Preparation of Polyclonal Antibodies to Barley Granule-Bound Amylopectin Synthase Ia and Their Application in the Characterization of Interacting Proteins
Qiyan Zhou,
Boai Xi,
Noman Shoaib,
Yan Gao,
Zhenbin Cheng,
Rizwan Ali Kumbhar,
Zongyun Feng,
Yajie Liu,
Hui Zhao,
Guowu Yu
Affiliations
Qiyan Zhou
State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China
Boai Xi
State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China
Noman Shoaib
Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
Yan Gao
State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China
Zhenbin Cheng
State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China
Rizwan Ali Kumbhar
State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China
Zongyun Feng
State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China
Yajie Liu
State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China
Hui Zhao
State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China
Guowu Yu
State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China
The production of amylose is facilitated by granule-bound starch synthase (GBSS). Despite its importance, the specific protein interactions involving barley grain-bound starch synthase Ia (HvGBSSIa) remain poorly understood. To elucidate this, we engineered a pET-32a-HvGBSSIa prokaryotic expression vector for specific expression in E. coli Rosetta cells. A rabbit anti-HvGBSSIa polyclonal antibody was generated and employed to enrich HvGBSSIa-binding proteins from barley grains through immunoprecipitation. The isolated complexes were then resolved through SDS-PAGE, and the constituent proteins were identified using mass spectrometry coupled with database searches. Our results confirmed the successful preparation of a highly specific polyclonal antibody against HvGBSSI. Furthermore, differential expression of HvGBSSIa was assessed across various barley tissues and developmental stages of the grain, revealing peak expression at 25 days post-flowering. Proteins interacting with HvGBSSIa, including sucrose synthase and starch branching enzyme, were identified through co-immunoprecipitation. This study lays the groundwork for further detailed analyses of the HvGBSSIa protein complex in barley.
