CN117126865A - 一种促进类胡萝卜素含量积累的LbaMYB44基因及应用 - Google Patents
一种促进类胡萝卜素含量积累的LbaMYB44基因及应用 Download PDFInfo
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Abstract
本发明公开了一种促进类胡萝卜素含量积累的LbaMYB44基因,所述LbaMYB44基因为枸杞转录因子基因,所述LbaMYB44基因的核苷酸序列,如序列表SEQ ID NO.1所示,所述LbaMYB44基因编码的氨基酸序列如序列表SEQ ID NO.2所示,所述LbaMYB44基因在增加番茄果实中玉米黄素含量积累中的应用。本发明利用酵母单杂交、双荧光素酶实验和凝胶迁移或电泳迁移率实验等技术验证了LbaMYB44与LbaBCH2启动结合,从而调控类胡萝卜素含量积累,对阐述番茄玉米黄素合成积累和提高果实品质培育高品质番茄新品种提供了重要基因资源,并具有潜在育种应用价值。
Description
技术领域
本发明涉及基因工程技术领域,尤其涉及一种促进类胡萝卜素含量积累的LbaMYB44基因及应用。
背景技术
R2R3-MYB转录因子在植物次生代谢物合成与积累发挥着重要作用,尤其是在调控植物类胡萝卜素代谢积累与合成方面。如首次在猩红猴面花中,通过集群分离分析法(BSA)定位到一个R2R3-MYB转录因子RCP1参与花类胡萝卜素调控(Sagawaetal.2016));在柑橘中,研究发现MYB转录因子CrMYB68通过负调控CrBCH2和CrNCED5,参与类胡萝卜素代谢途径(Zhuetal.2017);在猕猴桃中,研究发现MYB转录因子MYB7通过与AdLCY-β基因启动子结合,参与类胡萝卜素代谢调控((Ampomah-Dwamenaetal.2019);在番茄中,SIMYB72通过负调控LCYB,正调控PSY和ZISO,参与番茄果实发育过程类胡萝卜素代谢调控(Wuetal.2020);在番木瓜,两个MYB转录因子CpMYB1和CpMYB2参与调节细胞壁降解生理过程和类胡萝卜素调控,从而影响果实软化(Fuetal.2020)。上述研究证实,R2R3-MYB转录因子广泛参与调控植物类胡萝卜素,但是R2R3-MYB转录因子在枸杞中的功能尚不明确。
枸杞作为我国重要“药食同源”植物资源,其成熟的果实含有较为丰富的功效物质类胡萝卜素,具有“保肝明目、补肾益精”等多种保健功效。类胡萝卜素作为枸杞中最主要的保健和品质成分,其含量直接影响枸杞外观品质,尤其是果实色泽鲜艳程度。因此,挖掘枸杞类胡萝卜素生物合成调控基因、解析其分子调控机制对高类胡萝卜素新品种培育具有重要意义。
然而,枸杞遗传转化体系还未建立,功能验证研究进展较为缓慢。
发明内容
1.要解决的技术问题
本发明的目的是为了解决现有技术中枸杞遗传转化体系还未建立,功能验证研究进展较为缓慢的问题,而提出的一种促进类胡萝卜素含量积累的LbaMYB44基因及应用。
2.技术方案
为了实现上述目的,本发明采用了如下技术方案:
一种促进类胡萝卜素含量积累的LbaMYB44基因,所述LbaMYB44基因为枸杞转录因子基因,所述LbaMYB44基因基因的核苷酸序列,如序列表SEQ ID NO.1所示。
优选地,所述LbaMYB44基因编码的氨基酸序列如序列表SEQ ID NO.2所示。
本发明中还提出了一种促进类胡萝卜素含量积累的LbaMYB44基因的应用,所述LbaMYB44基因在增加番茄果实中玉米黄素含量积累中的应用。
3.有益效果
相比于现有技术,本发明的优点在于:
(1)本发明中,利用酵母单杂交、双荧光素酶实验和凝胶迁移或电泳迁移率实验等技术验证了LbaMYB44与LbaBCH2启动结合,从而调控类胡萝卜素含量积累。
(2)本发明中,通过在番茄中异源过表达LbaMYB44基因,通过农杆菌法将其稳定转化番茄果实,进而验证了LbaMYB44基因能够增加番茄果实中玉米黄素含量的积累,表明LbaMYB44基因能够调控番茄果实的颜色。LbaMYB44基因及其编码得到的枸杞转录因子LbaMYB44有利于提高培养高玉米黄素含量的番茄品种,进而增加玉米黄素的提取量。
