CN116143891A - 一种转录因子CeMyb108在调控芋淀粉合成中的应用 - Google Patents
一种转录因子CeMyb108在调控芋淀粉合成中的应用 Download PDFInfo
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Abstract
本发明涉及芋淀粉生物合成技术领域，尤其是一种转录因子CeMyb108在调控芋淀粉合成中的应用，转录因子CeMyb108的SANT和转录抑制结构域的氨基酸序列如SEQ ID NO：3‑4所示。转录因子CeMyb108在调控芋淀粉合成中具有重要作用，通过实时定量分析发现，CeMyb108转录因子与芋可溶性淀粉合成酶CeSSII表达模式相反，CeMyb108对CeSSII起负调控作用。
Description
技术领域
本发明涉及芋淀粉生物合成技术领域，具体领域为一种转录因子CeMyb108在调控芋淀粉合成中的应用。
背景技术
芋(Colocasia esculenta L.Schott)是天南星科芋属的多年生单子叶植物，具地下球茎，常作一年生栽培，中国芋的总产量居世界第二。芋的主要食用部位是地下球茎，因其富含淀粉，占总干物质的60～70％，在亚洲、非洲和印度等地区，常被作为主食之一；与马铃薯和甘薯相比，芋淀粉具有淀粉颗粒小、支链淀粉含量高、冷热稳定性好等典型特征，常用于制备婴儿食品、食品增稠剂或胶粘剂，在食品工业中的用途更为广泛。其独特优良的口感与芋淀粉品质，尤其是支链淀粉含量直接相关。因此，淀粉型芋必将成为今后芋重要的育种方向之一，明晰芋淀粉生物合成途径中重要合成酶的表达调控机制，解析芋淀粉合成的分子机理则显得尤为重要。
目前，关于淀粉合成机制的研究主要集中于拟南芥、水稻、小麦等植物。淀粉的合成是个复杂的过程，由一系列酶催化，而其中可溶性淀粉合成酶(Soluble starchsynthase，SS)，是淀粉合成酶重要组成成员之一，为植物生物合成淀粉的核心酶。SS对淀粉分支、淀粉颗粒的形成、直链淀粉的聚合度起决定影响作用，与总淀粉和支链淀粉积累速率呈极显著正相关，对稻米品质的形成起着决定作用。S主要有SSⅠ、SSII、SSⅢ和SSⅣ这4个亚型，SSII又可分为SSSIIa和SSSIIb。
但是，关于芋淀粉合成途径关键酶的研究较少，顾绘等研究表明子芋、孙芋SBE表达对支链淀粉合成积累可能起重要作用。Lin&Jeang克隆获得芋的SSSI基因，其在叶片中表达量高于球茎，在球茎中表现出高的蛋白活性。发明人课题组研究发现芋球茎中CeApS1的基因表达随着球茎的发育而升高(王立等，2016)；同时克隆了芋SS的2个基因，分别命名为CeSS I和CeSS II，其序列如SEQ ID NO：5-6所示，在芋的球茎中表达水平均较高，而在叶片、叶柄中次之，根中表达较低。基因总体表达水平与淀粉含量之间存在显著正相关。可溶性淀粉合成酶基因CeSS在芋淀粉合成中起着关键作用，揭示CeSS基因及其所在调控网络的作用机制，对于芋淀粉合成的调控机制研究有着十分重要的意义。
总体而言，芋球茎是生长于地下的一种变态茎，是芋淀粉合成和累积的主要部位，其分子水平的研究仍停留在淀粉合成途径少数几个基因的同源(分离)克隆以及单个基因的功能分析，在整个合成途径中起限速作用的关键酶的调控机制尚不明确，对淀粉结构和组分变异产生的机理没有进行系统性研究。可溶性淀粉合成酶SS主要参与支链淀粉合成，决定淀粉含量、淀粉颗粒大小等特性，前期研究虽然发现SS对芋淀粉合成起着关键作用，但具体调控机制还不明确。尽管植物淀粉合成的主要酶已经明确，但作为较特殊器官(地下生长变态茎)以及具有淀粉典型理化特征的芋球茎淀粉合成分子机理还有待明确。
发明内容
本发明的目的在于提供一种转录因子CeMyb108在调控芋淀粉合成中的应用。
近年来高通量测序技术、组学和系统生物学高速发展，网络分析技术(Networkanalysis)开始被应用于复杂生物学过程和功能研究中，基于基因间互作进行全基因组范围的网络(Deciphering genome-scale networks，DeGN)分析芋的时空表达模式，是解析芋CeSS基因调控网络的有效手段。
