CN111171129A - 小麦类胡萝卜素合成途径关键基因Lcye突变体及其应用 - Google Patents
小麦类胡萝卜素合成途径关键基因Lcye突变体及其应用 Download PDFInfo
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Abstract
本发明公开了小麦类胡萝卜素合成途径关键基因Lcye突变体及其应用。本发明提供了如下蛋白质：将Lcye‑D1蛋白的第253位的丝氨酸替换为苯丙氨酸后得到的蛋白质，或将其经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同能力的衍生蛋白质，或与其所限定的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质，或在其N端和/或C端连接标签后得到的融合蛋白。本发明不仅验证Lcye基因功能，也为面粉及其制品颜色性状改良提供了理论依据和种质资源。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种小麦类胡萝卜素合成途径关键基因Lcye突变体及其应用。
背景技术
面粉色泽是评价小麦粉品质的重要感官指标和市场指标。黄色素是小麦籽粒中最主要的天然色素，是面粉及其制品黄度形成的主要原因。籽粒黄色素含量与面粉、面团黄度的相关系数高达0.8-0.9，与面包和面条颜色的相关系数为0.69和0.76(Mares和Campbell，2001；Adom等，2003；Fratianni等，2005；Cong等，2010)。中式面食品，如白面条、馒头、包子和饺子等对面粉的白度要求比较高，细腻洁白的面粉及其制品备受青睐。
类胡萝卜素是构成黄色素的主要组分。类胡萝卜素，尤其是β-胡萝卜素，具有抗氧化、抗癌、维生素A原、预防眼睛老年性黄斑变性、延缓衰老及增强免疫等重要生理保健功能。人类和动物自身不能合成类胡萝卜素，必须从外界摄取(Cazzonelli和Pogson，2010)。近年来，随着人们营养和保健意识的增强，提高小麦籽粒类胡萝卜素含量、培育亮黄色的面粉和面制品小麦品种，逐渐成为新的育种目标。
植物类胡萝卜素生物合成涉及一个复杂的基因调控网络(Cazzonelli和Pogson，2010；Zhu等，2010)。番茄红素环化是类胡萝卜素合成代谢途径中的重要分支点。植物体内存在ε-番茄红素环化酶(Lycopene epsilon cyclase，LCYE)和β-番茄红素环化酶(Lycopene beta cyclase，LCYB)两种番茄红素环化酶。Howitt等(2009)克隆了普通小麦Lcye基因，应用Sunco/Tasman DH群体将该基因与3B染色体上黄色素含量QTL位点共分离，证实Lcye基因是影响籽粒黄色素含量的关键基因。董长海(2011)克隆了普通小麦3B和3D染色体上的Lcye基因全长，并针对B基因组序列差异开发了显性标记YP3B-1。Crawford和Francki(2013)克隆了Lcye-3A基因，并根据序列差异开发了功能标记e-LCY3A-3。综上所述，目前对小麦Lcye基因研究局限于QTL定位、基因克隆和分子标记开发，其基因功能和遗传调控机制尚不明确，严重制约了育种工作进展。因此，加强Lcye基因功能及其遗传调控机理研究，有助于深刻认识小麦黄色素含量形成分子机制，为培育黄色素含量符合市场需求的小麦新品种奠定理论基础。
定向诱导基因组局部突变技术(Targeting induced local lesions ingenomes，TILLING)是一种将诱发产生高频率点突变的化学诱变方法与PCR筛选和高通量检测方法有效结合，快速高效检测目标区域点突变，通过对其表型鉴定分析，获得基因功能的反向遗传学研究方法(Till等，2003a；闫智慧等，2014)。与转基因和分子标记辅助选择等分子育种技术相比，TILLING技术还具有诱变育种稳定快、仅改变单个目标性状、无需进行繁琐耗时的转基因及杂交、回交过程等优点，是一种高效定向的分子育种技术(侯彩玲等，2008；韩宁等，2013)。随着测序技术及作物基因组学的迅速发展，TILLING技术将在小麦基因功能分析、遗传调控及重要农艺、品质性状遗传改良中发挥重要作用(陈锋等，2010；潘娜等，2011；Zhai等，2016)。
