朝鲜淫羊藿EkCDPK基因的克隆及表达分析
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生物产业技术研发平台中央财政专项(851800501);


Cloning and Expression Analysis of EkCDPK Gene from Epimedium koreanum Nakai
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    摘要:

    钙依赖蛋白激酶(CalciumDependent Protein Kinase, CDPK)是在植物抗逆胁迫信号转导途径中起关键作用的酶。该研究采用RTPCR结合RACE技术,从朝鲜淫羊藿(Epimedium koreanum Nakai)中克隆获得一个全长2 036 bp的钙依赖蛋白激酶基因(EkCDPK)。结果显示:(1)EkCDPK基因cDNA为1 410 bp,5′UTR长387 bp, 3′UTR长239 bp,编码469 个氨基酸。EkCDPK蛋白保守结构域N端为S_TKc domain,C端为EFhand domain,该蛋白具有1个CDPKs典型结构Ser/Thr蛋白激酶活性位点、1个ATP位点、4个Ca2+ 结合位点、1个跨膜结构域、无信号肽的稳定亲水性蛋白质,二级结构主要由α螺旋(43.71%)和无规则卷曲(37.10%)构成。(2)系统进化分析表明,EkCDPK与菘蓝CDPK蛋白表现出较高相似性(95%)。(3)实时荧光定量PCR分析表明,EkCDPK基因在根、茎、叶与花组织中均有表达,根中表达量最高,茎次之;干旱胁迫后15 h内表达量显著高于对照组,之后表达量开始下降;盐胁迫5、15和25 h后EkCDPK基因表达量均明显高于对照组。(4)成功构建了pET28aEkCDPK原核表达载体,诱导出约50 kD蛋白质,与预测大小一致,表明重组蛋白EkCDPK表达成功,为采用基因工程手段提高朝鲜淫羊藿抗逆性能研究奠定了基础。

    Abstract:

    CalciumDependent Protein Kinase is a key enzyme and plays an important role in the signal transduction pathways of plant stress resistance. In this study, using reverse transcriptionPCR and rapid amplification of cDNA ends (RACE) techniques, we cloned EkCDPK gene from Epimedium koreanum Nakai, its fulllength about 2 036 bp. The results showed that: (1) EkCDPK gene cDNA is 1 410 bp, 5′UTR is 387 bp and 3′UTR is 239 bp,and the ORF encodes for 469 amino acid. Conserved domain of EkCDPK protein consists of S_TKc domain in N end and EFhand domain in C end. EkCDPK was predicted to be a stable hydrophilic protein, possessing a typical and conserved serine/threonine protein kinase domain and a transmembrane structure domain, also an ATP site, 4 Ca2+ binding sites, and without signal peptide. The secondary structure of EkCDPK is abundant in αhelices (43.71%) and random coils (37.10%). (2) The phylogenetic relationship analysis showed that EkCDPK is closely related to Isatis tinctoria (95%). (3) The qRTPCR results showed that EkCDPK gene expressed in root, stem, leaf and flower tissues, with the highest expression in root, followed by stem. After drought stress, EkCDPK expression was significantly higher than that in the control group in 15 h, then began to decrease, and its life index decreased too. After salt stress 5, 15 and 25 h, its expression was significantly higher than that of control group. (4) pET28aEkCDPK prokaryotic expression vector was constructed and induced the expected size protein, which is about 50 kD, indicating recombinant protein EkCDPK was expressed successfully. These results provided a basis for the further study on improving the plant stress resistance of E. koreanum Nakai using genetic engineering.

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王丹丹,鲍佳乐,张雨婷.朝鲜淫羊藿EkCDPK基因的克隆及表达分析[J].西北植物学报,2020,40(6):918-926

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  • 在线发布日期: 2020-07-30
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