茶树β-淀粉酶基因CsBAM3的克隆及其响应低温的表达模式

Cloning of β-amylase Gene (CsBAM3) and Its Expression Model Response to Cold Stress in Tea Plant

β-淀粉酶(BAM)是植物中参与淀粉水解的关键酶类,在应对非生物胁迫中发挥重要作用。从前期茶树冷驯化转录组分析中分离到1个参与淀粉代谢的差异表达基因,cDNA全长克隆及序列分析鉴定该基因为拟南芥BAM3同源基因,命名为CsBAM3。该基因编码548个氨基酸残基,与拟南芥的BAM1和BAM3一起归为第Ⅱ亚家族,推测为叶绿体定位、具有淀粉水解活性的β-淀粉酶编码基因。启动子克隆及序列分析显示该基因可能受生理节律、光、低温及多种激素等信号共同调控。CsBAM3在叶片中表达量最高,茎和花中表达量较低,根中基本不表达。CsBAM3在冷驯化初期被显著上调且一直保持相对较高的水平。成熟叶片及嫩芽(一芽二叶)中的CsBAM3均可以被4℃及0℃低温显著上调,且嫩芽中基因的表达量上调幅度明显高于成熟叶。模拟倒春寒低温条件下,检测不同萌发阶段新梢中CsBAM3的表达情况表明,CsBAM3在鲜叶初展的嫩芽中即可快速受低温诱导。以上结果表明,CsBAM3是茶树中调控淀粉水解的一个重要β淀粉酶编码基因,在茶树成熟叶和嫩芽受到不同低温胁迫时其表达可被快速诱导。

A major enzyme of starch breakdown in plant leaf,β-amylase(BAM),plays important roles in response to abiotic stresses.In tea plant,a differentially expressed gene involved in starch metabolism was identified from previous RNA-Seq analysis of cold acclimation.Its full-length cDNA was cloned and sequenced,showing that it is a homolog of BAM3in Arabidopsis(named CsBAM3).CsBAM3contains548amino acid residues and is grouped into subfamily II together with BAM1and BAM3from Arabidopsis.According to protein sequence and3D structure analyses,CsBAM3was considered as a chloroplast-targeted protein withβ-amylase activity.Promoter cloning and sequence analysis showed a couple of responsive elements related to circadian,light,cold,and phytohormone,indicating its complex regulating mechanism.CsBAM3showed the expression higher in leaf,relative lower in stem and flower and extremely low in root.The expression of CsBAM3in mature leaf was significantly up-regulated during the early time courses of winter cold acclimation and maintained at relative high level during the following period.With the treatments of4°C and0°C,the transcript levels of CsBAM3in both mature leaves and young shoots were dramatically increased,interestingly,much higher level was detected in young shoots.The cold environment similar to spring cold spell was created using climate chamber.And the expression changes of CsBAM3in shoots were determined then after the tea plants were moved into climatic chamber at different sprouting phrases.CsBAM3was quickly up-regulated by cold once the first leaf unfolded.Our study illuminated that CsBAM3is a keyβ-amylase coding gene involved in starch metabolism of tea plant,which can be stimulated in both mature leaves and young shoots by cold stress.