山杏种子中的苦杏仁苷极大限制其油脂及其他相关产品的开发利用,而多药和有毒化合物排出(multidrug and toxic compound extrusion,MATE)转运蛋白可能在苦杏仁苷转运过程中起重要作用。运用生物信息学方法对山杏种子PsMATE家族成员进行预测分析,采用qRT-PCR技术对其在发育种子中的表达模式进行分析测定;利用qRT-PCR技术对PsMATE40进行组织特异性表达分析,使用GFP融合蛋白表达法对其进行亚细胞定位分析。基于实验室前期获得的山杏种子转录组数据,利用功能注释及生物信息学分析确定17个山杏种子PsMATE基因家族成员,可分为3个亚家族:亚家族1、亚家族2与亚家族4;上述基因编码蛋白二级结构以α-螺旋为主,含多个磷酸化位点;除PsMATE23、PsMATE33、PsMATE35A与PsMATE35B蛋白为酸性蛋白外,其余均为碱性蛋白;15个PsMATE蛋白为强疏水性跨膜转运蛋白,定位于质膜。qRT-PCR分析显示,PsMATE家族成员在山杏发育种子中均有表达,但不同发育期表达量差异显著,其中PsMATE40表达量在该家族基因中最高;结合系统发育结果,PsMATE40与已被鉴定具有苦杏仁苷转运的MATE蛋白聚类在一起,据此推测PsMATE40基因在山杏种子苦杏仁苷的转运中起重要作用;成功克隆获得PsMATE40基因序列;qRT-PCR分析表明,PsMATE40基因在山杏种子中表达量最高而在根中最低,表明PsMATE40表达具有组织特异性;构建植物表达载体pBI121-GFP-PsMATE40并瞬时转化烟草叶片,荧光显微镜检测PsMATE40转运蛋白定位于质膜。研究结果为阐明山杏种子苦杏仁苷的跨膜转运原理及分子定向培育低苦杏仁苷含量的油料植物奠定重要基础。
在长期进化过程中,植物形成了一系列防御机制,抵抗各类病原物入侵,抗病R基因在其中发挥着关键作用.R基因的研究,在农作物中取得很大进展,已成为植物病理学的研究热点.相比之下,树木抗病基因研究较为落后,虽从苹果、杨树和柚子中已分离了几个与已知R基因具有类似结构与功能的基因,但还没有真正的树木抗病R基因被克隆出来;目前,大部分研究主要集中在利用分子标记构建连锁图谱,寻找抗病数量性状位点(Q uan titativetra it loc i,QTL)和与R基因紧密连锁或共分离的质量性状标记;其中一些标记已经应用在分子辅助选育中,并显示了诱人的应用前景.另外,利用已知抗病R基因的保守区域,从多种植物中已扩增出许多抗病基因类似序列,它们大多被转化为与R基因紧密连锁的标记或被当作抗病候选基因.
Populus tomentosa seedlings used for cold acclimation at -3℃ were pretreated with or without 10?mmol·L -1 CaCl 2, 3?mmol·L -1 of Ca 2+ chelator EGTA, 0 1?mmol·L -1 of Ca 2+ channel inhibitor LaCl 3,and 0 05?mmol·L -1 of CaM antagonist CPZ. The changes in the contents of total soluble protein and CaM, and freezing resistance in all pretreated seedlings in various periods ( viz: following cold acclimation, chilling stress and recovery) were investigated. Results showed that cold acclimation increased the contents of total soluble protein and CaM, and freezing resistance of seedlings, which could be strongly reduced by the pretreatments of EGTA CPZ and LaCl 3 Cold acclimation combined with CaCl 2 pretreatment enhanced the effect of cold acclimation on freezing resistance, and obviously increased the contents of total soluble protein and CaM, reduced the declining degree of the contents of total soluble protein and CaM caused by chilling stress as compared with cold acclimation, augmented the increase in the level of total soluble protein and CaM during the recovery periods. Further analysis found that an increase in total soluble protein content during cold acclimation with or without CaCl 2 pretreatment mainly resulted from the increase in content of heat stable protein in total soluble protein. It is suggested that Ca 2+ calmodulin may be involved in the synthesis of total soluble protein, and the induction of freezing resistance of seedlings.
