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首页> 外文期刊>Chinese science bulletin >Cloning and activity analysis of in vitro expression of plant NAD-IDH genes
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Cloning and activity analysis of in vitro expression of plant NAD-IDH genes

机译：植物AND-IDH基因体外表达的克隆及活性分析

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The plant NAD+-dependent isocitrate dehydro-gcnase (NAD-IDH) is an important multifunctional enzyme. The cDNAs encoding three different subunits of the NAD-IDH were cloned successfully from leaves of Arabidop-sis thaliana ecotypc Columbia gll and three lines (Shaan 2A, Shaan 2B and Ken Cl) of Brassica napus by RT-PCR method. By searching the sequences in GenBank Database, it is shown that these sequences from B. napus were novel. Their encoding regions of a functional protein were then inserted intothe multiple cloning sites of pMIDl vector for the expression of recomhinant protein. All the recombinants were successfully expressed in the IB vitro expression system. When the transcripts of subunits 0, 1 and 2 were added together to the in vitro system with 36 μg/mL of protein disulfide isomerase, the expressed products had NAD-IDH enzymatic activity. Comparison of different kinds of gene and molecular ratio of the transcripts showed that the NAD-IDH enzyme is composed of three subunits designatedsubunit 0, subunit 1 and subunit 2. All the three subunits are essential to catalytic activity. Missing subunit 0 could abolish activity. Missing either sub-unit 1 or subunit 2 would cause severe impact on activity. Deletions, which would cause frameshift mutation or nonsense mutation, were also found in some transcripts of sub-unit 1 gene from Shaan 2A and Shaan 2B of B. napus. The mutated subunit 1 lost its proper function. This may explain why there is difference of the NAD-IDH activity among three lines of B. napus.
机译：植物NAD +依赖性异柠檬酸脱氢葡聚糖酶（NAD-IDH）是一种重要的多功能酶。利用逆转录-聚合酶链反应（RT-PCR）方法成功地从拟南芥（Arabidop-sis thaliana ecotypc）Columbia gll和三株甘蓝型油菜（Shaan 2A，Shaan 2B和Ken Cl）的叶中成功克隆了编码NAD-IDH的三个不同亚基的cDNA。通过在GenBank数据库中搜索序列，表明来自甘蓝型油菜的这些序列是新颖的。然后将其功能蛋白的编码区插入pMID1载体的多个克隆位点以表达重组蛋白。所有重组体均在IB体外表达系统中成功表达。当将亚基0、1和2的转录本与36μg/ mL的蛋白质二硫键异构酶一起添加到体外系统中时，表达的产物具有NAD-IDH酶活性。比较不同基因的转录本和分子比率，发现NAD-IDH酶由三个亚基组成，分别称为亚基0，亚基1和亚基2。这三个亚基均对催化活性至关重要。缺少亚基0可能会取消活性。缺少亚基1或亚基2会对活动造成严重影响。在甘蓝型油菜的Shaan 2A和Shaan 2B的亚单位1基因的一些转录物中也发现了缺失，这些缺失会导致移码突变或无义突变。突变的亚基1失去了适当的功能。这可以解释为什么在三株甘蓝型油菜中NAD-IDH活性存在差异。

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