油菜钙依赖蛋白激酶BnCDPK1的克隆和表达分析
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强优势油菜杂交种的创制与应用(2010BAD01B01);油菜丰产高效栽培技术研究(2010BAD01B01)


Molecular Cloning and Expression Analysis of Calcium- dependent Protein Kinase BnCDPK1 in Brassica Napus
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    摘要:

    根据转录组测序获得的钙依赖蛋白激酶基因的EST序列设计特异引物,利用RT-PCR和RACE技术从油菜中克隆获得BnCDPK1基因全长序列,NCBI登录号为KF740477,其cDNA和gDNA全长分别为2115和2857bp,含有11个内含子和12个外显子。生物信息学分析表明其开放阅读框为1581bp,编码527个氨基酸,具有CDPKs 家族典型的特征,含有一个激酶区域和四个钙离子手型结构域,与拟南芥AtCDPK28同源性最高,属于第Ⅳ亚家族。BnCDPK1在油菜的根、茎、叶、花、种子中均有表达,但表达丰度存在差异,叶片中表达量最高,其次为茎,根和种子,花中的表达量最低;在高抗和高感油菜品种中,菌核菌胁迫均能够诱导BnCDPK1上调表达,但是在高抗品种比高感品种中反应更迅速,表达量更高。接种前、接种后12h、接种后24h,高抗品种的表达量相比高感品种分别为1.4倍、4倍和3倍,推测其可能参与油菜的生长发育以及油菜对菌核菌侵染的应答和防御反应。

    Abstract:

    The full length sequence was cloned from the leaves of brassica napus according to the EST of RNA-Seq through RT-PCR and rapid amplification of cDNA ends(RACE) techniques which had been deposited in GenBank database with the accession number of KF740477. The full length of BnCDPK1 amplified from cDNA is 2115 bp, compared to 2857 bp from gDNA which include 11 introns and 12 exons. Bioinformatics analysis showed that BnCDPK1 contain an entire opening reading frame of 1581bp, which encode a polypeptide of 527 amino acid residues possessing a serine/Threonine protein kinase catalytic domain(S_TKc ) and four EF-hand calcium-binding domains, and it shared high identity with AtCPK28 and belong to the CDPKs subgroup Ⅳ. Fluorescent quantitative PCR analysis displayed it was constitutively expressed in all tested organs, but the transcript abundance was relatively higher in leaves and stems , then in roots and seeds, and less was in flowers. It can be induced by Sclerotinia sclerotiorum infection in both high resistant and susceptible cultivars, however, the relative transcript level was higher in the former in all time periods , 1.4 fold before inoculation, 4 fold for12hpi and 3 fold for 24hpi.Thus it was deduced to involve in growth and development in oil seeds, and play an important role in the defense reaction to Sclerotinia sclerotiorum.

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张秋平,文李,王峰,等.油菜钙依赖蛋白激酶BnCDPK1的克隆和表达分析[J].植物遗传资源学报,2014,15(6):1320-1326.

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  • 收稿日期:2014-01-02
  • 最后修改日期:2014-02-08
  • 录用日期:2014-10-08
  • 在线发布日期: 2014-10-08
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