Isolation And Identification Of Proteins Involved In Quality Of The Strong-gluten Wheat By Two-dimensional Electrophoresis

Wheat powder can be processed into a variety of food,such as bread,noodles,steamed bread and biscuits.Thecomposition and quantity of storage protein are the critical factor for the processing quality of wheat.Xinmai26 is a quality of strong gluten cultivar by Xinxiang Academy of Agricultural Sciences in Henan,of which has super-parent advantage in baking quality and better than dark hard red spring wheat(DNS)of America.To date,the glutenin subunit composition hardly clear its molecular mechanism of the formation of processing high quality.In this study,We measured the quality data in xinmai26 and its parents,respectively.Establishment of a system of Two-dimensional Electrophoresis in mature wheat grain,and used it to investigate the proteins involved in quality of the strong-gluten wheat.The result of this project will not only demonstrate the molecular mechanism for the formation of strong gluten quality in xinmai26,but also provide theoretical,method and technical support for polymer molecular breeding of strong gluten wheat.The results were described as follows:1.Super-parent advantage of baking quality in Xinmai26: Quality analyzer was used to measure the physical and chemical properties of Xinmai26 and its parent.The results show that: The development time of Xinmai26 was over 20 minutes and stability time of Xinmai26 was over 30 minutes which were much higher than its parents;The weakening degree of Xinmai 26 was lower than its parent Ji’nan17,as well as its female parent xinmai18;The parameters of gluten peak of Xinmai26 were higher than its parent Ji’nan17,of which the PMT and AM were higer than its female parent Xinmai18.These characteristics show that the gluten quality in Xinmai26 surpassed their parents.2.Optimization of the two-dimensional electrophoresis system for wheat mature grain: Using mature seeds of Zhengmai366 as the material to establish two-dimensional electrophoresis system of the wheat grain.The results show that using the direct cleavage method extract the mature wheat grain protein,17 cm pH5-8 IPG prefabricated strip,and the loadingquantityofsample being 500μg are the most appropriate system.Two-dimensional electrophoresis patterns with clear and high protein resolution were obtained by using the system to verify the different wheat varieties.3.Isolation and identification of Xinmai26 and its parents: the differential proteins were separated from the mature grain of new wheat 26 and its parentsusing the two-dimensional electrophoresis system of mature grain of wheat,the results show that about 700 protein spots were obtained for each protein mass spectrum,154 significant different protein spots were found between Xinmai26 and Ji’nan17,152 significant different protein spots were found between Xinmai26 and Xinmai18.A total of 150 protein spots with significant differences were analyzed by mass spectrometry,and 135 protein points were successfully annotated.There are 77 points with significant differences between Xinmai26 and Ji’nan17 in mature grain,and 36 of them has strong expression in Ji’nan17,and 41 of them has strong expression in Xinmai26.There are 58 points with significant differences between Xinmai26 and Xinmai18 in mature grain,and 41 of them has strong expression in Ji’nan17,and 17 of them has strong expression in Xinmai26.4.Analyze preliminaryly on the molecular mechanism of excellent processing quality of Xinmai26: Proteins that related to quality mainly include glutenin and gliadin.The key quality related proteins were selected according to the functional annotation results of protein mass spectrometry to perform Real-time PCR verification.The results showed that the majority of gliadin expression was down regulated in Xinmai26,among them,the expression of gamma gliadin SSP6202 and SSP9409 was significantly decreased 45 times and 8 times;The majority of Glutenin expression was up-regulated,and the expression of glutenin subunit 1Ax1(SSP3905)was significantly increased 9 times.The results showed that the decrease of gliadin and the increase of glutenin might be the main cause of the super parent effect on the processing quality of Xinmai26.