拟南芥WUSCHEL基因在转基因烟草中的超表达

WUSCHEL(WUS)是近年报道的一个重要的干细胞调控基因.本实验用RT-PCR技术从拟南芥(Arabidopsisthaliana L.)中克隆到其cDNA并构建了双增强的CaMV3 5S启动子驱动的超表达载体pBKB.借助农杆菌(Agrobacterium tumefaciens)介导转化烟草(Nicotiana tabacum L.),获得转基因植株.PCR和RT-PCR鉴定分别证明,外源WUS已整合到烟草基因组并已表达.转基因烟草地上部分出现大量异位增生的突起,扫描电镜观察表明:突起部分的细胞与分生组织细胞相似,部分突起能够发育为叶芽、花芽,表明WUS超表达引起烟草细胞异常分裂并在已分化组织中重新启动了器官形成.茎尖和花的内两轮器官没有上述变化.结合拟南芥的有关研究,推测烟草中可能也存在类似拟南芥WUS和其阻抑蛋白CLAVATA3、AGAMOUS间的反馈调节机制.转基因烟草叶发育表型变化明显,与生长素极性运输受抑制引起的表型相似,因此,作为生长点调控基因,WUS可能通过生长素对叶的发育进行调控.本研究为WUS基因的功能分析和有关生物技术应用提供了有意义的信息.     

Isolation and characterization of a rice WUSCHEL-type homeobox gene that is specifically expressed in the central cells of a quiescent center in the root apical meristem

The Arabidopsis WUSCHEL (WUS) protein, which plays an important role in the specification of the stem cells in the shoot apical meristem (SAM), contains an 'atypical' homeodomain (HD) with extra residues in its loop and turn regions. We speculated that a WUS-type atypical HD protein might also be involved in the specification and maintenance of the root apical meristem (RAM) stem cells of rice. To investigate this possibility, we isolated and characterized a rice WUS-type homeobox gene designated quiescent-center-specific homeobox (QHB) gene. Using transformants carrying the QHB promoter-GUS and in situ hybridization, we found that QHB was specifically expressed in the central cells of a quiescent center (QC) of the root. During embryogenesis and crown root formation, QHB expression was observed prior to the morphological differentiation of the root. However, we detected different QHB expression patterns in the process of the RAM development, specifically between radicle and crown root formation, suggesting that the cell-fate determination of the QC may be controlled by different mechanisms. We also produced transformants that overexpress QHB or Arabidopsis WUS. These transformants did not form crown roots, but developed multiple shoots from ectopic SAMs with malformed leaves. On the basis of these observations, we propose that the WUS-type homeobox gene is involved in the specification and maintenance of the stem cells (QC cells) in the RAM, by a mechanism similar to that for WUS in the SAM.     

拟南芥WUSCHEL蛋白的原核表达、亲和纯化和多克隆抗体制备

本研究构建了带有His标签的拟南芥(Arabidopsis thaliana)WUSCHEL基因原核表达载体pET-31b(+)-WUS-His(6),优化了大肠杆菌(Escherichia coli)诱导表达体系,将亲和层析纯化后的WUS融合蛋白,经尿素梯度透析复性溶解,免疫新西兰大白兔,成功制备了WUS蛋白多克隆抗体。通过琼脂糖免疫扩散检测确定了抗血清效价和特异性,并以斑点杂交和Western blotting检验其灵敏性。结果表明,成功构建的拟南芥WUS原核表达载体,在E.coli中以0.5mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)28°C诱导表达10h后,融合蛋白得到高水平表达,亲和纯化后目标蛋白纯度达96%以上,所制备的多克隆抗体具有较高特异性和灵敏性,可用来检测纳克级蛋白抗原。