CAJ | 학술논문

为建立一套适合于牡丹试管苗茎基部蛋白的双向电泳技术,以便更好地利用蛋白质组技术研究牡丹试管苗不定根的发生机理,本研究比较了三种不同蛋白质提取方法对双向电泳结果的影响,并在蛋白质上样量方面进行了比较.结果表明,乙酸铵/甲醇酚提取法所得2-DE图谱的蛋白点很少,仅检测到45个,且较模糊,有明显的拖尾现象,分辨率很低;乙醇/乙醚丙酮法所得的蛋白点也较少(101个),较模糊,且横竖纹干扰较大;三氯乙酸/丙酮法所得蛋白点数较多,可检测到434个清晰的蛋白点,且形状规则,重复性好,适合后续分析,操作也较为简便.用三氯乙酸/丙酮法提取蛋白,采用800μg、1000 μg和1 200μg三个不同的上样量进行双向电泳,在上样量为1200μg时(IPG pH 3～10,24 cm),蛋白质在12%SDS-PAGE胶上得到了较好的分离,在2-DE图谱上可分辨出562个蛋白点.因此,三氯乙酸/丙酮法是较适合于牡丹试管苗茎基部蛋白质提取的方法,1200μg是较为合适的上样量.
위건립일투괄합우모단시관묘경기부단백적쌍향전영기술,이편경호지이용단백질조기술연구모단시관묘불정근적발생궤리,본연구비교료삼충불동단백질제취방법대쌍향전영결과적영향,병재단백질상양량방면진행료비교.결과표명,을산안/갑순분제취법소득2-DE도보적단백점흔소,부검측도45개,차교모호,유명현적타미현상,분변솔흔저;을순/을미병동법소득적단백점야교소(101개),교모호,차횡수문간우교대;삼록을산/병동법소득단백점수교다,가검측도434개청석적단백점,차형상규칙,중복성호,괄합후속분석,조작야교위간편.용삼록을산/병동법제취단백,채용800μg、1000 μg화1 200μg삼개불동적상양량진행쌍향전영,재상양량위1200μg시(IPG pH 3～10,24 cm),단백질재12%SDS-PAGE효상득도료교호적분리,재2-DE도보상가분변출562개단백점.인차,삼록을산/병동법시교괄합우모단시관묘경기부단백질제취적방법,1200μg시교위합괄적상양량.
In this paper, we compared the effects of different protein extraction methods and loading amount on 2-DE profile in order to establish a suitable and efficient 2-DE technique for proteome of basal part of stem of mudan in vitro and facilitate rooting mechanism research by using proteome approach. The results showed that samples prepared by ammonium acetate/methanol-phenol extraction only got 45 protien spots on the 2-DE profile, and the spots were indistinct, had obvious streak appearance and low resolution. Samples obtained by method of ethanol/ether-acetone precipitation got 101 protien spots that were not clear either and had an obvious interference of crosswise and uptight streaks. While samples acquired by TCA/acetone precipitation had more distinguishable and regularly-shaped protein spots (about 434), which was of good reproducibility and was suitable for subsequent analysis. And also TCA/acetone precipitation approach was more convenient for manipulation. Furthermore, we employed different loading amount proteins extracted by the method of TCA/acetone for 2-DE, that is, 800 μg,1 000μg and 1 200μg. Proteins of 1 200μg loading amount separated well on the 12% SDS-PAGE gel with IPG gel of pH 3～10, 24 cm, 562 protein spots were distinguished on the 2-DE profile. As a result, TCA/acetone precip-itation was much more suitable for protein extraction of basal part of stem of mudan in vitro and 1 200 μg was the optimum loading amount.