不同可溶性淀粉合成酶亚型在不同的组织中发挥相应的功能，而更多的可能是它们相互调节影响淀粉的合成。围绕芋淀粉合成的关键酶CeSS基因，对全生育期芋的球茎进行全基因组范围基因间转录水平相互关系分析，构建其调控网络，挖掘对其表达起关键调控作用的转录因子，分析其在芋生育周期淀粉合成的调控模式，为进一步解析芋淀粉合成的分子机理提供理论支撑，为芋淀粉研究奠定理论基础。
具体而言，本发明提供如下技术方案：
调控芋淀粉合成的转录因子CeMyb108的SANT和转录抑制结构域，其氨基酸序列如SEQ ID NO：3-4所示。其中，转录因子CeMyb108的核苷酸序列如SEQ ID NO：1所示；氨基酸序列如SEQ ID NO：2所示。
本发明所述的转录因子CeMyb108可用于调控芋淀粉合成。转录因子CeMyb108可调控可溶性淀粉合成酶基因CeSS，所述可溶性淀粉合成酶基因CeSS包括CeSS I和CeSS II，其序列如SEQ ID NO：5-6所示。其中，转录因子CeMyb108对CeSSII起负调控作用。
与现有技术相比，本发明的有益效果是：
本发明通过筛选获得了调控CeSS的转录因子CeMyb108，CeMyb108转录因子结构中包含了SANT和转录抑制结构。该转录因子在调控芋淀粉合成中具有重要作用，芋头地下球茎从初始到成熟的整个过程，通过实时定量分析CeMyb108转录因子有一个先下降到后期上升的过程，与芋可溶性淀粉合成酶CeSS，特别是CeSSII表达模式相反，CeMyb108对CeSSII起负调控作用。
本发明获得了芋淀粉合成起重要调控作用的转录因子CeMyb108，为进一步解析芋淀粉合成关键基因SS对淀粉品质形成的分子机理，并揭示淀粉合成的复杂调控机制奠定基础，为优质淀粉的芋资源发掘和品种改良提供技术支撑。
附图说明
图1为芋可溶性淀粉合成酶及其相关基因的表达模式。
图2为CeMyb108、CeSSII、CeSSⅠ和Actin实时定量引物的溶解曲线。
图3-5为芋球茎中CeMyb108、CeSSII以及CeSSⅠ的相对表达量。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1调控芋淀粉合成的转录因子CeMyb108
试验材料：芋基因组测序品种XH01，江苏省著名地方特色魁芋品种，国家地理标志农产品，于2020年3月31日种植于江苏省农业科学院六合动物科学基地，当年10月25日收获。芋植株于5月6日(播种后30d)出苗，分别于出苗后的第30d(6月9日)、60d(7月6日)、90d(8月6日)、120d(9月11日)、150d(10月13日)和180d(11月5日)，采集新生的地下球茎，每次3个重复，用液氮速冻后保存于-70℃冰柜中备用。
分别对芋全生育期的6个时期样品，进行转录组分析，对获得的基因，采用基因表达水平估算方法进行基因表达量的计算和差异基因的筛选。通过https://www.zhaolab.org/DeGNServer/基于基因间互作，应用co-expression和ContextLikelihood算法通过全基因组范围的调控解析模式DeGNserver计算基因间的相互作用，以芋CeSS基因为节点，抓取与之相互作用的基因，构建CeSS基因亚调控网络(构建了由159个基因2,146互作组成的亚调控网络(P>0.95))。从CeSS基因亚调控网络中筛选出参与调控的转录因子。通过http://planttfdb.cbi.pku.edu.cn/，http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi？id＝index，分析候选转录因子的结构和作用的靶标基因，筛选获得调控CeSS的候选转录因子CeMyb108。其核苷酸序列如SEQ ID NO：1所示；氨基酸序列如SEQ ID NO：2所示。
CeSSⅠ和CeSSII的序列如SEQ ID NO：5-6所示，CeSSII上游启动子序列如SEQ IDNO：9所示。CeSSII上游启动子中包含了两组Myb结合区，Myb结合位点序列如SEQ ID NO：7-8所示。CeMyb108转录因子结构中包含了SANT和转录抑制结构域，其氨基酸序列如SEQ IDNO：3-4所示。