发明内容
本发明的目的是提供小麦类胡萝卜素合成途径关键基因Lcye突变体及其应用。
第一方面，本发明要求保护一种蛋白质。
本发明所要求保护的蛋白质，可为如下任一：
(A1)将Lcye-D1蛋白的第253位的丝氨酸替换为苯丙氨酸后得到的(S253F)；
(A2)将(A1)所限定的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同能力的由(A1)衍生的蛋白质。
(A3)与(A1)-(A2)中任一所限定的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质；
(A4)在(A1)-(A3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
上述蛋白质中，所述标签是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测、示踪和/或纯化。所述标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
进一步地，所述(A1)所示蛋白质为由SEQ ID No.1所示的氨基酸序列组成的蛋白质。
第二方面，本发明要求保护编码前文第一方面所述蛋白质的核酸分子。
进一步地，所述核酸分子可为编码前文第一方面所述蛋白质的基因，所述基因可为如下任一所示的DNA分子：
(B1)SEQ ID No.2所示的DNA分子；
(B2)在严格条件下与(B1)限定的DNA分子杂交且编码前文第一方面所述蛋白质的DNA分子；
(B3)与(B1)或(B2)限定的DNA序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码前文第一方面所述蛋白质的DNA分子。
上述基因中，所述严格条件可为如下：50℃，在7％十二烷基硫酸钠(SDS)、0.5MNa3PO4和1mM EDTA的混合溶液中杂交，在50℃，2×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交，在50℃，1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交，在50℃，0.5×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交，在50℃，0.1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交，在65℃，0.1×SSC，0.1％SDS中漂洗；也可为：在6×SSC，0.5％SDS的溶液中，在65℃下杂交，然后用2×SSC，0.1％SDS和1×SSC，0.1％SDS各洗膜一次。
第三方面，本发明要求保护含有前文第二方面所述核酸分子的重组载体、表达盒、转基因细胞系或重组菌。
第四方面，本发明要求保护前文所述的蛋白质或所述的核酸分子或所述的重组载体、表达盒、转基因细胞系或重组菌在如下任一中的应用：
(C1)下调小麦Lcye基因总表达量；或制备用于下调小麦Lcye基因总表达量的产品；
(C2)下调小麦Lcye-B1基因和/或Lcye-D1基因表达量；或制备用于下调小麦Lcye-B1基因和/或Lcye-D1基因表达量的产品；
(C3)降低小麦籽粒黄色素含量；或制备用于降低小麦籽粒黄色素含量的产品。
在本发明的具体实施方式中，所述(C1)中下调小麦Lcye基因总表达量和所述(C2)中下调小麦Lcye-B1基因和/或Lcye-D1基因表达量具体体现在RNA水平。所述下调小麦Lcye基因总表达量为下调小麦籽粒中Lcye基因总表达量；所述下调小麦Lcye-B1基因和/或Lcye-D1基因表达量为下调小麦籽粒中Lcye-B1基因和/或Lcye-D1基因表达量。
第五方面，本发明要求保护一种降低小麦籽粒黄色素含量的方法。
本发明所要求保护的降低小麦籽粒黄色素含量的方法，可包括如下步骤：仅将受体小麦基因组中编码Lcye-D1蛋白的第253位的丝氨酸的密码子替换为编码苯丙氨酸的密码子(优选纯合突变)。
