CN116536336B - 一种龙眼果实大小、数目调控基因DlCYP71B10-like及应用 - Google Patents
一种龙眼果实大小、数目调控基因DlCYP71B10-like及应用 Download PDFInfo
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Abstract
本发明提供一种龙眼果实大小、数目调控基因DlCYP71B10‑like，其核苷酸序列如SEQ ID No.1所示。本发明对DlCYP71B10‑like基因进行克隆，分析该基因的序列结构、进化关系、组织表达等。同时构建过表达载体，并转化到Mico Tom番茄中进行功能分析。结果表明DlCYP71B10‑ like基因负调控果实发育，正调控果实数目。该结果不仅会为龙眼等果树果实重量/大小和数目调控理论研究的开展奠定重要基础，同时也为后续利用分子辅助育种开展大果型龙眼优质新品种选育提供重要基因资源和分子标记。
Description
技术领域
本发明涉及分子生物技术领域，具体涉及一种龙眼果实性状相关的调控基因DlCYP71B10-like及其应用。
背景技术
赤霉素(GAs)作为一种经典的植物激素调控植物生长发育的各个过程，其合成与代谢途径已被广泛深入的研究(He J,Xin P,MaX,Chu J,Wang G.Gibberellin metabolismin flowering plants:An update and perspectives.Frontiers in plant science,2020:532.)，它与植物体内其他激素信号交互作用，协同调节植物的生长发育以及对环境变化的应答。一旦细胞分裂阶段结束，GAs将成为细胞扩张阶段的主要调节因子，调控果实的膨大(Kumar R,Khurana A,Sharma A.Role of plant hormones and their interplayin development and ripening of fleshy fruits.Journal of Experimental Botany,2014.65:4561-4575.)。GAs在果实发育中的主要作用是促进细胞分裂和维持细胞伸长(McAtee P,Karim S,Schaffer R,David K.A dynamic interplay betweenphytohormones is required for fruit development,maturation,andripening.Frontiers in Plant Science,2013,4:79.)。目前已经从植物、真菌和细菌中鉴定出了136种GAs，具有生物活性GAs主要有GA1、GA3、GA4和GA7(宋松泉,刘军,黄荟,伍贤进,徐恒恒,张琪,李秀梅,梁娟.赤霉素代谢与信号转导及其调控种子萌发与休眠的分子机制.中国科学:生命科学,2020,50:599–615)，在绝大多数植物中，GA1和GA4是最主要的活性赤霉素结构，并且GA4的活性显著高于GA(Salazar-Cerezo S,Martínez-Montiel N,García-Sánchez J,Pérez-Y-Terrón R,Martínez-Contreras D.Gibberellin biosynthesis andmetabolism:a convergent route for plants,fungi and bacteria.Microbiologicalresearch,2018,208:85-98.)。在龙眼果实中活性GA主要是GA4和GA7(李建国,黄旭明,周碧燕,周贤军.人工疏果对龙眼果实大小、内源激素和细胞壁成分的影响.热带作物学报,2000,21(3):28-33.)。目前的研究表明CYP p450成员可以通过调控赤霉素活性影响作物种子发育。比如拟南芥中CYP714A1是使赤霉素失活的基因(Nomura T,Magome H,Hanada A,TakeE#￥da-Kamiya N,Mander L,Kamiya Y,Yamaguchi S.Functional analysis ofArabidopsis CYP714A1 and CYP714A2 reveals that they are distinct gibberellinmodification enzymes.Plant Cell Physiology,2013,54(11):1837-1851.)。