通过SDS-PAGE分析，在二倍体、四倍体和六倍体冰草[Agropyron cristatum (L.) Gaertn.]中都检测到2个表达的高分子量麦谷蛋白亚基（HMW-GS），但不同倍性的材料之间，以及相同倍性材料的不同种子之间的HMW-GS组成均有所不同。利用PCR技术对冰草的HMW-GS基因进行克隆，结果从二倍体、四倍体和六倍体冰草中分别克隆了3个、1个和3个y型HMW-GS基因的全长编码区序列。序列分析表明，只有来自六倍体冰草中的Bsy2基因具有完整的开放阅读框，编码1个有487氨基酸残基、分子量约为53 kD的HMW-GS，大小相当于SDS-PAGE图谱中的大亚基。而其余6个基因均在中间重复区发生了无义突变。本文对冰草HMW-GS基因的结构特点和进化关系进行了分析。
Wheat processing quality is closely correlated with composition and quantity of gluten proteins, in particular with high-molecular-weight glutenin subunits (HMW-GS) encoded by the Glu-1 loci (Glu-A1, Glu-B1 and Glu-D1). In the related species of wheat, there are abundant HMW-GS alleles which are possibly valuable genetic resource for improvement of the wheat processing quality. Agropyron is a closely related genus to wheat and some species in it have been successfully crossed with common wheat and used for variety improvement. However, the HMW-GS in Agropyron was rarely studied. In the present study, SDS-PAGE and PCR methods were used to study the composition and the coding genes of HMW-GS in diploid (PP), tetraploid (PPPP) and hexaploid (PPPPPP) A. cristatum (L.) Gaertn. The results of SDS-PAGE indicated that the composition of HMW-GS varied not only in different ploidy types (2x, 4x and 6x) but also in different seeds belonging to the same ploidy type since Agropyron cristatum is a cross-pollinated species. There were two expressed HMW-GS totally in each ploidy type. The molecular weigh (MW) of all the subunits in the three types of A. cristatum with different ploidy levels was smaller than that in Chinese Spring. As results of PCR, three y-type HMW-GS genes named as Bty1, Bty2 and Bty3 were isolated from diploid A. cristatum with the length of 1 227 bp, 1 350 bp and 1 530 bp, respectively. One y-type gene named Bfy1 was isolated from tetraploid A. cristatum with the length of 1 227 bp. And three y-type genes named Bsy1, Bsy2 and Bsy3 were isolated from hexaploid A. cristatum, with the length of 1 227 bp, 1 530 bp and 1 896 bp, respectively. The sequence comparison showed that the homologous relationship of the seven genes was very close. When the ORFs of the seven genes were analyzed, it showed that only Bsy2 in hexaploid A. cristatum possessed an ORF which encoded a protein consisting of 487 amino acid residues with an estimated MW of 53 kD which was similar with the MW of the larger HMW-GS in SDS-PAGE pattern. The nonsense mutations of C to T or G to A in the repetitive domains of the other six genes silenced them all. The mutation of C to T was the main type of nonsense mutation occurring in HMW-Gs genes of A. cristatum, just as occurring in wheat. The amino acid sequences analysis indicated that the sequences of N-terminal, C-terminal and repetitive domain of the seven subunits were similar to the y-type subunits in wheat. But some differences were also found, such as a decapeptide deletion in subunit Bty2, no irregular tripeptide in subunits Bty1, Bty2, Bfy1 and Bsy1, an extra Cys in the repetitive domain of unexpressed subunits of Bty3 and Bsy3, etc. Finally, the evolutional relationship of the seven subunits was analyzed using Neighbor-Joining tree and the results showed that Bty1, Bfy1 and Bsy1 were most close to each other and secondarily close to Bty2, Bty3 and Bsy2 were close to Ey subunit in Elytrigia elongata, and Bsy was close to Dy10 and Dy12 subunits in common wheat. The N-terminal of the seven subunits was close to the y-type subunits in wheat, and the C-terminal of the six subunits except Bsy3 was more close to the x-type subunits. It could be concluded that the six HMW-GS (Bty1, Bty2, Bty3, Bfy1, Bsy1 and Bsy2) in A. cristatum are not typical y-type HMW-GS subunits and they may represent an osculant subunits creating in evolution history.
全 文 ：