进一步地，所述将受体小麦基因组中编码Lcye-D1蛋白的第253位的丝氨酸的密码子替换为编码苯丙氨酸的密码子为将所述受体小麦基因组中编码Lcye-D1蛋白的基因替换为编码由SEQ ID No.1所示的氨基酸序列组成的蛋白质的基因。
更进一步地，所述编码由SEQ ID No.1所示的氨基酸序列组成的蛋白质的基因为SEQ ID No.2所示DNA分子。
第六方面，本发明要求保护如下任一应用：
(D1)前文所述的蛋白质或所述的核酸分子或所述的重组载体、表达盒、转基因细胞系或重组菌或所述应用或所述方法在小麦面粉或面制品颜色改良中的应用；
(D2)利用前文所述方法培育得到的籽粒黄色素含量降低的小麦品种在小麦育种中的应用。
本发明应用TILLING技术筛选甲基磺酸乙酯(Ethyl methane sulfonate，EMS)诱变群体，根据小麦Lcye序列设计特异引物，通过非变性聚丙烯酰胺凝胶电泳技术检测突变位点，获得不同等位变异基因的突变植株，对不同等位基因与表型进行关联分析，鉴定各突变位点对LCYE功能的影响。在2491份M2代EMS诱变群体中共检测到21个Lcye基因的点突变，包含6个错义突变，2个同义突变和13个内含子突变，Lcye基因在群体中的突变频率为1/266.1kb。PARSENP软件预测分析显示，两个错义突变M090815(C2202T)和M091648(G3284A)可能严重影响蛋白质功能。MEME分析结果表明，M090815和M092230(G2195A)突变位点位于Lcye基因保守结构域内。6个错义突变植株与野生型杂交构建的F2代群体中，M090815突变位点显著降低籽粒黄色素含量，证实该位点对LCYE功能具有重要影响。qRT-PCR结果显示，M090815突变位点显著降低Lcye基因表达水平，且Lcye-B1和Lcye-D1基因表达降低趋势相似，而Lcye-A1在花后14-28天表现出补偿效应。本发明不仅验证Lcye基因功能，也为面粉及其制品颜色性状改良提供了理论依据和种质资源。
附图说明
图1为Lcye的突变类型。
图2为突变体M090815的F2群体中不同基因型植株籽粒Lcye及其同源基因的相对表达量。*和**分别代表0.05显著水平和0.01显著水平。(a)Lcye-all，(b)Lcye-A1，(c)Lcye-B1，(d)Lcye-D1。
图3为突变体M090815的F2群体中三种基因型植株籽粒黄色素含量。*代表0.05显著水平。
图4为Lcye功能结构域预测。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、小麦类胡萝卜素合成途径关键基因Lcye功能研究
一、材料与方法
1、EMS诱变群体构建
EMS诱变群体的构建参照Slade等人(2005)方法。首先筛选一批稳定纯合、大小一致、籽粒饱满的济麦20(马少康等.不同水氮处理对济麦20蛋白组分和加工品种的影响.麦类作物学报,2010,30(3):477-481)和济麦22(刘佳等.高产小麦品种济麦22旗叶叶绿素和活性氧清除系统酶活性的变化.山东农业科学,2012,44(8)：31-34)种子，利用1.2％浓度的化学诱变剂EMS(Solarbio，E8150)进行诱变处理，产生一系列的点突变；将处理后的种子温室种植获得突变群体M1代；M1代种子大田种植获得2491份M2代植株(济麦20：1251份；济麦22：1240份)，籽粒单株收获保存。
2、应用TILLING技术筛选EMS突变体库
参照Till等(2006)的方法，进行EMS突变体筛选。具体步骤如下：
(1)突变体DNA池构建：应用CTAB法(Doyle和Doyle，1987)分别提取每个M2突变体植株的基因组DNA，将其存放于96孔板中。利用NanoDrop-2000超微量分光光度仪(ThermoScientific)测定DNA的浓度和质量。按8个样品一组将DNA进行等量混合，构建8倍DNA混合池，4℃保存备用。
(2)Lcye特异性引物设计：基于小麦Lcye同源基因序列间的差异，设计A、B和D基因组特异性引物。利用中国春缺体-四体材料(倪志勇等.小麦转录因子TaDREB6基因的克隆及鉴定.麦类作物学报,2008,28(3)：357-363)及PCR产物测序方法，进行引物基因组特异性验证；应用CODDLE(Codons Optimized to Discover Deleterious Lesions；http://www.proweb.org/coddle/)软件分析扩增区域是否对基因功能起重要作用。筛选获得4对Lcye的特异性引物(表1)。
