D was also confirmed by TEM.During method D,the crude extracts showed high catalase activity,whereas the purified chloroplast fractions did not show any significant catalase activity.The result indicates that it was once again the higher integrity rate and higher purity that allowed for the chloroplast isolated by method D.There was a higher integrity rate,higher isolation rate,and higher material utilization for the chloroplast isolated with the help of the percoll density gradient centrifugation method D.The extraction yields of the chloroplast protein extracted by phenol extraction and modified phenol extraction were（1.961 ±0.017） mg·g^-1 and（0.442±0.013） mg·g^-1,respectively.Compared with the phenol extraction,more protein strips was separated by SDS-PAGE than from the chloroplast protein extracted by modified phenol extraction.The protein spots of the chloroplast protein extracted by phenol extraction and modified phenol extraction were 75±3and 433±14 respectively,which were separated by 2-DE and tested by the software,Image Master 2D Platinum 7.0.Many procedures of the 2-DE operating system were improved（during the experiments below,the protein spots of chloroplast protein was separated by 2-DE and the gel map was tested by the software,Image Master 2D Platinum 7.0）.Three types of precast IPG strips（pH 4-7,7 cm;pH 4-7,11 cm;pH 4-7,18 cm） were used for the 2-DE,and the separated protein spots were 83±8,109±3 and 433±14,respectively.Three loaded samples（250 μg,350 μg,and 450 μg） were used for the 2-DE,and the separated protein spots were 205±8,433±14 and 365±6,respectively.The total focusing power of the improved program of isoelectric focusing could achieve 50 625 Vh,more than the total focusing power of the two programs provided by GE Healthcare.The improved program of isoelectric focusing was as follows：Step and Hold,500 V,2 h;Gradient,1 000 V,1.5 h;Gradient,8 000 V,4 h;Step and Hold,8 000 V,3.5h;Step and Hold,500 V,6 h.The gels of the two types of concentration（12%,15%） were used for the 2-DE,and the separated protein spots were 268±8 and 433±14,respecitvely.Compared with the gel of 12%concentration,in the 2-DE map of the gel of 15%concentration,the background showed great clarity and the protein spots were fully separated.The improved 2-DE steps were as follows：IPG strips pH 4-7,18 cm,loaded protein samples 350 μg,the improved program of isoelectric focusing,and the gel of 15%concentration.The chloroplast protein sample in apple leaves,with the help of the improved 2-DE operating system,could obtain a clear 2-DE map and more separated protein spots.[Conclusion]The chloroplast protein,which was extracted from apple leaves with the help of percoll density gradient centrifugation method D and modified phenol extraction,were both suitable for the analysis of 2-DE.The chloroplast proteins from the apple leaves,by means of the improved 2-DE system,were able to obtain a clear 2-DE map and showed more separated protein spots.The method and system provided in the research,was suitable for the analysis of chloroplast proteome in apple leaves.It contributed to unearth functionality protein and promote the processes of molecular mark assisted breeding selection（MAS） and germplasm resources evaluation in apples for developing the research of chloroplast proteomics in apple leaves.