IDENTIFICATION AND EXPRESSION OF THE CHLOROPLAST CLPP GENE IN THE CONIFER PINUS-CONTORTA
IDENTIFICATION AND EXPRESSION OF THE CHLOROPLAST CLPP GENE IN THE CONIFER PINUS-CONTORTA
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DOI：
10.1007/bf00028853
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发表时间：
1994-11-01
影响因子：
5.1
通讯作者：
LIDHOLM, JA
中科院分区：
文献类型：
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作者：
CLARKE, AK;GUSTAFSSON, P;LIDHOLM, JA
通过与烟草叶绿体ClpP基因的第二外显子进行异源杂交，从烟草叶绿体基因组文库中鉴定并分离到针叶松叶绿体ClpP基因。对两个重叠克隆的DNA测序显示，该克隆有一个不间断的615bp的开放阅读框，与其他五个叶绿体基因组和大肠杆菌中的ClpP基因有41%到65%的相似性。该615个碱基的序列包含与大肠杆菌中GlpP酶的丝氨酸和组氨酸活性部位完全匹配的基序。利用叶绿体基因组的物理图谱确定了ClpP基因的位置，发现ClpP基因位于psbE/F和vpoB基因之间的10kb区域。对与ClpP基因相邻的区域进行测序，发现rps12基因的第一个外显子位于135bp的下游。与ClpP基因相关的rps12基因的第一个外显子的基因组位置对于到目前为止已发现的所有其他叶绿体ClpP基因都是保守的。Northern印迹分析表明，该基因存在于不同长度的转录本中，大小在0.8~2.4kb之间。在P.conorta中的两个较长的转录本还包括rps12基因的第一外显子。然而，通过引物延伸定位ClpP转录本的5‘端，发现在第一个ATG密码子的上游有一个53bp的单一转录起始点。对生长在黑暗和中等光照条件下(250mU·m~(-2)S(~(-1)的两种松树的总RNA分析表明，ClpP基因的表达水平没有显著差异。这些结果表明，针叶树中的ClpP基因是一个操纵子的一部分，该操纵子包括rps 12的第一个外显子和整个rp120基因，并且以光不依赖的方式作为多顺反子前体表达，然后经过转录后加工得到成熟的单顺反子ClpP mRNA。
The clpP gene from the conifer Pinus contorta was identified and isolated from a chloroplast genomic library by heterologous hybridisation to the second exon of the chloroplast clpP gene in tobacco. DNA sequencing of two overlapping clones revealed an uninterrupted 615 bp open-reading frame with 41 to 65% similarity to the clpP genes in five other chloroplast genomes and Escherichia coli. The 615 bp sequence in P. contorta contained perfectly matched motifs for the serine and histidine active sites of the GlpP protease in E. coli. The location of the clpP gene was determined using a physical map of the P. contorta chloroplast genome, and was found to lie within a 10 kb region between the psbE/F and vpoB genes. Sequencing of the regions adjacent to the clpP gene revealed the first exon of the rps12 gene located 135 bp downstream. The genomic position of the first exon of the rps12 gene in relation to the clpP gene is conserved for all other chloroplast clpP genes identified so far. Northern blot analysis showed that the clpP gene in both P. contorta and P. sylvestris was present in several transcript of different length, ranging from 0.8 to 2.4 kb. The two longer transcripts in P. contorta also included the first exon of the rps12 gene. Mapping of the 5' end of the clpP transcripts by primer extension, however, revealed a single transcription initiation site 53 bp upstream of the first ATG codon. Analysis of total RNA isolated from The two pine species grown in darkness or moderate light conditions (250 mu mol photons m(-2) s(-1)) showed no significant difference in the level of expression of the clpP gene. The results suggest that the clpP gene in conifers is part of an operon which includes the first exon of the rps12 and the entire rp120 gene, and is expressed in a light-independent manner as a polycistronic precursor which later undergoes post-transcriptional processing to give the mature monocistronic clpP mRNA.