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作物学报 ›› 2017, Vol. 43 ›› Issue (10): 1417-1425.doi: 10.3724/SP.J.1006.2017.01417

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

茶树β-淀粉酶基因CsBAM3的克隆及其响应低温的表达模式

郝心愿1,**,岳川1,2,**,唐湖1,钱文俊1,王玉春1,王璐1,王新超1,*,杨亚军1,*   

  1. 1中国农业科学院茶叶研究所 / 国家茶树改良中心 / 农业部茶树生物学与资源利用重点实验室，浙江杭州31008；2福建农林大学园艺学院，福建福州350002
  • 收稿日期:2017-02-13 修回日期:2017-05-10 出版日期:2017-10-12 网络出版日期:2017-05-23
  • 通讯作者: 王新超, E-mail: xcw75@tricaas.com Tel: 0571-86653162; 杨亚军: E-mail: yjyang@tricaas.com
  • 基金资助:

    本研究由浙江省自然科学基金项目(LY16C160001), 国家现代农业产业技术体系建设专项(CARS-23)和中国农业科学院农业科技创新工程(CAAS-ASTIP-2014-TRICAAS)资助。

Cloning of β-amylase Gene (CsBAM3) and ItsExpression ModelResponseto Cold Stress in Tea Plant

HAO Xin-Yuan1,**,YUEChuan1,2,**,TANG Hu1,QIAN Wen-Jun1,WANG Yu-Chun1,WANG Lu1, WANG Xin-Chao1,YANG Ya-Jun1,*   

  1. 1 Tea Research Institute, Chinese Academy of Agricultural Sciences / National Center for Tea Improvement / Key Laboratory of Tea Plant Biology and Resources Utilization, Ministry of Agriculture, Hangzhou 310008, China; 2College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002
  • Received:2017-02-13 Revised:2017-05-10 Published:2017-10-12 Published online:2017-05-23
  • Contact: 王新超, E-mail: xcw75@tricaas.com Tel: 0571-86653162; 杨亚军: E-mail: yjyang@tricaas.com
  • Supported by:

    This study was supported by the Zhejiang Provincial Natural Science Foundation (LY16C160001), the Earmarked Fund for China Agriculture Research System (CARS-23), and the Chinese Academy of Agricultural Sciences through an Innovation Project for Agricultural Sciences and Technology (CAAS-ASTIP-2014-TRICAAS).

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摘要：

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。

关键词: β淀粉酶, 茶树, 基因克隆, 低温胁迫, 表达调控

Abstract:

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 BAM3 in Arabidopsis (named CsBAM3). CsBAM3 contains 548 amino acid residues and is grouped into subfamily II together with BAM1 and BAM3 from Arabidopsis. According to protein sequence and 3D structure analyses, CsBAM3 was considered as a chloroplast-targeted protein with β-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. CsBAM3 showed the expression higher in leaf, relative lower in stem and flower and extremely low in root. The expression of CsBAM3 in 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 of 4℃ and 0℃, the transcript levels of CsBAM3 in 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 CsBAM3 in shoots were determined then after the tea plants were moved into climatic chamber at different sprouting phrases. CsBAM3 was quickly up-regulated by cold once the first leaf unfolded. Our study illuminated that CsBAM3 is a key β-amylase coding gene involved in starch metabolism of tea plant, which can be stimulated in both mature leaves and young shoots by cold stress.

Key words: β-amylase, Tea plant, Gene clone, Cold stress, Expression regulation

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