发布日期：2019-08-15     浏览人数：732

3 山东省农业科学院蔬菜花卉研究所/山东省设施蔬菜生物学重点实验室/国家蔬菜改良中心山东分中心，山东济南，250100

  摘要：CBL 基因在植物逆境应答和生长发育中具有重要功能，但在甜瓜中尚未见报道。本文利用比较基因组学的方法，从甜瓜基因组中鉴定出7个CBL基因，并对它们的基因组分布、基因结构、系统进化和顺式调控元件进行了分析。结果显示，它们分布在不同的染色体上，都含有8个外显子，编码区在642-738bp,编码的蛋白都含有3个结合钙离子的EF手结构和能够与CIPK互作的FPSF位点。而且，在它们的上游基因序列中存在多个环境应答元件，且各不相同，暗示它们可能具有多种生物学功能。本文为进一步研究甜瓜CBL的功能奠定了基础。
关键词：甜瓜；CBL； 结构；进化；顺式元件
Identification and characterization of CBL genes in melon
Liu Mingyu1, Ji Fuqin2, Ma Qiang1, Hu Xiangshu1，李利斌3，董玉梅3§ 
1 Dongping County Seeds Limited Cooperation of Shandong Province, Dongping County of Shandong Province, 271500
2 Ningyang Wanfeng Seeds Limited Cooperation of Shandong Province, Ningyang County of Shandong Province, 271400
3 Vegetable and Flower Research Institute of Shandong Academy of Agricultural Sciences;Key Laboratory of Greenhouse Vegetables Biology of The Shandong Province;National Improvement Center for Vegetables, Shandong Branch,Jinan, 250100
Abstract  Plant CBL genes have very important roles in response to environmental stress and development. However,there is no investigation report about them in melon at pesent. In this paper, 7 CBL genes in melon genome were identified and their genome distribution, gene structure, phylogeny and cis-elements were systematically analyzed. The results showed that all these genes distribute on different chromosomes respectively, and all of them have 8 exons. And their conding seuquence length is from 642bp to 738bp.There are 3 EF- hands that can bind calcium ions, and  FPSF motif which interacts which CIPK kinases. What’s more, there are different cis-elements responsive to multiple hormones and stimuli in their upstream sequences, which suggests they have multiple biological roles. This text will lay the foundation for further functional elucidation of these genes in melon.
Key words: Cucumis melo L.; CBL; structure; phylogeny; cis-element
  作者简介：刘明毓，女，农艺师，主要从事作物育种和种子生产检测。电话：0538-2821990
E-mail: 503541083@qq.com.

  为通讯作者。董玉梅，女，博士，研究员，主要从事西瓜和甜瓜遗传育种研究。E-mail：dongyumeijn@163.com。
  基金项目：山东省蔬菜良种工程项目；山东省农业重大应用技术创新项目“设施西瓜、甜瓜优质安全高效生产关键技术研究与示范” 。

表1 甜瓜CBL的命名染色体分布及结构特征

  图1 甜瓜CBL基因的外显子-内含子结构 （图中方框代表外显子，方框之间的线段代表内含子）
  2.2 甜瓜CBL基因的系统进化和蛋白结构分析
  基因的遗传进化分析可为基因功能研究提供重要的线索。分析结果显示，甜瓜的CBL基因分为三类（图2）：CmCBL2/3/4/6为一类；CmCBL1单独为一类；而CmCBL5/7为第三类。CmCBL1与AtCBL10直系同源，CmCBL2与AtCBL1直系同源；CmCBL3与AtCBL5直系同源；CmCBL4与AtCBL4直系同源；CmCBL5/7与AtCBL2/3直系同源；CmCBL6与AtCBL9直系同源。序列比对分析发现，甜瓜CBL蛋白含有多个保守的氨基酸残基和位点（图3），其中包括能与蛋白激酶CIPK互作的位点FPSF。蛋白基序分析发现，它们都含有3个EF手结构，1～2个磷酸化位点；多数还含有糖基化位点和豆蔻酰化位点（表2）。只有CmCBL2含有一个酰胺化位点。从结构上看，它们能与钙离子结合，且能与CIPK互作。

图2 拟南芥和甜瓜CBL的系统进化（At代表拟南芥，Cm代表甜瓜）

图 3 甜瓜CBL预测编码蛋白的序列比对
表 2 甜瓜CBL蛋白基序分析

  2.3 甜瓜CBL基因的顺式调控元件分析
  顺式调控元件在基因表达调控中具有重要功能。分析发现，在甜瓜CBL基因的上游序列中存在多个逆境和激素应答元件，且在种类和数目上各不相同（表3）。其中，CmCBL2/4/6/7 含有脱落酸应答元件ABRE, CmCBL2/3/4/5/6具有真菌激发子应答元件Box-W1；CmCBL3/6/7 具有茉莉酸甲酯应答元件CGTCA-motif；它们都含有赤霉素应答元件GARE-motif或者p-box；CmCBL1/2/3/6/7有热胁迫应答元件HSE；CmCBL4还有一个冷胁迫应答元件LTR；CmCBL3/4/6有干旱诱导元件MBS；除CmCBL4外，还都有逆境和防卫应答元件TC-rich repeats；CmCBL2/3/4/5/6都有水杨酸应答元件；CmCBL2/3/4/6有防卫应答元件。
  表 3 甜瓜CBL基因的顺式应答元件

  注：ABRE为脱落酸应答元件；Box-W1，真菌病原物应答元件；CGTCA-motif茉莉酸甲酯应答元件；GARE-motif和p-box为赤霉素响应元件；HSE为热胁迫应答元件；LTR为冷胁迫应答元件；MBS为干旱胁响应元件；TC-rich repeats为防卫和逆境应答元件；TCA-element为水杨酸应答元件；W box为防卫应元件。
  3 结论和讨论
  本文从全基因组水平对甜瓜CBL基因进行分析，结果显示甜瓜基因组中存在7个CBL基因，分别位于不同的染色体上。它们在进化上分为三个不同的类群，在外显子数目上保守，都是8个。它们编码的蛋白都具有EF手结构、与蛋白激酶CIPK的互作位点FPSF和多个保守的氨基酸残基，可能这些位点为其行使功能所必须。在它们的上游序列中，都存在多个激素和逆境应答元件，且各不相同。它们可能不仅应答非生物逆境，而且也可应答生物逆境。在水稻和番茄中最近研究的成果也间接证实了这一点[9,21]。根据系统进化和顺式调控元件分析的结果以及拟南芥CBL研究的进展，我们认为甜瓜CBL基因具有多样的生物学功能：CmCBL1可响应赤霉素信号和热胁迫、盐胁迫及生物逆境；CmCBL2可响应ABA、赤霉素和水杨酸信号以及参与植物防卫应答反应；CmCBL3参与干旱、热以及病原物胁迫应答；CmCBL4参与ABA信号及盐、热、干旱等胁迫应答；CmCBL5/7参与种子发育和盐胁迫应答；CmCBL6参与钾离子吸收和ABA等信号应答。本文为进一步研究甜瓜CBL的功能奠定了基础。
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