EXPRESSION OF 2 RELATED VACUOLAR H+-ATPASE 16-KILODALTON PROTEOLIPID GENES IS DIFFERENTIALLY REGULATED IN A TISSUE-SPECIFIC MANNER
EXPRESSION OF 2 RELATED VACUOLAR H+-ATPASE 16-KILODALTON PROTEOLIPID GENES IS DIFFERENTIALLY REGULATED IN A TISSUE-SPECIFIC MANNER
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DOI：
10.1104/pp.108.4.1395
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发表时间：
1995-08-01
期刊：
影响因子：
7.4
通讯作者：
WILKINS, TA
中科院分区：
文献类型：
--
作者：
HASENFRATZ, MP;TSOU, CL;WILKINS, TA
16-kD蛋白脂质亚基是液泡H+-ATP酶（V-ATP酶）复合物的主要膜蛋白，该复合物形成负责跨脂质双层转运质子的质子通道。利用两个简并合成的寡核苷酸COT 11和COT 12扩增了棉花V-ATP酶蛋白脂亚基的部分cDNA。聚合酶链反应（PCR）。利用这些PCR产物从棉花花后-3d胚珠文库中分离到两个全长cDNA。两个克隆，CVA16.2和CVA16.4，分别由816和895 bp，编码的16 kD的蛋白脂亚基的V-ATP酶。在核苷酸水平上，两个克隆的完整序列显示73.5%的同一性，但在编码区内共享约95%的同一性，尽管两个多肽仅相差一个氨基酸。蛋白脂亚基的推导氨基酸序列的比较显示，四个跨膜结构域和两个胞质膜外结构域是高度保守的，在所有真核生物。Southern杂交分析表明，这些克隆属于二倍体和异源四倍体植物中的小基因家族。北方印迹分析表明，三个主要的V-ATP酶亚基（69，60和16 kD）的协调调节，部分，在转录水平上。RNA分析和逆转录PCR建立了16-kD蛋白脂质转录差异积累在不同的组织和急剧增加的组织经历快速扩张，特别是在花药，胚珠和花瓣。CVA 16.4蛋白脂质转录是最普遍的两个蛋白脂质的消息在扩大胚珠收获开花后10天。相反，两种蛋白脂质mRNA在发育中的花瓣中积累到相似的水平。
The 16-kD proteolipid subunit is the principal integral membrane protein of the vacuolar H+-ATPase (V-ATPase) complex that forms the proton channel responsible for translocating protons across lipid bilayers. Two degenerate synthetic oligonucleotides, COT11 and COT12, corresponding to highly conserved transmembrane domains in all 16-kD subunits sequenced so far, were used to amplify a partial cDNA of the V-ATPase proteolipid subunit from cotton (Gossypium hirsutum L.) by polymerase chain reaction (PCR). These PCR products were used to isolate two full-length cDNAs from a -3 d postanthesis cotton ovule library. Both clones, CVA16.2 and CVA16.4, consisting of 816 and 895 bp, respectively, encode the 16-kD proteolipid subunit of the V-ATPase. At the nucleotide level, the complete sequences of the two clones show 73.5% identity, but share about 95% identity within the coding region, although the two polypeptides differ by only one amino acid. Comparison of deduced amino acid sequences of the proteolipid subunits revealed that the four transmembrane domains and the two cytosolic extramembrane domains are highly conserved in all eukaryotes. Southern blot analysis of cotton genomic DNA showed that these clones belong to small gene families in related diploid and allotetraploid species. Northern blot analysis suggested that the three major V-ATPase subunits (69, 60, and 16 kD) are coordinately regulated, in part, at the transcriptional level. RNA analysis and reverse-transcription PCR established that 16-kD proteolipid transcripts differentially accumulate in different tissues and increase dramatically in tissues undergoing rapid expansion, particularly in anthers, ovules, and petals. The CVA16.4 proteolipid transcript is the most prevalent of the two proteolipid messages in expanding ovules harvested 10 d postanthesis. In contrast, the two proteolipid mRNAs accumulate to similar levels in developing petals.