Molecular identification of ω-secalin gene expression activity in a wheat 1B/1R translocation cultivar  

作　　者：CHAI Jian-fang ZHANG Cui-mian MA Xiu-ying WANG Hai-bo 

机构地区：[1]Institute of Genetics and Physiology,Hebei Academy of Agriculture and Forestry Sciences/Plant Genetic Engineering Center of Hebei Province

出　　处：《Journal of Integrative Agriculture》2016年第12期2712-2718,共7页农业科学学报（英文版）

基　　金：supported by the Natural Science Foundation of Hebei Province,China (C2014301005)

摘　　要：ω-Secalin was an important factor influencing processing quality of wheat 1BL/1RS translocations. On the basis of four ω-secalin gene sequences cloned from Lankao 906 （a wheat cultivar with 1BL/1RS translocation） with putative transcription activity, a pair of primers with suitable restriction endonucleases added at their 5＂ ends were designed to amplify the mature protein-coding regions of the four genes. After digestion with restriction endonucleases, the amplified products were ligated into the prokaryotic expression vector pET30a（＋）. The prokaryotically expressed recombinant proteins and gliadin isolated from the Lankao 906 seed were analyzed on the same acid polyacrylamide gel electrophoresis. All four prokaryotically expressed secalin bands had corresponding seed-expressed gliadin bands. The four corresponding gliadin bands were confirmed to be the expression products of the four ω-secalin genes by liquid chromatography tandem mass spectrometry （LC-MS/MS）. This conclusion was further confirmed because the four ω-secalin bands could be detected in all 14 wheat 1BL/1 RS translocation cultivars used in the study, although there was some interference for the detection of one ω-secalin band from nearby wheat gliadin bands. The sequence information of ω-secalin genes with expression activity will be helpful for improving the processing quality of wheat with 1BL/1RS translocations by using RNA interference method to silence the expression of the ω-secalin genes.ω-Secalin was an important factor influencing processing quality of wheat 1BL/1RS translocations. On the basis of four ω-secalin gene sequences cloned from Lankao 906 （a wheat cultivar with 1BL/1RS translocation） with putative transcription activity, a pair of primers with suitable restriction endonucleases added at their 5＂ ends were designed to amplify the mature protein-coding regions of the four genes. After digestion with restriction endonucleases, the amplified products were ligated into the prokaryotic expression vector pET30a（＋）. The prokaryotically expressed recombinant proteins and gliadin isolated from the Lankao 906 seed were analyzed on the same acid polyacrylamide gel electrophoresis. All four prokaryotically expressed secalin bands had corresponding seed-expressed gliadin bands. The four corresponding gliadin bands were confirmed to be the expression products of the four ω-secalin genes by liquid chromatography tandem mass spectrometry （LC-MS/MS）. This conclusion was further confirmed because the four ω-secalin bands could be detected in all 14 wheat 1BL/1 RS translocation cultivars used in the study, although there was some interference for the detection of one ω-secalin band from nearby wheat gliadin bands. The sequence information of ω-secalin genes with expression activity will be helpful for improving the processing quality of wheat with 1BL/1RS translocations by using RNA interference method to silence the expression of the ω-secalin genes.

关 键 词：Triticum aestivum processing quality storage proteins prokaryotic expression LC-MS/MS 

分 类 号：S512.1[农业科学—作物学] S531.03