用电喷雾质谱和特异PCR初步鉴定粳稻GluB蛋白突变体

Characterization of a glutelin B mutant from novel japonica rice varieties using electrospray ionization mass spectrometry and specific PCR

用十二烷基硫酸钠.聚丙烯酰胺凝胶电泳(SDS-PAGE)方法对采集于中国、日本、印尼、老挝的42份特异粳稻资源进行了分析。结果显示,在编号为 B104的粳稻中有谷蛋白亚家族 GluB 基因沉默现象。将对照材料 B90的对应蛋白进行回收染色、原位酶解,然后利用电喷雾-四极杆飞行时间质谱(ESI-Q-TOF-MS)技术进行鉴定分析。用 MASCOT 数据库进行比对的结果显示,该蛋白的推测分子量为54kD,被包括 GluB2、GluB4、GluB7等基因在内的 GluB 亚家族基因编码。为了深入探讨 GluB 蛋白的突变机制,设计了2对特异引物,以 B90为对照,用基因组 PCR 方法来钓取并鉴定其中的 GluB4和 GluB7基因。鉴定结果表明 GluB7基因在 DNA 水平上未发生变化,而 GluB4基因发生了点突变,导致一个氨基酸的变异。研究证实,电喷雾电离质谱(ESI-MS)可以用来辅助解析植物蛋白质的复合体。

The seeds storage protein analyses on 42 novel japonica rice varieties from China, Japan, Indonesia and Laos were performed using the sodium dodecyl sulphate-polyacrylamide gel electrophoresis （SDS-PAGE）. The result showed that the protein subunits in GluB region were absent in line B104. The counterpart protein bands of the control line B90 were dissected, and then analyzed using the electrospray ionization quadrupole time-offlight mass spectrometry （ESI-Q-TOFMS）. The database searching of peptide segments using MASCOT program showed that the 54kD protein consisted of glutelin subunits coded by GluB genes cluster including genes GluB2, GluB4 and GluB7. In order to determine the molecular basis of the GluB protein mutant, two pairs of specific primers were designed to target the nucleotide sequence of GlulM and GluB7 genes by genomic PCR analysis. The coding regions of GluB4 and GluB7 sequences from line B104 were compared to those of the control line B90. The results suggested that no nucleotide changes occurred in the coding region of GluB7 gene, while there was a nucleotide replacement in GluB4 coding region, which may be responsible for the protein silence in combining with other modifications of gene transcription or translation. In addition, the present study indicated that the electrospray ionization mass spectrometry （ESI-MS） could be served as a new assistant approach to precise identification of a plant protein complex.