Characterization and expression analysis of waxy alleles in barley accessions
Characterization and expression analysis of waxy alleles in barley accessions
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大麦种质蜡质等位基因的表征和表达分析
DOI：
10.1007/s10709-013-9721-x
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发表时间：
2013-06-01
期刊：
影响因子：
1.5
通讯作者：
Zheng, You-Liang
中科院分区：
文献类型：
--
作者：
Ma, Jian;Jiang, Qian-Tao;Zheng, You-Liang
大麦蜡质基因编码的颗粒结合淀粉合成酶Ⅰ（GBSS Ⅰ）在淀粉粒发育过程中对直链淀粉的积累起重要作用。在本研究中，我们分离并鉴定了三个蜡质玉米的蜡质等位基因，（GSHO 908、GSHO 1828和NA 40）和两个非糯大麦种质（PI 483237和CIho 15773），通过实时定量PCR（RT-qPCR）估计蜡质基因的表达模式，通过分析启动子-GUS表达研究启动子活性，并通过扫描电子显微镜（SEM）研究了蜡质等位基因对大麦淀粉粒形态的可能影响。在糯大麦基因组中检测到内含子1中193 bp的插入片段，编码区中15 bp的插入片段，以及一些单核苷酸多态性位点。此外，在糯大麦中还发现了一个397 bp的缺失，包括TATA盒、转录起始点、外显子1和部分内含子1。RT-qPCR分析表明，糯性材料的蜡质基因表达量低于非糯性材料。瞬时表达分析表明，非糯性材料中由1,029 bp启动子驱动的GUS活性高于糯性材料中由822 bp启动子驱动的GUS活性。扫描电镜观察表明，糯性和非糯性材料淀粉粒形态无明显差异。我们的研究结果表明，397 bp的缺失鉴定的糯大麦种质可能是负责减少糯转录本，导致较低浓度的GBSS I蛋白，从而降低直链淀粉含量。
Granule Bound Starch Synthase I (GBSS I) encoded by the waxy gene plays an important role in accumulating amylose during the development of starch granules in barley. In this study, we isolated and characterized waxy alleles of three waxy (GSHO 908, GSHO 1828 and NA 40) and two non-waxy barley accessions (PI 483237 and CIho 15773), estimated the expression patterns of waxy genes via Real-time quantitative PCR (RT-qPCR), investigated promoter activity by analyzing promoter-GUS expression, and examined possible effects of waxy alleles on starch granule morphology in barley accessions by scanning electron microscopy (SEM). A 193-bp insertion in intron 1, a 15-bp insertion in the coding region, and some single nucleotide polymorphic sites were detected in the waxy barley accessions. In addition, a 397-bp deletion containing the TATA box, transcription starting point, exon 1 and partial intron 1 were also identified in the waxy barley accessions. RT-qPCR analysis showed that waxy accessions had lower waxy expression levels than those of non-waxy accessions. Transient expression assays showed that GUS activity driven by the 1,029-bp promoter of the non-waxy accessions was stronger than that driven by the 822-bp promoter of the waxy accessions. SEM revealed no apparent differences of starch granule morphology between waxy and non-waxy accessions. Our results showed that the 397-bp deletion identified in the waxy barley accessions is likely responsible for the reduction of waxy transcript, leading to lower concentrations of GBSS I protein thus lower amylose content.