(3)本发明中,对阐述番茄玉米黄素合成积累和提高果实品质培育高品质番茄新品种提供了重要基因资源,并具有潜在育种应用价值。
附图说明
图1为本发明提出的LbaMYB44基因的克隆图,其中M:DNAmarker DL2000;1,、2和3:LbaMYB44扩增片段;
图2为本发明提出的LbaMYB44与拟南芥R2R3-MYB系统进化树构建图;
图3为本发明提出的LbaMYB44蛋白亚细胞定位图,Bright:明场;GFP:绿色荧光;RFP:红色荧光;Merge:合并;
图4为本发明提出的LbaMYB44截断体转录激活活性验证图;
图5为本发明提出的LbaMYB44基因及部分结构基因荧光定量检测结果图;
图6为本发明提出的LbaBCH2基因启动子PCR扩增电泳图;
图7为本发明提出的LbaMYB44与LbaBCH2启动子酵母单杂交流程图,其中A:LbaBCH2基因启动子含有MYB结构域元件示意图;B:LbaMYB44与LbaBCH2酵母单杂交载体构建结构示意图;C:LbaMYB44与LbaBCH2单杂交验证实验;
图8为本发明提出的LbaMYB44过表达株系的阳性苗PCR鉴定结果图,其中M:markerDL2000;WT:野生型番茄;P:重组质粒phellsGate2-LbaMYB44;OE:过表达株系;
图9为本发明提出的番茄果实中超表达LbaMYB44的影响结果图,其中A:转基因番茄果实表型。WT,野生型;OE-2/8,2个独立LbaMYB44超表达系;MG,绿熟期;BR,破色期;RR,成熟期。B:野生型与过表达株系类胡萝卜素含量检测;C:类胡萝卜结构基因在野生型和过表达株系中表达量。
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。
实施例1:
一种促进类胡萝卜素含量积累的LbaMYB44基因及应用,LbaMYB44基因为枸杞转录因子基因,LbaMYB44基因的核苷酸序列,如序列表SEQ ID NO.1所示。
本发明中,LbaMYB44基因编码的氨基酸序列如序列表SEQ ID NO.2所示,LbaMYB44基因在增加番茄果实中玉米黄素含量积累中的应用。
本发明中,利用酵母单杂交、双荧光素酶实验和凝胶迁移或电泳迁移率实验等技术验证了LbaMYB44与LbaBCH2启动结合,从而调控类胡萝卜素含量积累。
本发明中,通过在番茄中异源过表达LbaMYB44基因,通过农杆菌法将其稳定转化番茄果实,进而验证了LbaMYB44基因能够增加番茄果实中玉米黄素含量的积累,表明LbaMYB44基因能够调控番茄果实的颜色。基因LbaMYB44及其编码得到的枸杞转录因子LbaMYB44有利于提高培养高玉米黄素含量的番茄品种,进而增加玉米黄素的提取量。
本发明中,对阐述番茄玉米黄素合成积累和提高果实品质培育高品质番茄新品种提供了重要基因资源,并具有潜在育种应用价值。
实施例2:
LbaMYB44基因的克隆及其编码蛋白特性分析:
本实施例中,以‘宁夏黄果’果实为材料,利用多糖多酚植物总RNA提取试剂盒(TIANGEN,北京)提取果实的总RNA。用EasyScript One-StepgDNARemovalandcDNASynthesisSuperMix试剂盒反转录合成cDNA。反转录体系为(20μL):RNA模板2μL,2×ESReactionMix10μL,EasyScriptRTMix1μL,gDNARemover1μL,剩余的用ddH2O补足至20μL。反转录程序为42℃30min,85℃5s。利用特异性引物LbaMYB4-F(5'-ATGGCTGCAATTATACAGAGAAAAG-3')和LbaMYB44-R(5'-TCACTCAATCTTGCTAATGCCC-3')通过PCR扩增获得LbaMYB44基因片段(图1)。PCR扩增体系为(25μL)cDNA模板2μL,正向引物和反向引物各1μL,Mix12.5μL,ddH2O8.5μL。PCR扩增程序为95℃5min,9510s,58℃30s,72℃1min,34个循环,72℃5min。通过测序比对分析,该基因长度为942bp,编码313个氨基酸,分子量为34.39kDa,理论等电点为8.80。
实施例3:
LbaMYB44蛋白的进化树构建与序列比对分析:
