VIGS诱导MdHB-1沉默对苹果果实成熟的影响

为研究MdHB-1是否可调控苹果果实MdACO1表达及果实成熟进程,以‘澳洲青苹’果实为试材,通过病毒诱导基因沉默(Virus-induced gene silencing,VIGS)技术抑制果实MdHB-1表达,分析其对MdACO1表达情况和果实成熟相关指标的影响。结果显示,VIGS技术成功抑制果肉MdHB-1表达,使MdACO1表达量下调,果实的呼吸强度和乙烯释放速率受到显著抑制,果实硬度、可溶性固形物和可滴定酸质量分数的下降延缓。说明:MdHB-1不仅可以正向调控苹果果实MdACO1表达,还参与果实的成熟衰老过程。

苹果ACO1转录因子MdHB-1基因的克隆及其真核表达载体的构建

【目的】通过生物信息学分析,将苹果的一段EST序列初步定性为转录因子MdHB-1基因,对该基因进行克隆和真核表达载体构建,为进一步研究其分子生物学功能奠定基础。【方法】根据番茄LeACO1转录因子LeHB-1的基因序列,对苹果EST库进行Blast,获得一段EST序列,根据该EST序列设计1对特异性引物,以"皇家嘎啦"苹果花器总RNA为材料,经RT-PCR得到1条长约1 000bp的cDNA片段,T/A克隆后进行序列测定及分析。将基因MdHB-1与真核表达载体pPICZA连接,构建真核表达载体pPICZA-MdHB-1,并导入毕赤酵母菌种GS115,通过PCR鉴定该载体的导入情况。【结果】克隆到苹果MdHB-1基因(GenBank登录号为JQ678788),其开放阅读框为1 011bp,编码336个氨基酸。Blastn和Blastp序列比对分析发现,MdHB-1与已登录的其他物种的HB-1基因相似性达50%～60%。该基因编码的氨基酸中存在保守序列HD-Zip结构域,该保守序列与已经报道的番茄LeHB-1、拟南芥AtHB-1的保守序列的同源性均大于85%。重组表达载体PICZA-MdHB-1构建正确并已成功导入酵母基因组中。【结论】成功克隆了苹果转录因子MdHB-1基因全长,并正确构建了其真核表达载体。

苹果ACO1基因及转录因子MdHB-1基因的表达模式

研究苹果Md ACO1基因及转录因子Md HB-1基因在不同器官和不同发育阶段的表达量及其对外源乙烯的反应,以期探讨两基因之间的可能关系。以苹果品种'皇家嘎拉'为试材,利用实时荧光定量聚合酶链式反应技术分析不同器官和果实不同发育阶段基因的表达,以及乙烯利处理果实对基因表达的影响。在不同组织器官中,Md HB-1在花瓣中表达量较高,而Md ACO1在成熟果中表达量较高。在果实不同发育阶段中Md HB-1在未成熟果中(盛花期40 d后)表达量最高,Md ACO1在成熟果中尤其是花后100 d表达量最高。外源乙烯利(500?mg/L)处理的果实放置0~1 d时抑制Md HB-1的表达,但在7 d后其表达量增高;Md ACO1表达量在外源乙烯利处理后迅速升高,并随着放置时间的延长而大幅度增高。转录因子Md HB-1可能参与了苹果幼嫩果实和花器官中其他相关基因表达的调控,这一过程似与Md ACO1无关。Md ACO1参与了果实的成熟过程,但并无受Md HB-1调控的迹象。外源乙烯会诱导成熟果实ACO1基因的过量表达,这一反馈调节过程似与Md HB-1无关。

Identification and functional characterization of the MdHB-1 gene promoter sequence from Malus×domestica

Homeobox 1 in Malusxdomestica （MdHB-1） is a transcription factor that belongs to homeodomain-leucine zipper I （HD-Zip I） protein subfamily. According to previous reports, MdHB-1 could regulate ethylene synthesis by binding with the MdAC01 promoter, but other functions of MdHB-1 are still unknown. To reveal more clues concerning the characters of the MdHB-1 gene promoter and the functions of MdHB-1, the promoter region of MdHB-1 was cloned from the Royal Gala apple genome and recombined with the 13-glucuronidase （GUS） gene in this study. This research was conducted in Nicotiana tabacum and supported by Agrobacterium-mediated transient transformation and bioinformatics analysis. Deletion analysis of the MdHB-1 promoter showed that the GUS gene could be activated by serially deleted promoters, and the activity promoted by 680 nucleotides （nt） was the lowest. The region, which is 266 nt upstream of the initiation code （ATG）, was effective for GUS expression. Meanwhile, the activity of the MdHB-1 promoter （-1 057 nt）, which was stronger than MdHB-1 promoter （-1 057 to -266 nt） and lack the 5＂-untranslated region （5＂-UTR）, showed that 5＂-UTR may have a positive effect on gene transcription. After the sequence analysis, the cis-acting elements that respond to hormones and environmental stresses were identified in the promoter region. The MdHB-1 promoter （1 057 nt） activity in Nicotiana tabacum was positively induced by ethrel and darkness, and it was suppressed by gibberellic acid （GA）, whereas abscisic acid （ABA）, salicylic acid （SA）, wounding, and Pseudomonas syringae pv. tomato （DC3000） treatments revealed a slight auxo-action. These results reveal that the MdHB-1 promoter receive internal or external signals, and MdHB-1 may refer to many biological activities in apple, such as its stress response, development, and ripening.

苹果MdHB-1基因启动子中的乙烯响应元件对外源乙烯的响应分析

通过PlantCARE和PLACE软件在线分析预测表明,在苹果Md HB-1启动子中存在一个乙烯响应元件(距ATG上游576 bp)。为了探究该启动子中的乙烯响应元件对不同浓度乙烯的响应,以苹果品种‘皇家嘎拉’(Malus domestica Borkh.cv.Royal Gala)基因组DNA为模板,用PCR方法扩增得到长度分别为680 bp(含有乙烯响应元件)和485 bp(不含有乙烯响应元件)的启动子序列,构建GUS报告基因瞬时表达载体,依靠农杆菌介导转化烟草叶片,检测经过不同浓度乙烯利(0、500、1 000和1 500mg·L^(-1))处理的转基因烟草叶片的GUS蛋白活性。结果表明,该乙烯响应元件受到外源乙烯正调控作用,随着处理的外源乙烯浓度的增大,正调控效应先升高达到最大值后逐渐减弱,500 mg·L^(-1)乙烯利能最大程度地提升乙烯响应元件活性。