表1利用TILLING技术筛选Lcye突变体的引物信息
(3)PCR扩增与异源双链生成：以DNA池为模板进行PCR扩增，反应体系：DNA50ng，SuperMix(全式金，AS111-01)7.5μL，上、下游引物(10μmol·L-1)各1μL，用ddH2O补足至15μL；反应程序：首先95℃ 5min；然后95℃ 30s，由66℃开始，每个循环退火温度降低0.3℃，退火时间45s，72℃ 1.5min，共35个循环；72℃延伸10min；然后99℃ min，85℃ 1min，由85℃开始，每个循环退火温度降低0.5℃，退火时间30s，共99个循环；最后16℃保温备用。如果DNA混合池样本目标片段序列中含有突变位点，扩增产物经过反复变性、复性形成野生型和突变体扩增片段的异源双链(即含有错配碱基)。
(4)CEL I酶切：用特异性识别并切割错配碱基的核酸内切酶CELI剪切异源双链核酸分子。CEL I内切酶的提取参照Till等(2006)的方法。确定CELI酶具有酶切活性后，参照潘娜等(2012)方法，分析CELI酶浓度、酶切时间、酶切温度和酶切缓冲液浓度等因素对酶切效果的影响，优化酶切体系。确定最适宜的酶切反应体系为10×消化缓冲液2μL、CEL I 1μL、15μL异源双链DNA，ddH2O补足至20μL，45℃反应20min，随后加入EDTA(0.25M，生工生物工程股份有限公司，ET0895)5μL终止酶切反应。
(5)非变性聚丙烯酰胺凝胶电泳检测：利用非变性聚丙烯酰胺凝胶电泳检测技术，筛选获得含有突变位点的阳性DNA池。对阳性DNA池中的每个DNA逐一与野生型DNA进行混合，重复上述步骤，筛选阳性突变体单株。
(6)突变样本克隆测序：对突变个体PCR产物进行克隆测序，鉴定突变类型和位置。
3、突变位点对蛋白功能影响预测
利用PARSESNP(Proiect aligned related sequences and evaluate SNPs；http://www.proweb.org/parsesnp/)软件分析突变体植株DNA序列的突变类型，预测突变位点对LCYE功能是否造成影响。当PSSM值大于10和SIFT(Sorting intolerant fromtolerant)值小于0.05的氨基酸改变被认为可能对蛋白质功能造成重要影响(Ng和Henikoff，2003；Taylor和Greene，2003)。
4、突变位点功能分析
为了减小其他突变背景影响，将含有Lcye基因错义突变位点的纯合M3植株与野生型植株进行杂交，构建F2代群体(将6个含有Lcye基因错义突变位点的M2代植株收获籽粒大田种植，克隆测序鉴定M3植株基因型，分别选取3株纯合突变型植株与对应野生型植株杂交，构建F2群体。三个杂交穗收获籽粒为F0，F0自交收获籽粒F1，F1再播种获得F2群体)，用于分析突变位点对基因表达水平及蛋白质功能的影响。F2群体于2017-2018年度种植于山东济南，行长3m，行距25cm，每行30株，每个F2群体种15行，田间管理采用常规方法。
利用克隆测序的方法，鉴定F2群体中每个株系Lcye基因型(纯合突变型、杂合突变型和野生型)。每种基因型各选取10个生长发育进程一致的生物学重复，记录开花期，分别采集花后7、14、21和28天籽粒，立即置于液氮中，–80℃保存，用于Lcye基因表达水平分析。单株收获，成熟籽粒-20℃保存，用于黄色素含量测定。