CYP714A1编码细胞色素P450 CYP714A1催化GA12转变为16-羧化GA12(16-carboxy-16β,17-dihydroGA12)，GA12是赤霉素合成的重要中间物质，GA12的改变导致合成的GA4没有活性。CYP714A1过表达植株表现出活性GA4水平显著降低，沉默CYP714A1，活性GA4水平增加(Nomura T,Magome H,Hanada A,Takeda-Kamiya N,Mander L,Kamiya Y,Yamaguchi S.Functionalanalysis of Arabidopsis CYP714A1 and CYP714A2 reveals that they are distinctgibberellin modification enzymes.Plant Cell Physiology,2013,54(11):1837-1851.)，最近研究表明CYP714A1基因主要在拟南芥发育中的角果和种子中表达(He J,XinP,MaX,Chu J,Wang G.Gibberellin metabolism in flowering plants:An update andperspectives.Frontiers in plant science,2020:532.)。Jiang等(Jiang S,An H,LuoJ,Wang X,Shi C,Xu F.Comparative analysis of transcriptomes to identify genesassociated with fruit size in the early stage of fruit development in Pyruspyrifolia.International Journal of Molecular Science,2018,19:2342.)通过转录组测序在小果梨‘GH81H’上也鉴定出了CYP714A1基因，在花后20d和30d上调CYP714A1可能阻碍果实的发育。
发明内容
本发明目的在于提供一种龙眼果实大小、数目的调控基因。
本发明另一目的在于提供上述龙眼果实大小、数目调控基因表达的蛋白。
本发明又一目的在于提供上述龙眼果实大小、数目调控基因的应用。
本发明目的按如下技术方案实现：
一种龙眼果实大小、数目调控基因DlCYP71B10-like，其核苷酸序列如SEQ IDNo.1所示。
一种龙眼龙眼果实大小、数目调控蛋白，其氨基酸序列如SEQ ID No.2所示。
本发明还提供了含有前述编码基因的载体。
本发明还提供了含有前述载体的工程菌。
本发明进一步提供了前述基因在龙眼果实大小、数目方面的应用。
进一步地，所述应用为将所述工程菌侵染植株，获得龙眼果实大小、数目调控的转基因植株。
本发明具有如下有益效果：
已公开的报道龙眼CYP p450家族成员中共有327个基因，本发明以DlCYP71B10-like基因为对象，克隆了该基因的ORF全长，分析该基因的序列结构、进化关系、组织表达情况等。qRT-PCR分析表明该基因在F1代中大果株系FD105和小果株系FD21的不同发育阶段果实表现出差异表达。
本发明对DlCYP71B10-like基因进行克隆，分析该基因的序列结构、进化关系、组织表达等。同时构建过表达载体，并转化到Mico Tom番茄中进行功能分析。结果表明，DlCYP71B10-like基因含有CYP蛋白保守的p45 domain结构域，说明DlCYP71B10-like是典型的细胞色素p450蛋白，与来自茶树等果树的CYP71B10-like亚家族成员关系更近，具有组织表达特异性，在果肉中表达较低，在茎和幼果中的表达量最高；在F1代的大果株系FD105果实发育后期80～90d内显著下调表达，过表达转基因株系的果实也明显小于野生型MicoTom番茄的果实，果实数目显著增多。以上结果表明DlCYP71B10-like基因负调控果实大小和重量，正调控果实数目。该结果不仅会为龙眼等果树果实重量/大小理论研究的开展奠定重要基础，同时也为后续利用分子辅助育种开展大果型龙眼新品种选育提供重要基因资源和分子标记。
附图说明
图1：DlCYP71B10-like基因PCR扩增图。
图2：DlCYP71B10-like与龙眼(“红核子”)基因组CYP71B10-like(Dlo_002096.1)的碱基和氨基酸序列对比。红色箭头表示碱基或氨基酸不同处。
图3：不同物种间CYP P450蛋白序列比对图。框部分表示CYP蛋白保守的p45domain结构域氨基酸序列。
