Mapping of barley (Hordeum vulgare L.) beta-amylase alleles in which an amino acid substitution determines beta-amylase isoenzyme type and the level of free beta-amylase
Mapping of barley (Hordeum vulgare L.) beta-amylase alleles in which an amino acid substitution determines beta-amylase isoenzyme type and the level of free beta-amylase
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DOI：
10.1006/jcrs.2001.0398
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发表时间：
2002-01-01
影响因子：
3.8
通讯作者：
Scoles, GJ
中科院分区：
文献类型：
--
作者：
Li, CD;Langridge, P;Scoles, GJ
利用限制性片段长度多态性（RFLP）分析将栽培大麦的3个β-淀粉酶基因（Bmy 1、2和3）分别定位于染色体4 HL、2 HL和4 HL。在264个DH系中未检测到Bmy 1和Bmy 3之间的重组。Chebec（Sd 2）×哈灵顿（Sd 1）双单倍体群体中与染色体4 HL上Bmy位点共分离的β-淀粉酶同工酶Sd 1和Sd 2的多态性该位点还解释了双亲间游离酶水平变异的90.5%。利用RT-PCR技术从哈灵顿（Sd 1）和Galleon（Sd 2）籽粒发育过程中分离到两个β-淀粉酶基因的cDNA。推导的氨基酸序列的比对鉴定了β-淀粉酶的Sd 2和Sd 1形式之间的三个氨基酸取代（Arg 115 - Cys，Asp 165 - Glu和Val 430- Ala）。基于这三个氨基酸替换的三个等位基因特异性PCR引物对用于扩增16个大麦品种/系基因组DNA中的β-淀粉酶基因。只有Arg 115（Sd 2）/Cys（Sd 1）取代与同工酶形式一致。这种氨基酸替换降低了Sd 1 β-淀粉酶的pI，这与以下事实一致：当通过IEF分离时，Sd 2形式比Sd 1形式更具碱性。Sd 1形式中从Arg 115到Cys的突变还提供了一个更多的-SH基团以形成S-S-桥。由于结合的β-淀粉酶通过二硫桥与胚乳的不溶性蛋白质及其抑制剂连接，这可以解释Sd 1与Sd 2相比表现出更高水平的结合。因此，单个氨基酸取代决定同工酶类型和β-淀粉酶结合。(C)2002 Elsevier Science Ltd.
The three beta-amylase genes (Bmy1, 2 and 3) in cultivated barley were mapped to chromosomes 4HL, 2HL and 4HL respectively using RFLP analysis. No recombinants between Bmy1 and Bmy3 were detected among 264 DH lines. Polymorphism of the Sd1 and Sd2 isoenzymes of beta-amylase co-segregated with the Bmy loci on chromosome 4HL in a doubled-haploid population of the cross Chebec (Sd2) x Harrington (Sd1). This locus also explained 90.5% of the variation in the level of free enzyme between the two parents. Two cDNAs of beta-amylase were isolated by RT-PCR from the developing grains of Harrington (Sd1) and Galleon (Sd2). Alignment of the deduced amino acid sequences identified three amino-acid substitutions between the Sd2 and Sd1 forms of beta-amylase (Arg 115 - Cys, Asp 165 - Glu, and Val430 - Ala). Three allele-specific PCR primer pairs based on the three amino acid substitutions were used to amplify the beta-amylase genes in genomic DNA of sixteen barley cultivars/lines. Only the Arg115(Sd2)/Cys(Sd1) substitution was consistent with the isoenzyme form. This amino acid replacement reduced the pI of the Sd1 beta-amylase consistent with (lie fact that (lie Sd2 form is more basic than the Sd1 form when separated by IEF The mutation from Arg115 to Cys in the Sd1 form also provides one more -SH group to form S-S-bridges. As bound beta-amylase is linked to the insoluble proteins of the endosperm and its inhibitor via disulphide bridges this could explain the higher level of binding exhibited by Sd1 vs Sd2. Thus a single amino acid substitution determines both the isoenzyme type and beta-amylase binding. (C) 2002 Elsevier Science Ltd.