利用实时定量PCR(Quantitativereal-timePCR，qRT-PCR)技术，测定F2群体中纯合突变型、杂合突变型和野生型植株籽粒不同发育时期(花后7、14、21和28天)Lcye基因及其各同源基因表达量，分析突变位点对Lcye基因表达水平的影响。具体步骤为：应用RNAprep Pure植物总RNA提取试剂盒(TIANGEN Biotech，DP441)提取籽粒总RNA，纯合突变型、杂合突变型和野生型植株各3个生物学重复；利用PrimeScriptTMRT Reagent试剂盒(Takara Bio Inc.，RR047A)将其反转成cDNA，置于-20℃保存备用；基于小麦Lcye同源基因cDNA序列间的保守性和差异性，设计Lcye基因的保守性引物和A、B、D基因组特异性引物(表2)，对其进行溶解曲线分析和qRT-PCR产物克隆测序，验证引物保守性和特异性，普通小麦β-actin基因(AB181991)作为内参基因；应用qRT-PCR技术，检测F2群体中纯合、杂合和野生型植株籽粒不同发育时期Lcye基因表达量，每个样本3次技术重复，基因相对表达量用平均值±标准误差(Standard error，SE)表示。反应体系：LightCycler FastStart DNA MasterSYBR Green(Roche Applied Sciences，No.03003230001)10μL，上下游引物0.5μM，cDNA50ng，ddH2O补充至20μL。反应程序：95℃ 10min；95℃ 15s，60℃20s和72℃ 20s，共40个循环；应用公式2-ΔΔCT计算目标基因相对表达量(Livak和Schmittgen，2001)。首先，利用同一样本中β-actin基因转录水平来校正目标基因相对表达水平；其次将野生型植株花后28天籽粒目标基因相对表达量设为1，计算不同基因型植株籽粒不同发育时期目标基因的相对表达量。
表2 Lcye基因的qRT-PCR引物信息
测定F2群体中纯合突变型、杂合突变型与野生型植株籽粒黄色素含量，分析突变位点对LCYE蛋白功能的影响。籽粒黄色素含量的测定参照AACC方法14-50，稍作改动。称取1g全麦粉，用水饱和正丁醇溶液(体积比5:1)振荡提取1h；5000rpm离心10min。应用吸收光酶标仪(Absorbance Microplate Reader；http://www.moleculardevices.com)测定上清液在436.5nm处吸光值，计算黄色素含量。每个样本3次技术重复，黄色素含量用平均值±SE表示。
5、LCYE功能结构域预测
利用NCBI(National center for biotechnology information；http://www.ncbi.nlm.nih.gov/)数据库，获得27个物种(表3)Lcye基因的cDNA序列，利用MEMESuite 5.1.0(http://meme-suite.org/)预测LCYE功能结构域，分析各突变位点在结构域中的分布情况。
表3 27个物种Lcye基因cDNA序列信息
6、统计分析
应用Student’s t检验对F2群体中纯合突变型、杂合突变型和野生型植株籽粒Lcye基因表达差异和黄色素含量进行显著性分析。
二、结果
1、EMS突变体库筛选
应用TILLING技术，在2491份M2代EMS诱变群体中共检测到21个Lcye基因的突变植株(表4)。克隆测序和序列分析显示，核苷酸从C到T的突变频率为57.14％，G到A的突变频率为38.10％，还检测到1个T到C的特异突变位点。根据突变位置分类，8个位于外显子区，13个分布于内含子区。外显子区域的点突变又分为6个错义突变和2个同义突变(图1)。
表4利用TILLING技术筛选获得Lcye突变体信息
注：Hom纯合突变型；Het杂合突变型。表中的各突变体，对于Lcye基因而言，仅存在对应的一处突变，即一个突变体不存在Lcye基因内的多处突变。
本发明采用常规聚丙烯酰氨凝胶分离CEL I酶切产物，片段两端0.15kb内的错配将超出检测范围，因此Lcye基因筛选的有效目标片段长度累计为2.24kb(表1)。筛选群体包含2491个M2株系，推断Lcye基因在该EMS诱变群体中的突变密度为1/266.1kb。
2、突变位点对蛋白质功能影响预测
PARSESNP软件分析结果显示，错义突变体M090815(C2202T)和M091648(G3284A)的PSSM值分别为26.9和27.7，SIFT值均为0，推断这些突变位点可能对蛋白功能产生严重影响(表4)。
表4突变位点对蛋白功能影响严重度预测
注：错义突变体M090815(C2202T)中突变后的Lcye-D1基因的基因组序列如SEQ IDNo.2所示，SEQ ID No.2编码SEQ ID No.1所示的突变后Lcye-D1蛋白。
