ZHAO Yongfei,CHEN Xiaolu,PENG Futian,et al.Cloning and Arabidopsis Ectopic Expression of PpSnRK1βλ1 From Prunus persica[J].Northern Horticulture,2017,41(11):98-106.[doi:10.11937/bfyy.201711021]
桃PpSnRK1βλ1基因的克隆及在拟南芥中异源转基因的功能分析
- Title:
- Cloning and Arabidopsis Ectopic Expression of PpSnRK1βλ1 From Prunus persica
- 关键词:
- 桃树; PpSnRK1βλ1; 基因克隆; 表达分析; 功能研究
- 文献标志码:
- A
- 摘要:
- 以实生毛桃为试材，采用PCR和实时荧光定量技术，克隆桃树SnRK1蛋白激酶（蔗糖非发酵-1-型相关蛋白激酶-1）的调节亚基βλ，并分析其组织表达特性。构建p35S::PpSnRK1βγ1重组载体，获得超表达PpSnRK1βλ1基因拟南芥植株用于分析其生物功能。结果表明：克隆获得桃树SnRK1βλ1，命名为PpSnRK1βλ1。该cDNA全长为1 476 bp，编码492个氨基酸。序列分析表明，其包含CBM和4个CBS结构域；PpSnRK1βλ1与果梅的SnRK1βλ蛋白亲缘关系最近；PpSnRK1βλ1基因在实生毛桃的根、茎、叶均有表达，其中在茎中表达量最低。超表达PpSnRK1βγ1基因拟南芥株系A-βγ1的花期比野生型晚2.19 d，单株莲座叶数量比野生型多1.17片；叶片的叶绿素、可溶性糖和可溶性蛋白质含量均显著高于野生型拟南芥，分别比野生型提高了13.7%、12.9%和23.3%，但淀粉含量却没有显著性差异。在氧化胁迫条件下，转基因植株与野生型的生长均受到抑制，但前者具有更好的萌发率和主根长度，以保证植株正常生长。因此，PpSnRK1βλ1基因参与调控植株的碳氮代谢，并影响植物的花期，以及在防御氧化胁迫过程中起重要作用。
- Abstract:
- Wild peach was used as test material，PpSnRK1βλ1，the regulatory subunit βλ of SnRK1 (Sucrose non-fermenting-1-related protein kinase-1)，was cloned and its expressions in different organs was analyzed.And to assay its function，transgenic plants were obtained successfully by transgenic technology.The results indicated that the reading frame of PpSnRK1βλ1 possessed 1 476 bp and encoded 492 amino acid residues deduced from the DNA sequence.PpSnRK1βλ1 contained CBM and four CBS domain.The deduced PpSnRK1βλ1 protein was highly homologous to other PpSnRK1βλ1proteins from different species，and PpSnRK1βλ1 was very closely related to SnRK1βλ of GuoMei.Real-time PCR results showed that the PpSnRK1βλ1 expressed in root，stalk and leaf of peach seedling.The expression level of PpSnRK1βλ1 was very low in stalk of peach seedling.Via statistical studies，the results indicated that transgenic plant flowered 2.19 days later than WT，and produced around 1.17 rosette leaves than WT.The contents of chlorophyll，soluble sugar，and soluble proteins of transgenic plant were significantly higher than WT，which increased by 13.7%，12.9% and 23.3%，respectively.There was no difference between transgenic plant and WT in starch content.Under the condition of oxidative stress，the growth rate of the wild-type Arabidopsis was severely suppressed，while the transgenic Arabidopsis showed less impact.Transgenic plants showed better seed germination rate and relatively longer root length than wild type，which could support plant growth under stress condition.In a word，the results of study indicated that PpSnRK1βλ1 participated in regulating carbon and nitrogen metabolism and had effect on flower time，and played a role in the resistant of oxidative stress.
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备注/Memo
第一作者简介：赵永飞（1990-），女，硕士研究生，研究方向为果树分子生物学。E-mail：zhao781990zhaoliu@163.com.责任作者：彭福田（1969-），男，博士，教授，博士生导师，研究方向为果树营养生理与施肥技术。E-mail：pft@sdau.cn.基金项目：国家现代农业产业技术体系建设专项资金资助项目（CARS-31）。