Sequence of the S9-RNase cDNA and PCR-RFLP system for discriminating S1- to S9-allele in Japanese pear
Sequence of the S9-RNase cDNA and PCR-RFLP system for discriminating S1- to S9-allele in Japanese pear
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DOI：
10.1023/b:euph.0000014907.50575.d0
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发表时间：
2004-01-01
期刊：
影响因子：
1.9
通讯作者：
Nakanishi, T
中科院分区：
文献类型：
--
作者：
Takasaki, T;Okada, K;Nakanishi, T
在 6 个日本梨品种“Shinkou”、“Shinsei”、“Niitaka”、“Amanokawa”、“Nangetsu”和“Nansui”中发现了一个新的 S-9 等位基因。通过3'和5'RACE从'Shinkou'和'Shinsei'的雌蕊中克隆了编码S-9-RNase（S-9等位基因的花柱产物）的cDNA。 S-9-RNase基因具有684个核苷酸的开放阅读框，编码228个氨基酸残基。 S-9-RNase 具有与 S-1- 至 S-8-RNase 不同的高变 (HV) 区域，并且与苹果 S-3-RNase 的相似性 (95.2%) 高于与 8 个日本梨 S-RNase 的相似性 (从 61.0% 到 70.7%)。使用引物“FTQQYQ”和“抗（I/T）IWPNV”进行的基因组 PCR 提供了 S-1- 至 S-9- 扩增子（产物），但无法区分大约 100% 的 S-2 和 S-9。 1.3 KB。 S-2和S-9分别通过AflII和BstBI消化来区分。用九种 S 等位基因特异性限制性内切酶 SfcI、AflII、PpuMI、NdeI、AlwNI、HincII、AccII、NruI 和 BstBI 进行消化，区分 S-1 至 S-9，确定该 PCR-RFLP 系统可用于携带 S-1 至 S-9 等位基因的日本梨的 S 基因型分配。被指定为 S4S9 的“Shinkou”、“Shinsei”、“Nangetsu”和“Nansui”被确定为交叉不兼容。
A new S-9-allele was discovered in 6 Japanese pear cultivars, 'Shinkou', 'Shinsei', 'Niitaka', 'Amanogawa', 'Nangetsu' and 'Nansui'. cDNA encoding S-9-RNase, a stylar product of S-9-allele, was cloned from pistils of 'Shinkou' and 'Shinsei' by 3' and 5' RACE. The S-9-RNase gene had an open reading frame of 684 nucleotides encoding 228 amino acid residues. S-9-RNase had a hypervariable (HV) region different from S-1- to S-8-RNase and shared higher similarity (95.2%) with apple S-3-RNase than with 8 Japanese pear S-RNases (from 61.0% to 70.7%). Genomic PCR with primers 'FTQQYQ' and 'anti-(I/T) IWPNV' provided S-1- to S-9-amplicon (product), but could not discriminate the S-2 from the S-9 of ca. 1.3 kb. The S-2 and S-9 were distinguished by digestion with AflII and BstBI, respectively. The digestion with nine S-allele-specific restriction endonucleases, SfcI, AflII, PpuMI, NdeI, AlwNI, HincII, AccII, NruI and BstBI, distinguished S-1 to S-9, establishing that this PCR-RFLP system is useful for S-genotype assignments in Japanese pear harboring S-1- to S-9-allele. 'Shinkou', 'Shinsei', 'Nangetsu' and 'Nansui' assigned as S4S9 were determined to be cross incompatible.