本实施例中,从TAIR(https://www.arabidopsis.org/)下载拟南芥R2R3-MYB蛋白序列。利用MEGA将其与LbaMYB44和AtR2R3-MYB蛋白序列合并构建系统发育树,设置Bootstrap为1000,最大似然法,其余参数采用默认值。利用iTOL(https://itol.embl.de/)在线网站对构建好的系统发育树进行美化。系统进化树显示,LbaMYB44蛋白被划分到S22亚家族,与拟南芥AtMYB44亲缘关系最近(图2),前期研究表明AtMYB44参与调控植物次生代谢合成,因此可推断LbaMYB44可能具有调控次生代谢物积累功能。
实施例4:
LbaMYB44蛋白的亚细胞定位:
(1)重组载体构建
分别设计带XbaI和KpnI酶切位点的LbaMYB44的上下游特异性引物,其中上游引物序列为5'-ACACGGGGGACGAGCTCGGTACCATGGCTGCAATTATACAGAGAAAAG-3',下游引物序列为5'-CTCACCATGGTGTCGACTCTAGACTCAATCTTGCTAATGCCC-3'。PCR扩增体系为(25μL):cDNA模板2μL,正向引物和反向引物各1μL,Mix12.5μL,ddH2O8.5μL。PCR扩增程序为95℃5min,9510s,58℃30s,72℃1min,34个循环,72℃5min。经过胶回收,测序获得正确的LbaMYB44-GFP质粒,以次质粒为模板进行双酶切。酶切体系为(50μL):Buffer5μL,XbaI和KpnI各1μL,质粒20μL,ddH2O23μL。酶切时间为37℃2h。
最后通过同源重组法构建获得35S::LbaMYB44-GFP亚细胞定位载体。
(2)亚细胞定位
将构建好的35S::LbaMYB44-GFP载体和GFP空载体(对照)通过农杆菌介导法注释侵染本氏烟草叶片,进行瞬时表达,在共聚焦显微镜下观察GFP荧光型号。结果显示:空载体的荧光信号分布在细胞膜、细胞质和细胞核上,而35S::LbaMYB44-GFP的应该信号只分布在细胞核上,表明LbaMYB44定位于细胞核(图3)。
实施例5:
LbaMYB44转录自激活验证:
本实施例中,LbaMYB44全长及截断ORF片段构建的PGBKT7诱饵载体同pGBKT7-53+pGADT7、pGBKT7分别转至AH109菌株中,最后将转化产物按1、1:10、1:100分别涂布于固体筛选培养基SD/-Trp、SD/-Trp-Ade、SD/-Trp-Ade/X-a-gal,30℃培养3-5d,观察每个培养基上生长情况。结果发现SD/-Trp上均能生长,而在SD/-Trp-AdepGBKT7,pGBKT7-LbaMYB44(1-324)不能正常生长,而在SD/-Trp-Ade/X-a-gal培养基上也不变蓝(图4)。说明LbaMYB44全长及361-942片段均具有转录激活活性,而1-324片段没有转录激活活性。
实施例6:
LbaMYB44与枸杞类胡萝卜素生物合成结构基因的表达模式分析:
本实施例中,利用qRT-PCR检测LbaMYB44、LbaPSY1,LbaPDS,LbaZDS,LbaBCH2和LbaVDE在‘宁夏黄果’不同组织(根、叶、茎、花、果实)表达情况。qRT-PCR扩增体系为(15μL):cDNA模板2μL,上下游引物各0.3μL,Mix7.5μL,ddH2O4.9μL。扩增程序如下:95℃3min;95℃20s,58℃20s,72℃30s,35个循环;72℃5min。荧光定量所需引物如表1,以Actin基因作为内参基因(Liuetal.2014)。
表1荧光定量引物信息
本实施例中,qRT-PCR检测结果表明:LbaMYB44基因在果实转色时期表达量最高,类胡萝卜素生物结构基因LbaPSY1、LbaPDS和LbaBCH2也均在果实中高表达,而LbaZDS基因则在盛花期表达量较高,LbaVDE在叶片表达量最高(图5)。因此,这些数据结果表明LbaMYB44、LbaPSY1、LbaPDS和LbaBCH2在果实类胡萝卜素合成积累中发挥着重要作用。
实施例7:
LbaMYB44蛋白与LbaBCH2启动子互作分析:
(1)LbaBCH2基因启动子克隆
