中国樱桃花芽休眠相关MADS-box基因的克隆与功能初探

梅忠1, 朱友银1,*, 刘向蕾1, 李永强2, 赵华1
1金华职业技术学院农业与生物工程学院, 浙江金华321007; 2浙江师范大学浙江省特色经济植物生物技术研究重点实验室, 浙江金华321004

通信作者:朱友银;E-mail: zhuyouyin@jhc.edu.cn

摘 要:

中国樱桃(Prunus pseudocerasus)花芽具有典型的休眠现象, 足够的低温积累被认为是诱导其休眠解除的关键因素之一。本研究利用3′ RACE技术克隆了一个MADS-box转录因子编码基因。利用生物信息学方法分析了该基因的开放阅读框、氨基酸理化性质以及保守基序等, 发现该基因编码4个保守基序, 其中一个为高度保守的MADS-box, 位于N端, 属于MADS-box转录因子家族典型结构, 且氨基酸序列与桃(P. persica)和梅(P. mume)的DAM6 (dormancy-associated MADS-box)以及拟南芥(Arabidopsis thaliana)的SVP和AGL24具有较高的相似性, 据此, 将其命名为PpcDAM6。利用Real-time PCR分析发现, 在低温积累过程中, PpcDAM6基因明显受低温诱导表达, 在需冷量满足之前上调表达, 在365 CU处表达量最高, 随后下调, 在生态休眠期表达量较低。为进一步研究该基因对植物生长发育的影响, 构建了35S启动子驱动PpcDAM6基因表达的超表达载体。利用沾花法将该基因转入拟南芥中, 发现超量表达PpcDAM6基因的种子萌发明显受到抑制。基因表达分析和种子萌发实验结果表明, PpcDAM6基因在樱桃花芽休眠及休眠解除过程中可能发挥重要的调控作用。

关键词:中国樱桃; 花芽; 休眠; MADS-box基因; 功能分析

收稿:2018-03-27   修定:2018-07-01

资助:浙江省自然科学基金(LQ17C150004和LQ15C150001)。

Isolation and functional analysis of dormancy-associated MADS-box gene in cherry (Prunus pseudocerasus) flower bud

MEI Zhong1, ZHU You-Yin1,*, LIU Xiang-Lei1, LI Yong-Qiang2, ZHAO Hua1
1College of Agricultural and Biological Engineering, Jinhua Polytechnic, Jinhua, Zhejiang 321007, China; 2Zhejiang Provincial Key Laboratory of Biotechnology on Specialty Economic Plants, Zhejiang Normal University, Jinhua, Zhejiang 321004, China

Corresponding author: ZHU You-Yin; E-mail: zhuyouyin@jhc.edu.cn

Abstract:

The flower bud of Chinese cherry (Prunus pseudocerasus) display a typical dormancy in the winter. Sufficient chilling accumulation was considered as one of factors inducing dormancy break. A gene encoding MADS-box transcription factor was cloned from cherry flower bud using 3′ RACE PCR. Open reading frame (ORF) and conserved motifs of the amino acid were predicted and analyzed via various bioinformatics methods. Four highly conserved motifs identified from amino acid sequence. One of motifs known as MADS box was found as a typical and unique domain for the members of the MADS-box family of transcription factors. According to the result of sequence alignment, the amino acid sequence was highly similar to the DAM6 (dormancy-associated MADS-box) of peach (P. persica) and plum (P. mume), SVP and AGL24 of Arabidopsis thaliana. Therefore, the gene cloned from Chinese cherry flower bud was named as PpcDAM6. Real-time PCR results indicated that the expression of PpcDAM6 was significantly up-regulated during endodormancy release and then down-regulated during ecodormancy. In order to reveal the function of PpcDAM6, the gene driven by 35S promoter was transferred into A. thaliana plant. Seed germination of transgenic Arabidopsis was inhibited significantly by expression of PpcDAM6. All of results shown above demonstrated that the PpcDAM6 might regulate the dormancy maintenance and release by response to low temperature accumulation.

Key words: Prunus pseudocerasus; flower bud; dormancy; MADS-box gene; functional analysis

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