3、突变体Lcye基因表达和黄色素含量分析
将纯合错义突变体与野生型植株杂交，构建6个F2代群体，分析突变位点对Lcye基因表达水平和籽粒黄色素含量的影响。结果表明，6个F2代群体中，仅M090815(C2202T)突变位点显著降低了Lcye基因表达和黄色素含量(图2和图3)，表明该突变位点对LCYE蛋白功能产生重要影响。
Lcye及其同源基因表达水平如图2所示，花后7-21天，纯合突变型植株籽粒Lcye基因总表达量降低至野生型的9％-83％，杂合型降低至71％-91％，花后28天各基因型表达差异不显著。Lcye-B1和Lcye-D1基因表达显示相似的趋势，花后各时期，纯合突变型植株表达量降低至野生型的56％-92％，杂合型降低至75％-90％；但Lcye-B1纯合突变型在花后28天显著高于野生型。Lcye-A1在花后7天，各基因型表达量未见显著性差异，花后14-28天，表现出补偿效应，纯合突变型比野生型表达量高33％-70％，杂合型高1％-48％。相应地，在突变体M090815构建的F2群体中，纯合突变型植株成熟籽粒黄色素含量显著低于杂合突变型和野生型(图3，1.63vs 1.90和2.02)。
4、LCYE功能结构域预测
基于拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa)、大麦(Hordeumvulgare)等27个物种已知的Lcye基因cDNA序列，利用MEME Suite 5.1.0预测LCYE功能结构域，共检测到3个结构域(图4)。6个错义突变中，M090815(C2202T)和M092230(G2195A)突变位点位于结构域1内，且M090815突变位点在27个物种中保守存在，M092230突变位点存在G、A和U三种碱基变异。
三、讨论
1、LCYE调控小麦籽粒类胡萝卜素合成
小麦籽粒类胡萝卜素含量影响面制品营养品质和表观色泽。类胡萝卜素含量和组成在发育过程中是动态变化的，显示出复杂的网络调控机制(Howitt等，2009)。番茄红素环化是类胡萝卜素合成途径中重要的分节点，经两条途径形成不同的产物：一条经β-β途径生成玉米黄质、环氧玉米黄质和堇菜黄质等类胡萝卜素；另外一条β-ε途径最终生成叶黄体素。普通小麦籽粒发育早期β-β途径类胡萝卜素产物占有较高表达水平，随着籽粒生长发育，表达量及所占比例逐渐降低，成熟籽粒中几乎不含有玉米黄质、环氧玉米黄质和堇菜黄质等类胡萝卜素。相反，叶黄体素的积累在籽粒发育各时期均表现较稳定的状态，是构成成熟籽粒类胡萝卜素的主要成分(Howitt等，2009)。
通过调节LCYB和LCYE的相对活性和相对含量可以决定底物转化为α-胡萝卜素和β-胡萝卜素的比例。在番茄Delta突变体中(Hornero-Mendez等，2000)，因Lcye转录水平的提高，果实内δ-胡萝卜素含量大量增多；而在番茄Beta突变体中，因Lcyb过表达，果实中累积大量的β-胡萝卜素。对比两种番茄突变体的差异进一步证实番茄果实中类胡萝卜素的积累主要是相关基因在转录水平上差异表达的结果(Ronen等，2000)。
TILLING技术实现了高频率点突变与现代分析技术有效结合，不仅可获得一系列的点突变等位基因-，而且仅需较小的突变群体就可筛选到目标基因的突变体(Till等，2003b)。Richaud等(2018)应用TILLING技术，筛选硬粒小麦Lcye突变体，W437*(Lcye-A1)突变位点显著增加突变体叶片中β-胡萝卜素和类胡萝卜素的含量，但对籽粒中类胡萝卜素含量无显著影响。本发明利用TILLING技术筛选普通小麦EMS突变体库，获得Lcye基因一系列等位变异，研究其对基因表达和籽粒黄色素含量的影响，为基因功能研究和面制品颜色性状改良奠定理论基础和提供重要种质资源。2、Lcye突变体筛选
TILLING技术有效结合了化学诱变的高频率点突变及快速简便的突变体检测技术，能够快速有效地从突变群体中检测目的基因的点突变，逐渐成为植物功能基因组学、作物遗传育种以及自然资源遗传多样性评估等研究的重要手段(Gilchris和Haughn，2005；Till等，2018)。目前，该技术已广泛应用于水稻(Till等，2007)、玉米(Till等，2004)、小麦(Acevedo-Garcia等，2017；Kim等，2018)、大麦(Gottwald等，2009)、大豆(Hoshino等，2010)、高粱(Nida等，2016)等20多种作物。