图4：DlCYP71B10-like在龙眼不同组织中的相对表达量图。不同字母靶标表示差异达到显著水平。
图5：DlCYP71B10-like在不同F1后代果实发育中的相对表达量图。
图6：DlCYP71B10-like转基因番茄果实发育表型图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1目的基因的克隆
材料和方法
1.1植物材料
选取3组在长势和树龄(9年龄)一致的‘四季蜜’龙眼为取样树，取‘四季蜜’龙眼的花、花芽、叶、果皮、果肉、根、种子、茎和幼果(花后60d整果)等器官为材料进行组织表达分析。选取3组在长势和树龄(10年龄)一致的F1代大果型株系FD105和小果型株系FD21龙眼为取样树(F1代父母本是‘凤梨朵’(母本)ב大乌圆’(父本))，取花后60、70、80、90和100d的龙眼果肉为材料进行果实发育分析。采集FD21龙眼叶片作为基因克隆模板材料。所有的试验设3次重复，取样后立即放入液氮速冻并转入-80℃冰箱中保存、备用。
1.2DlCYP71B10-like基因序列的克隆及生物信息分析
从龙眼基因组数据库(NCBI Sequence Read Archive，SRA315202)中获得CYP71B10-like基因(Dlo_002096.1)的碱基序列和氨基酸序列信息。利用Primer Premier5.0根据DlCYP71B10-like基因的ORF序列设计引物CYP71B10-like-S和CYP71B10-like-A(表1)，委托天一辉远生物科技有限公司(广州)合成。用北京华越洋生物公司的植物RNA提取试剂盒提取FD21龙眼叶片的RNA，采用Takara公司的PrimeScript RT-PCR试剂盒，具体操作步骤参照说明书，反转录cDNA作为模板，进行PCR扩增克隆DlCYP71B10-like基因。扩增条件为：94℃预变性5min；94℃变性30s，60℃退火30s，72℃延伸90s，35个循环(变性-延伸)；72℃延伸10min，4℃保存。扩增产物进行切胶回收和纯化连接到pMD18-T载体上，转化DH5α感受态细胞，PCR筛选阳性克隆，挑取阳性单克隆送天一辉远生物科技有限公司(广州)进行测序。
利用在线软件SMART程序(http://smart.emblheidelberg.de/)预测蛋白结构域，利用ExPASy(http://expasy.org/tools/)分析蛋白的等电点和分子量。根据克隆所得的cDNA序列，利用BLASTp对氨基酸序列进行同源性对比，同时利用MEGA 11软件进行氨基酸序列同源性分析及系统发育分析，构建Neighbor-Joining进化树，1000次重复，其它均为默认设置。
1.3表达分析
根据克隆所得的DlCYP71B10-like基因序列设计qRT-PCR引物qCYP71B10-like-S和qCYP71B10-like-A(表1)，并在NCBI里利用BLASTn检验以确保引物的特异性。以龙眼的Actin基因(Dlo_028674)为内参基因，具体引物序列见表1。
表1所用引物信息Tab.1Information of primers used
qRT-PCR反应所用仪器为Roche的LightCycler 480，PCR反应酶为Takara公司的SYBR Green Master Mix。反应体系为20mL，其中模板cDNA 40ng，上、下游引物各250nM，SYBR Green Master Mix 10μL，其余用ddH2O补齐。反应程序：94℃预变性5min；94℃10s，59℃20s，72℃30s，40个循环后作熔解曲线(95→65℃，0.1℃/s)。利用2-ΔΔCt计算DlCYP71B10-like基因的相对表达量。所有样品进行3次重复，均设阴性对照。采用Excel软件进行平均数统计，以SPSS软件进行单因素方差分析目的基因在不同组织和材料里的变化的差异显著性(P<0.05)，并使用SigmaPlot 12.5软件作图。
实施例3过表达载体构建及转基因番茄功能验证
