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籽粒硬度是小麦品质改良的重要目标性状。根据已报道的谷类作物中硬度主效基因 Pina和 Pinb的 DNA序列 ,设计了两对简并引物 ,以我国软质小麦品种京 4 11基因组 DNA为模板 ,进行 PCR扩增 ,分别获得了约 4 5 0 bp的Pina和 Pinb基因的全长特异性片段。将其克隆到 p GEM- 3Zf(+)上 ,重组子和酶切鉴定正确后 ,进行序列测定和分析。结果表明 ,与国外已克隆的软质小麦 Heron中 Pina基因相比较 ,京 4 11中 Pina与其核苷酸同源性为 98.9% ,氨基酸的同源性为 98% ,其全长为 4 4 7bp,编码 14 9个氨基酸残基 ,具有谷类作物中 Pina所特有 19个氨基酸的信号肽序列和 WWKWWK的色氨酸结构域。同样 ,与国外已克隆的软质小麦 Heron中 Pinb基因相比较 ,京 4 11中 Pinb与其核苷酸同源性为 99.8% ,氨基酸的同源性为 99.3% ,其全长为 4 4 7bp,编码 14 9个氨基酸残基 ,具有谷类作物中 Pinb所特有 19个氨基酸的信号肽序列和 KWWK的色氨酸结构域。硬度主效基因的分离克隆为进行我国小麦品种硬度的基因工程改良奠定了基础。
Grain hardness is an important target trait of wheat quality improvement. According to reported DNA sequences of major genes Pina and Pinb in grain crops, two pairs of degenerate primers were designed, and the genomic DNA of Chinese soft wheat Jing 4 11 was used as a template to obtain PCR products of about 4 Full-length specific fragments of 50 bp Pina and Pinb genes. After cloning into p GEM-3Zf (+), the recombinants and restriction enzymes were identified correctly and then sequenced and analyzed. The results showed that the Pina gene in Jing 4 11 was 98.9% homologous to that of Pina gene in soft wheat Heron. The amino acid sequence of Pina was 98% with a total length of 477 bp , Encoding a peptide sequence of 14 amino acids with a signal peptide sequence of 19 amino acids unique to Pina in cereals and the tryptophan domain of WWKWWK. Similarly, Pinb gene in Jing411 was 99.8% homologous to Pinb gene in soft wheat Heron, which was 99.4% homologous to that of Pinb in soft wheat. The full length of Pinb was 447bp, Which encodes a peptide sequence of 14 amino acids with a signal peptide sequence of 19 amino acids unique to Pinb in cereals and a tryptophan domain of KWWK. Isolation and cloning of the main genes of hardness laid the foundation for genetic engineering improvement of the hardness of wheat varieties in our country.