Ma Jian, Jiang Qian-Tao, Zhao Quan-Zhi, Zhao Shan, Lan Xiu-Jin, Dai Shou-Fen, Lu Zhen-Xiang, Liu Chunji, Wei Yu-Ming, Zheng You-Liang
Triticeae Research Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan, China.
Genetica. 2013 Jun;141(4-6):227-38. doi: 10.1007/s10709-013-9721-x. Epub 2013 May 21.
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.
由蜡质基因编码的颗粒结合淀粉合酶I（GBSS I）在大麦淀粉颗粒发育过程中直链淀粉的积累中起重要作用。在本研究中，我们分离并鉴定了三个蜡质大麦品种（GSHO 908、GSHO 1828和NA 40）以及两个非蜡质大麦品种（PI 483237和CIho 15773）的蜡质等位基因，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）检查蜡质等位基因对大麦品种淀粉颗粒形态的可能影响。在蜡质大麦品种中检测到内含子1中有193 bp的插入、编码区中有15 bp的插入以及一些单核苷酸多态性位点。此外，在蜡质大麦品种中还鉴定出一个包含TATA框、转录起始点、外显子1和部分内含子1的397 bp缺失。RT-qPCR分析表明，蜡质品种的蜡质表达水平低于非蜡质品种。瞬时表达分析表明，非蜡质品种1029 bp启动子驱动的GUS活性强于蜡质品种822 bp启动子驱动的活性。扫描电子显微镜显示蜡质和非蜡质品种之间淀粉颗粒形态没有明显差异。我们的结果表明，在蜡质大麦品种中鉴定出的397 bp缺失可能是蜡质转录本减少的原因，导致GBSS I蛋白浓度降低，从而直链淀粉含量降低。