实施例2CeMyb108转录因子在芋地下球茎发育全过程的表达模式
(1)芋可溶性淀粉合成酶及其调控基因的表达模式分析
通过芋球茎发育过程的6个时期的转录组动态数据分析，发现CeSSII、CeSSⅠ以及Ser/thr kinase有相同的表达模式，均表现为T2时期表达量达到最高，随后呈下降趋势，T4期降至最低，T5-T6期略有反弹，其中CeSSII的表达丰度明显高于CeSSⅠ，CeSSII是芋球茎中可溶性淀粉合成酶3种亚型中表达量比较高的一个亚型。通过共表达分析找到的转录因子CeMyb108则表现出与CeSS完全相反的表达模式，如图1所示。
(2)实时荧光定量分析验证
①方法
采用
反转录试剂盒(TaKaRa，大连)合成cDNA，对芋CeSS基因及其候选的转录因子，使用Primer 5.0(Premier BIosoft International，USA)设计引物，选择DaActin作为内参基因进行RT-qPCR。引物及内参基因的序列如SEQ ID NO：10-17所示。
CeMyb108-F:TAGAGAACTGCAGCCCTTGC；
CeMyb108-R:CAGTTAGCCAACGCGGAGT；
CeSSⅠ-F:CCTCGGACCAGCACATACAA；
CeSSⅠ-R:CCAACCCATCCACGGAACTT；
CeSSII-F:GTCCGGTGCAGGTTGAATTG；
CeSSII-R:ACATCCACGTCGCCCATATC；
Actin-F:TCTGGCCACCACACCTTCTAC；
Actin-R:GACACACCGTCACCAGAGTC；
反应程序：95℃30min；95℃5s，58℃30s，72℃30s，40个循环，通过定量反应的溶解曲线(如图2所示)，验证引物的特异性满足试验要求。RT-qRCR反应在ABI7500荧光定量PCR检测系统(Thermofisher，美国)中进行，依据2-△△Ct算法计算基因的相对表达量，如图3-5所示。
②实时荧光定量分析结果
通过定量分析验证4个基因的表达丰度和表达模式，CeSSII是球茎中控制芋可溶性淀粉酶合成的主效基因，在芋球茎的形成初期表达快速上升、并且在快速膨大期达到最大值，随着芋球茎的成熟表达量逐步下降。CeMyb108则随着芋球茎的发育呈现先下降到后期上升的过程，与芋可溶性淀粉合成酶CeSS，特别是CeSSII表达模式相反，CeMyb108对CeSSII起负调控作用。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
Claims (6)
1.调控芋淀粉合成的转录因子CeMyb108的SANT和转录抑制结构域，其特征在于：氨基酸序列如SEQ ID NO：3-4所示。
2.根据权利要求1所述的转录因子CeMyb108的SANT和转录抑制结构域，其特征在于：转录因子CeMyb108的核苷酸序列如SEQ ID NO：1所示。
3.根据权利要求2所述的转录因子CeMyb108的SANT和转录抑制结构域，其特征在于：转录因子CeMyb108的氨基酸序列如SEQ ID NO：2所示。
4.转录因子CeMyb108在调控芋淀粉合成中的应用。
5.根据权利要求4所述的转录因子CeMyb108在调控芋淀粉合成中的应用，其特征在于：转录因子CeMyb108可调控可溶性淀粉合成酶基因CeSS，所述可溶性淀粉合成酶基因CeSS包括CeSS I和CeSS II，其序列如SEQ ID NO：5-6所示。
6.根据权利要求5所述的转录因子CeMyb108在调控芋淀粉合成中的应用，其特征在于：转录因子CeMyb108对CeSS II起负调控作用。
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| CN116769002B (zh) * | 2023-08-11 | 2023-11-03 | 云南师范大学 | 转录因子StERF75及调控马铃薯支链淀粉合成用途 |
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