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Establishment of identification method for self-incompatibility genotype in Japanese pears

日本梨自交不亲和基因型鉴定方法的建立

基本信息

批准号：
08660027
负责人：
HIRATSUKA Shin
金额：
$ 1.47万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08660027/
关键词：
Japanes pear self-incompatibility S-genotype S-protein

项目摘要

Stylar proteins associated with self-incompatibility alleles, S_1 to S_5, were identified by isoelectricfocusing polyacrylamide gel electrophoresis (IEF-PAGE), and self-incompatibility genotypes of several cultivars including new ones were established by using the IEF-PAGE technique. The established S-genotypes were confirmed by pollination experiments in the orchard. Present study also suggests the usefulness of Concanavalin-A (Con-A) staining of S-protein bands for predicting S-genotypes of cultivars, because each S-protein was proved to be glycoprotein. The trial to make mono-clonal antibodies of respective S-proteins resulted in failure by the following reasons ; 1)two S-proteins in the cultivar were contaminated each other because of their very close pI points, 2)the recovery was trace level when each protein was purified.The S_4-gene was amplified by PCR techniques using 5'TTTACGCAGCAATATCAG3' and 5'G(C/T)GGGGGCA(A/G)T(T/C)TATGAA3'as primers. However, it was not amplified when genomic DNAs from young leaves of 'Shinsui'(S_4S_5) and 'Seigyoku'(S_3S_4) were used as templates. Therefore, probalble probe for S_4-gene could not be established in this study. The cDNAs were synthesized from stylar mRNA or rRNA by RT-PCR techniques, and PCR was conducted using the cDNAs as templates to select each S-gene. We could detect several candidates for each S-gene using RAPD primer, but probes for respective genes were not established yet.

用等电聚焦聚丙烯酰胺凝胶电泳法鉴定了与自交不亲和等位基因S_1至S_5相关的花柱蛋白，并利用等电聚焦聚丙烯酰胺凝胶电泳法建立了包括新品种在内的几个品种的自交不亲和基因型。通过果园授粉试验对所建立的S基因型进行了验证。本研究还表明，S蛋白条带的刀豆蛋白A(ConA)染色可用于品种S基因分型的预测，因为每种S蛋白都被证明是糖蛋白。以5‘TTTACGCAGCAATATCAG3’和5‘G(C/T)GGGGGCA(A/G)T(T/C)T(T/C)TATGAA3’为以‘新遂’(S_4S_5)和‘世谷’(S_3S_4)幼叶基因组DNA为模板，均未扩增出DNA片段。因此，本研究不能建立S_4基因的可能探针。以花柱mRNA或rRNA为模板，通过RT-PCR法合成cDNA，并以此为模板进行聚合酶链式反应，筛选出S基因。利用随机扩增技术，我们可以检测到S基因的多个候选基因，但针对各个基因的探针尚未建立。