DNA提取:称取枸杞幼嫩叶片0.1g置于2mL离心管中,加液氮研磨至粉末;之后加入800μL预热(65℃)2%CTAB提取液,轻摇混匀;65℃水浴1h,每隔10min轻摇一次;冷却至室温后加入800μL氯仿异戊醇(V:V=24:1),混匀20min;1000rpm离心10min;吸取上清液移到1.5mL的离心管中,加入600μL预冷的异丙醇,混匀后置于4℃沉淀1h或-20℃30min;1000rpm离心10min;弃上清液,用70%乙醇洗涤3次,自然晾干;加入100μLddH2O和1μLRNAase,37℃水浴30min;待充分溶解后,用0.8%琼脂糖凝胶电泳检测DNA的浓度和纯度;稀释成工作液-20℃保存备用。
LbaBCH2基因启动子克隆:利用特异性引物LbaBCH2pro-F(5'-AGTTTCAATACCCAAGTTATTCTTGAAG-3')和LbaBCH2pro-R(5'-GGACGGTAGCCTTAAAAGTGAAG-3')通过PCR扩增获得LbaBCH2基因启动子片段(图6)。PCR扩增体系为(25μL)DNA模板2μL,正向引物和反向引物各1μL,Mix12.5μL,ddH2O8.5μL。PCR扩增程序为95℃5min;9510s,58℃30s,72℃1min,40个循环,72℃5min。将PCR产物纯化回收,克隆至pHIS2载体后,转化大肠柑橘Top10感受态细胞,阳性克隆送公司测序。
本实施例中,重组载体构建及酵母转化:将LbaBCH2启动子克隆到pHIS2载体中,构建诱饵载体,然后将诱饵质粒线性化后,转化到Clontech酵母基因组中,产生诱饵酵母。将LbaMYB44转录因子的CDS序列克隆到pGADT7载体中,与GAL4激活域(AD)融合,产生猎物载体AD-LbaMYB44。将猎物载体和空载体分别转化诱饵酵母细胞。酵母转化按照如下步骤进行:①从YPDA平板挑取Y187单菌落接种于YPDA液体培养基4ml,30℃,225rpm,振荡培养18-20h(过夜),至OD600>1.5,一般为4左右;②转接YPDA液体培养基,培养体积为50ml,使初始OD600=0.2,30℃,225rpm,振荡培养4-5h,至OD600=0.6。③离心收菌,室温,1,000rpm,5min;④用20ml无菌水重悬菌体,混匀,离心收菌,室温,1,000rpm,5min,弃上清;⑤用5ml0.1MLiAc重悬菌体,混匀,离心收菌,室温,1,000rpm,5min,弃上清。⑥用500μL0.1MLiAc重悬菌体,混匀,分装至1.5ml离心管,50μL每管(一个转化),备用。⑦每个1.5ml离心管中依次加入50%PEG3350240μL,1MLiAc36μL,ssDNA(10mg/ml)5μL,质粒DNA各5μL;30℃水浴孵育,30min;42℃水浴热激,25min;30℃水浴复苏,1h;离心收菌,室温,700rpm,5min,弃上清;每个转化用200μL无菌水悬浮菌体,尽量温和地混匀,涂布相应的缺陷型筛选平板;30℃恒温培养3-4天。转化后的细胞按100、10-1和10-2依次稀释,各取5μL涂在含有0mM3AT和75mM3AT缺陷型培养基上,30℃恒温培养3-5天。YIH检测结果发现,仅含有重组载体AD-LbaMYB44的诱饵酵母细胞能添加3AT的缺陷型培养基上生长,说明LbaMYB44蛋白能与LbaBCH2启动子上的MBS元件互作(图7C)。
实施例8:
异源过表达LbaMYB44基因能够提高番茄果实类胡萝卜素含量:
(1)LbaMYB44过表达载体构建
以‘宁夏黄果’cDNA为模板,利用特异性引物进行PCR检测。特异性上游引物序列为(5'-CATTTGGAGAGGACACGCTCGAGATGGCTGCAATTATACAGAGAAAAG-3'),下游引物序列(5'-TCT CATTAAAGCAGGACTCTAGATCACTCAATCTTGCTAATGCCC)PCR扩增体系为(25μL)cDNA模板2μL,正向引物和反向引物各1μL,Mix12.5μL,ddH2O8.5μL。PCR扩增程序为95℃5min;9510s,58℃30s,72℃1min,40个循环,72℃5min。PCR扩增产物用1%琼脂糖凝胶电泳检测,检测是否符合预期。将PCR扩增产物回收后,经XbaI和XhoI双酶切,与phellsGate2载体上,构建phellsGate2-LbaMYB44重组表达载体。最后将构建好的载体和空载,通过农杆菌介导的遗传转化,进行番茄遗传转化,具体方法参考已发表文献(Wangetal,2022)。待转基因幼苗生根后,将其移栽到土壤中,在温室或大田中生长,同时种植未转化的番茄植株作为阴性对照。
