华山新麦草籽粒硬度吲哚蛋白基因的分离与序列分析

Isolation and Sequence Analysis on Puroindoline Genes of Grain Hardness in Psathyrostachys Huashanica

采用AS-PCR技术,从华山新麦草基因组DNA中分离获得了其籽粒硬度吲哚蛋白Pin a(HM 448475)和Pin b(HM448476)的基因序列。DNA序列分析显示,这2条序列相似性仅为69.25%,它们与小麦、山羊草、黑麦、燕麦和大麦亲缘种属植物等的Pin a和Pin b基因序列具有93%以上的一致性;推导的氨基酸序列分析显示,序列HM 448475(Pin a)和HM 448476(Pin b)都具有籽粒硬度吲哚蛋白基因的19个氨基酸残基组成的信号肽,与脂类易于结合的色氨酸结构域WRWWKWWK(Pin a)或WPTKWWK(Pin b),以及位置固定的10个半胱氨酸残基等典型结构特征,同时,这2条序列与小麦、山羊草等亲缘植物的Pin a和Pin b基因氨基酸相比,分别有15个和8个主要位点的差异;系统进化树分析显示,序列(HM 448475)可能为Pin a基因家族中的一个新类型,而序列(HM 448476)与智利大麦的Hordoindoline b基因具有相对较近的亲缘关系。研究表明,华山新麦草含有与麦类作物差异较大的控制籽粒硬度的等位基因。该研究对保护利用华山新麦草、丰富小麦籽粒硬度基因资源具有一定的理论意义和应用价值。

The Puroindoline a （ HM 448475 ） and Puroindoline b （ HM 448476 ） genes of Psathyrostachys huashanica were isolated by AS-PCR method. Nucleotide sequence analysis showed that the similarity in these two sequences was only 69.25% ,and the identity of these two sequences with the Puroindoline gene of relative species, such aTriticum,Aegilops, Secale ,Arena, and Hordeum so on, were more than 93%. Deduced amino acid sequence analysis demonstrated that the sequence HM 448475 （Pin a） and HM 448476 （Pin b） all had the typical structural features of Puroindoline gene in cereal crop, containing the signal peptide of 19 amino acid residues,the tryptophan motif of WRWWKWWK （Pin a） or WPTKWWK （Pin b） , and the location fixed 10 cysteine residues. Meanwhile, compared with the amino acid sequence of Puroindoline gene in relative species, the two sequences HM 448475 （Pin a） and HM 448476 （Pin b） had fifteen and eight major sites difference, respectively. Phylogenetic tree analysis indicated that the sequence （ HM 448475 ） might be a new type of Pin a gene family, and sequence （ HM 448476 ） might has a relatively near homologous with Hordoindoline b gene （ GU 214830） of Hordeum chilense. This result suggested that P. huashanica contain the different Puroindoline gene alleles to control the grain hardness. The studies had important significance for enriching and improving wheat resources of grain hardness, and for protecting and exploiting the advantageous genes of the endangered species P. huashanica.