使用特异PCR引物OECYP71B10-like-S/OECYP71B10-like-A(表1)，以龙眼cDNA为模板，进行PCR扩增。该引物5’端分别加有BamH I酶切位点，反引物5’端分别加有Sac I酶切位点。获得的PCR产物与pMD19-T载体连接并进行测序。最后，提取测序正确的质粒，用BamHI和Sac I分别双酶切pBI121和测序正确的质粒，通过T4 DNA连接酶构建含有DlCYP71B10-like目标基因的植物表达载体，并命名为pBI121-DlCYP71B10-like。通过液氮冻融法将所构建的过量表达载体pBI121-DlCYP71B10-like转入农杆菌菌株GV3101，参照文献(ArshadW,Waheed M T,Mysore K S,et al.Agrobacterium-mediated transformation of tomatowith rolB gene results in enhancement of fruit quality and foliar resistanceagainst fungal pathogens[J].PLoS One,2014,9(5):e96979.)，通过农杆菌侵染法将DlCYP71B10-like基因转入番茄(Micro-Tom)，获得T0代种子。在含30ug/ml的MS固体培养基上筛选阳性番茄，同时用pBI121质粒特异引物检测阳性转基因番茄幼苗。将T3代转基因植株分别和野生型在相同环境中培养，并比较它们的果实发育表型。
实施例4结果与分析
1.DlCYP71B10-like基因的克隆及生物信息学分析
以FD21龙眼叶片cDNA为模板，用CYP71B10-like-S/CYP71B10-like-A(表1)引物扩增出1300bp左右的片段(图1)。测序结果显示。该基因(Dlo_002096.1)大小为1308bp，编码435个氨基酸，其分子量为49.96kDa，理论等电点为5.76。我们获得的DlCYP71B10-like基因与已知龙眼(“红核子”)基因组数据库(NCBI Sequence Read Archive，SRA315202)中CYP71B10-like基因(Dlo_002096.1)相比，在碱基序列中存在4处不同(461处的T-G；479处的T-C；497处的G-A；508处的A-C)，导致氨基酸序列存在3处不同(154处的C-F；160处的T-I；166处的E-G)(图2)。根据与其他作物CYP p450家族成员的亲缘关系，命名为DlCYP71B10-like。氨基酸序列分析表明DlCYP71B10-like与茶树CsCYP71B10-like(Camelliasinensis,XP_028069352.1)；苦楝MaCYP(Melia azedarach,KAJ4719235.1)和蒜头果MoCYP71E(Malania oleifera,QDA69651.1)一样，含有CYP p450蛋白保守的p450 domain结构域，说明DlCYP71B10-like是典型细胞色素p450蛋白(图3)。3、DlCYP71B10-like基因组织表达特性分析
qRT-PCR结果表明DlCYP71B10-like基因在被检测的9种龙眼组织中都有表达，但表达具有组织特异性，其中在果肉中表达较低，在茎和幼果中的表达量最高(图4)。该结果说明DlCYP71B10-like基因特异参与龙眼果实发育。4、DlCYP71B10-like基因在花、果发育过程中的表达模式
利用qRT-PCR技术，我们分析了DlCYP71B10-like在F1代大果型株系FD105和小果型株系FD21果实发育过程中的表达模式。结果显示，在果实发育早期60～70d，DlCYP71B10-like基因在FD21和FD105中都呈显著上升趋势。在果实发育后期80～90d，DlCYP71B10-like基因在FD105中呈下调趋势(图5)。该结果表明，DlCYP71B10-like基因表达与果肉器官发育呈负相关。5、转DlCYP71B10-like基因的番茄表型分析
转基因表型结果显示，过表达DlCYP71B10-like基因的番茄植株的果实相对于野生型显著变小(降低25.31％)，横经和纵经也显著降低(分别降低34.33％和32.99％)。但同时，果实数目显著增多近1.47倍(图6)，该结果说明DlCYP71B10-like基因过量表达会显著降低果实大小和重量，同时显著增加果实数目，可以作为大果优质龙眼新品种选育候选基因。
Claims (2)
1.过表达龙眼基因DlCYP71B10-like在降低龙眼果实大小、增加果实数目方面的应用，所述基因DlCYP71B10-like核苷酸序列如SEQ ID No.1所示。
2.通过含有龙眼基因DlCYP71B10-like的载体制备得到的工程菌在侵染龙眼，获得降低龙眼果实大小、增加果实数目的转基因龙眼中的应用，所述基因DlCYP71B10-like核苷酸序列如SEQ ID No.1所示。
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