【作者】 安帅；

【导师】 王小纯；

【作者基本信息】 河南农业大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 2007—2009年采用大田盆栽的方法,较系统地研究了氮素形态对豫麦34不同生育期各组织器官GS同工酶表达及亚基组成的影响;回收鉴定了不同种类的GS同工酶,主要结果如下:苗期叶片GS同工酶的表达受生育期影响较大,均存在三种同工酶,一叶一心期较其它三个时期GS同工酶的活性低,同时发现一种新的同工酶GSX。开花灌浆期叶片共有有四种同工酶的表达,又发现一种新的同工酶GSX2,,且GSX2只在开花期和花后7天的生育时期出现,铵盐对其有诱导作用。同时期茎节中也有四种同工酶的表达,但以GS1为主。籽粒中只有GS1同工酶的表达。开花期旗叶粗酶液Native-PAGE蛋白杂交证实新发现的GSX和GSX2是GS同工酶,开花期旗叶粗酶液SDS-PAGE蛋白杂交显示,大田盆栽条件下GS同工酶亚基存在除以往报道的43.6KD和38.4KD亚基之外的87.8KD、68.6KD、37KD、19.05KD、17.85KD亚基。通过Native-PAGE对拔节期GS1、GS2、GSX及开花期GS1、GS2、GSX、GSX2的分别回收鉴定,发现GS1、GS2、GSX亚基构成在不同生育期也发生变化,相对拔节期开花期GS1亚基构成种类增加了43.6KD的亚基而缺失了57.4KD和37KD亚基;GS2亚基构成种类增加了38.4KD亚基而缺失了57.4KD亚基;GSX亚基构成种类缺失了87.8KD亚基而增加了37KD亚基。开花期GSX2则与同时期GS1亚基组成接近。对不同N素形态下豫麦34拔节期GS同工酶亚基组成变化的研究得到:拔节期硝态氮对于豫麦34 GS同工酶各类亚基的含量有促进作用。铵盐能够促进部分组织亚基种类丰富,及尿素处理相对较低。对硝态氮处理下不同生育期豫麦34 GS同工酶亚基构成及含量变化的研究得到:不同生育期间GS同工酶亚基构成种类及含量呈先升后降的趋势,受生育期影响较大,开花期GS同工酶种类及含量最高。生育期内不同组织器官GS同工酶种类及含量由高到低依次为叶、鞘、节、根。籽粒和颖壳内GS同工酶亚基构成种类较为独特。提取硝态氮处理下豫麦34旗叶片全蛋白进行双向电泳,结果显示:捕捉分离到GS2和GS2a两个同工酶,并捕获到未知蛋白4个。更多还原

【Abstract】 From 2007 to 2009,with the methods of pot experiment, the effects of N forms on the expression and Subunit composition of GS isozymes in different organs during different phase of yumai34 were studied, and the GS isozymes In Wheat were Separated and sublimated, the subunits composition of GS protein were Characterized and evaluated.the main results were as follows:By the pot experiment in field, simulating field, the effects of N forms on the expression of GS in leaves of yumai34 were studied. The results indicated: the expression of GS isozymes in leaves of the seeding stage was little affected by N forms and genotypes, but affected greatly by different growth stages. There are three types of GS isozymes in all phases of seeding stage, but compared the other three phases, the GS activity was lower in the stage of one leave and one heart. Compared to the expression of GS isozymes in the seeding stage, a new isozyme GSX2 was found in the leaves of anthesis, the ammonium has certain function in inducing the appearance of GSX2. It appears in anthesis and 7DAF only. There are also four types of isozymes expressed in the stem in the same stage, the GS1 isozyme occupy the dominant position, and only GS1 isozyme expresses in the grain.By the method of Native-PAGE, it is confirmed that the newly discovered protein hybrid GSX and GSX2, from the crude extract of flowering of flag leaf, are GS isozymes.By the pot experiment in field , simulating field, the crude extract of flowering of flag leaf is studied by the method of SDS-PAGE. It is reported that the GS isozyme subunit exist 87.8KD, 68.6KD, 37KD, 19.05KD, 17.85KD subunit except the previously reported 43.6KD and 38.4KD subunits.By the method of Native-PAGE, the proteins of GS1,GS2,GSX from the jointing stage and the proteins of GS1,GS2,GSX,GSX2 from the period of flowing are recovered respectively, the results indicated that the GS1,GS2,GSX subunits have changed at different growth stages of the wheet .Compared to the expression of GS isozymes in the jointing stage, the GS1 subunits increased the types of 43.6KD subunits and is lack of the 57.4KD and 37KD subunit in the flowering;GS2 subunits increased the 38.4KD subunits and missing the 57.4KD subunits;GSX subunits missing the types of 87.8KD subunits and increased the 37KD subunits.The constitute of GSX2 subunits in the flowing are approaching to the GS1 subunits in the same period.The effects of different N forms on the changement of GS isozyme subunits during the jointing stage of yumai 34 were studied, the result showed that nitrate can promote the content of various types of subunits of GS isozyme from yumai 34. Ammonium salt can facilitate the types of GS isozyme subunits of parts of organizations.Compared to the nitrate and ammonium, the effect of urea on the GS isozymes was relatively lower.The effects of nitrate on the composition and the changements of contents of GS isozymes in the different growth stages of yumai 34 were studied, it was found that the tendency of GS isozymes’types of subunits composition and content during different growth was first increased and then decreased. The GS isozymes were affected greately by different growth stages. In the flowering , the types and contents of GS isozymes were highest .During the different phases of yumai 34, reproductive organs of GS isozymes and content of order were leaves, sheath, section, root. The types of GS isozymes of grain and glume were more unique.By the method of two-dimensional electrophoresis, the whole proteins of flag leaves with the treatment of nitrate of yumai 34 were extracted and studied, the result showed that two kinds of GS isozymes, GS2 and GS2a, were captured and isolated. And 4 kinds of unknown proteins were captured, too.更多还原