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Journal of Zhejiang University SCIENCE B
ISSN 1673-1581(Print), 1862-1783(Online), Monthly
2016 Vol.17 No.2 P.100-109
Tissue-specific expression, developmentally and spatially regulated alternative splicing, and protein subcellular localization of OsLpa rice
Abstract: The OsLpa1 gene (LOC_Os57400) was identified to be involved in phytic acid (PA) metabolism because its knockout and missense mutants reduce PA content in rice grain. However, little is known about the molecular characteristics of OsLpa rice and of its homologues in other plants. In the present study, the spatial pattern of OsLpa1 expression was revealed using OsLpa1 promoter::GUS transgenic plants (GUS: β-glucuronidase); GUS histochemical assay showed that OsLpa1 was strongly expressed in stem, leaf, and root tissues, but in floral organ it is expressed mainly and strongly in filaments. In seeds, GUS staining was concentrated in the aleurone layers; a few blue spots were observed in the outer layers of embryo, but no staining was observed in the endosperm. Three OsLpa1 transcripts (OsLpa1.1, OsLpa1.2, OsLpa1.3) are produced due to alternative splicing; quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis revealed that the abundance of OsLpa1.3 was negligible compared with OsLpa1.1 and OsLpa all tissues. OsLpa1.2 is predominant in germinating seeds (about 5 times that of OsLpa1.1), but its abundance decreases quickly with the development of seedlings and plants, whereas the abundance of OsLpa1.1 rises and falls, reaching its highest level in 45-d-old plants, with abundance greater than that of OsLpa both leaves and roots. In seeds, the abundance of OsLpa1 continuously increases with seed growth, being 27.5 and 15 times greater in 28-DAF (day after flowering) seeds than in 7-DAF seeds for OsLpa1.1 and OsLpa1.2, respectively. Transient expression of chimeric genes with green fluorescence protein (GFP) in rice protoplasts demonstrated that all proteins encoded by the three OsLpa1 transcripts are localized to the chloroplast.
Key words: OsLpa1, Low phytic acid, Expression pattern, Alternative splicing, Subcellular localization
创新点：确定了OsLpa1存在的三种剪切方式，明确了三种转录本在不同组织和发育时期丰度的变化；揭示了OsLpa1表达的组织和时空差异，确定其在根、种子糊粉层细胞和花丝中高度表达；明确了三种转录本编码的蛋白均定位于亚叶绿体。
方法：通过培育OsLpa1启动子与β-葡萄糖醛酸糖苷酶（GUS）杂合基因的转基因植株，通过不同组织的GUS组织化学染色确定OsLpa1表达的组织特异性；通过设计特异性引物确定OsLpa1存在的转录方式，采用实时荧光定量聚合酶链式反应（PCR）分析三种转录本在不同组织和发育时期的丰度；采用OsLpa1三种转录本与绿色荧光蛋白（GFP）基因构建杂合基因并在水稻原生质体中的瞬时表达，在共聚焦显微镜下观察蛋白的亚细胞定位。
结论：OsLpa1在根、茎、叶和花丝有强烈的表达。它存在三种可变剪切方式，产生的三种转录本存在明显的时空和组织差异，但其编码的蛋白均定位于叶绿体。
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DOI:
10.1631/jzus.B1500205
CLC number:
Q946.2
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On-line Access:
2016-02-01
Received:
2015-08-29
Revision Accepted:
2015-11-30
Crosschecked:
2016-01-13