MOLECULAR-CLONING, CHARACTERIZATION AND EXPRESSION OF MN-SUPEROXIDE DISMUTASE FROM THE RUBBER TREE (HEVEA-BRASILIENSIS)
MOLECULAR-CLONING, CHARACTERIZATION AND EXPRESSION OF MN-SUPEROXIDE DISMUTASE FROM THE RUBBER TREE (HEVEA-BRASILIENSIS)
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DOI：
10.1007/bf00029003
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发表时间：
1993-10-01
影响因子：
5.1
通讯作者：
GAYNOR, JJ
中科院分区：
文献类型：
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作者：
MIAO, ZH;GAYNOR, JJ
利用烟叶锰超氧化物歧化酶（Nicotiana plumbaginifolia MnSOD）的异源cDNA探针，从噬菌体EMBL3构建的巴西胶合树基因组文库中分离到一个编码含锰超氧化物歧化酶（SOD, EC 1.15.1.1）的基因组克隆。测定基因组克隆4968 bp的核苷酸序列。根据推测的翻译起始密码子和终止密码子，设计了PCR引物，用于从总mRNA中克隆全长cDNA。在分离的MnSOD的两个不同的cdna中，MnSOD- a与核基因的外显子完全匹配，而MnSOD- b具有90.2%的同源性，并且在假定的传递肽区比MnSOD- a长6个核苷酸。核基因由6个外显子和5个内含子组成，全长3211bp。测定了起始密码子上游1400 bp和终止密码子下游320 bp的序列。用基因组片段对Hevea基因组DNA进行Southern分析表明，Hevea中至少存在两个MnSOD基因。Northern blot分析显示，MnSOD转录本存在于所有被检测的组织中（叶、叶柄、根、胶乳、愈伤组织），其中幼叶的MnSOD转录本在完整植株中含量最高。胚性愈伤组织的转录本水平是成熟叶片的近50倍。此外，在蔗糖、乙烯利和Murashige-Skoog盐的作用下，MnSOD转录本可诱导3- 5倍。
A genomic clone encoding manganese-containing superoxide dismutase (SOD; EC 1.15.1.1) was isolated from a Hevea brasiliensis genomic library made in A phage EMBL3 by using a heterologous cDNA probe of MnSOD from Nicotiana plumbaginifolia. The nucleotide sequence of 4968 bp from the genomic clone was determined. Based on the putative translation initiation codon and stop codon, PCR primers were design ed and utilized for cloning the full-length cDNA from total mRNA. Of the two distinct cDNAs of MnSOD isolated, MnSOD-A has a perfect match with exons of the nuclear gene, while MnSOD-B has a 90.2% homology and is 6 nucleotides longer than MnSOD-A in the putative transit peptide region. The nuclear gene comprises 6 exons and 5 introns, giving a total length of 3211 bp. The sequences of 1400 bp upstream of the initiation codon and 320 bp downstream of the stop codon were also determined. Southern analysis of genomic DNA from Hevea probed with a genomic fragment indicated there are at least two genes of MnSOD in Hevea. Northern blot analysis showed that MnSOD transcripts were present in all tissues examined (leaf, petiole, root, latex, callus) with young leaves showing the highest levels in intact plants. The transcript level in embryogenic callus was nearly 50-fold higher than in mature leaves. In addition, transcripts of MnSOD could be induced 3- to 5-fold in response to sucrose, ethephon and Murashige-Skoog salts.