(2)转基因株系阳性苗鉴定
利用通用引物对获得转基因株系进行PCR鉴定。PCR鉴定体系为(15μL):DNA模板2μL,上下游引物各0.3μL,Mix7.5μL,ddH2O补足至15μL。PCR鉴定程序为95℃5min;95℃20s,58℃30,72℃1min10s,40个循环;72℃5min。经鉴定获得14个阳性植株。
(3)类胡萝卜素含量提取与检测
样品前处理:取出低温保存的冻干样本,用球磨仪研磨(30Hz,1min)至粉末状;称取50mg的研磨后的样本,加入适量内标,用含0.01BHT(g/mL)的正己烷/丙酮/乙醇混合液(1:1:2,v/v/v)进行提取;室温下旋涡20min,重复提取一次后离心合并上清液;将得到的提取液浓缩后用甲醇/甲基叔丁醚混合液(3:1,v/v)复溶,过0.22μm滤膜后,保存于棕色进样瓶中,用于LC-MS/MS分析。
样品检测:数据采集仪器系统主要包括超高效液相色谱(Ultra-PerformanceLiquidChromatography,UPLC)和串联质谱(TandemMassSpectrometry,MS/MS)。采用YMCC30(3μm,100mm×2.0mmi.d)色谱柱检测,流动相为:A相,甲醇/乙腈(1:3,v/v)加入0.01%BHT和0.1%甲酸;B相,甲基叔丁基醚加入0.01BHT;梯度洗脱程序:0minA/B为100:0(v/v),3min为100:0(v/v),5min为30:70(v/v),9min为5:95(v/v),10min为100:0(v/v),11min为100:10(v/v);流速0.8mL/min:柱温28℃;进样量2μL。
通过HPLC检测转基因株系中类胡萝卜素含量的变化。结果发现,过表番茄株系主要检测到的类胡萝卜素种类有新黄质(Neoxanthin)、紫黄质(Violaxanthin)、玉米黄质(Zeaxanthin)、叶黄素棕榈酸酯(Luteinpalmitate)、番茄红素(Lycopene)、叶黄素(Lutein)和β-胡萝卜素(β-carotene)。与野生型相比,LbaMYB44超表达系中新黄质、玉米黄质和叶黄素含量显著增加,番茄红素含量也增加但没有达到显著性差异(图9B)。
(4)荧光定量检测
进一步采用qRT-PCR的方法检测野生型和转基因番茄果实中类胡萝卜素合成基因表达。qRT-PCR扩增体系为(15μL):cDNA模板2μL,上下游引物各0.3μL,Mix7.5μL,ddH2O4.9μL。扩增程序如下:95℃3min;95℃20s,58℃20s,72℃30s,35个循环;72℃5min。荧光定量所需引物如表2,以SlActin基因作为内参基因。qRT-PCR结果显示,LbaMYB44超表达株系果实中,PSY2,PDS,LCYB,BCH2,VDE及ZEP基因表达量显著高于野生型(图9C),说明在番茄中异源表达LbaMYB44能够增番茄果实类胡萝卜素含量,改变番茄果实类胡萝卜素代谢。
表2过表达番茄荧光定量检测引物信息
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (3)
1.一种促进类胡萝卜素含量积累的LbaMYB44基因,其特征在于,所述LbaMYB44基因为枸杞转录因子基因,所述LbaMYB44基因的核苷酸序列,如序列表SEQ ID NO.1所示。
2.根据权利要求1所述的一种促进类胡萝卜素含量积累的LbaMYB44基因,其特征在于,所述LbaMYB44基因编码的氨基酸序列如序列表SEQ ID NO.2所示。
3.根据权利要求1所述的一种促进类胡萝卜素含量积累的LbaMYB44基因的应用,其特征在于,所述LbaMYB44基因在增加番茄果实中玉米黄素含量积累中的应用。
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