小麦基因突变频率远远高于拟南芥、水稻和玉米等植物。TILLING技术在小麦等麦类作物中的应用潜力更为巨大、前景更为光明。对于基因组复杂的多倍体物种，基因多拷贝是限制TILLING技术高效应用的一个瓶颈。设计特异性引物至关重要，否则会影响检测效率。小麦Lcye基因A、B和D同源基因间序列相似性非常高(89.1％-97.0％)，设计特异性标记非常困难。结合CODDLE程序给出的EMS诱导植株产生有害突变的可能范围，最终筛选到4对引物用于Lcye突变体检测(表1)，共筛选到21个Lcye突变位点，G/C变为A/T的突变频率为95.24％，T到C的突变频率为4.76％。
酶切产物的检测方法有多种，一般采用高效液相色谱技术、聚丙烯酰胺凝胶电泳、琼脂糖凝胶电泳以及双色红外荧光检测技术(Colbert等，2001；Dong等，2009；Uauy等，2009；Colasuonno等，2016)。本发明采用非变性聚丙烯酰胺凝胶电泳检测技术，既不使用荧光标记引物，又不使用昂贵的变性凝胶电泳成像系统，有效地简化了实验流程，降低了实验成本，在一定程度上扩大TILLING的应用范围和工作效率。
3、影响LCYE功能的重要调控位点
基于拟南芥、水稻、大麦等27个物种Lcye的cDNA序列，利用MEME预测LCYE功能结构域，结果显示M090815(C2202T)和M092230(G2195A)突变位点恰好位于结构域1内(图4)。M092230(G2195A)突变位点在自然界物种进化中存在G、A和U三种碱基变异，因此推断G到A核苷酸突变对LCYE功能未产生影响。而M090815(C2202T)突变位点在27个物种中非常保守，推断该位点对LCYE功能具有重要影响。M090815构建的F2群体中，与野生型植株相比，纯合突变型植株籽粒黄色素含量显著下降(图3)，表明M090815(C2202T)突变位点对LCYE功能具有重要影响。
PARSESNP软件预测显示M090815(C2202T)和M091648(G3284A)突变位点可能对蛋白功能产生严重影响(表4)。进一步证实M090815(C2202T)突变位点对LCYE蛋白功能的重要性。然而在M091648构建的F2群体中纯合突变型与野生型植株相比籽粒黄色素含量差异并不显著。这种不一致性可能是由于PARSESN软件是基于序列同源性及氨基酸物理特性进行突变位点对蛋白功能影响严重度的预测，而有些位点可能并不参与蛋白功能调控(Ng和Henikoff，2003；Taylor和Greene，2003)。
对M090815构建的F2群体进行Lcye及其同源基因表达水平分析显示，与野生型植株相比，纯合突变型植株籽粒Lcye基因总表达量显著降低(图2)。籽粒发育各时期，除Lcye-B1纯合突变型表达水平在花后28天高于野生型外，Lcye-B1和Lcye-D1基因表达水平均呈现下降趋势。而Lcye-A1在花后7天，各基因型表达量未见显著性差异，花后14-28天，表现出补偿效应。推测Lcye-B1和Lcye-D1基因表达协同调控，而Lcye-A1表达调控机制可能与之不同。鉴于上述结果只通过一个F2群体获得，因此该推测仍需进行后续验证。序列分析发现，Lcye-B1和Lcye-D1基因序列相似性达97.0％，而Lcye-A1与Lcye-B1和Lcye-D1的序列相似性为89.1％和89.2％，为上述推测提供间接支持。
4、分子育种
种质资源缺乏成为小麦面粉及其制品颜色改良的“瓶颈”，严重影响我国小麦品种面粉颜色性状的遗传改良。EMS诱变可以创造大量的等位变异，且在后代稳定遗传，由于不涉及转基因操作，获得的优异突变体可以直接用于育种实践(侯彩玲等，2008；Slade等，2012)。在我国，面制品以蒸煮为主，细腻洁白的面粉备受消费者青睐。本研究采用TILLING技术筛选获得的显著降低籽粒黄色素含量的M090815突变体，可作为面粉颜色遗传改良的重要种质资源，此外，济麦20为高产优质面包、面条兼用型强筋小麦；济麦22为超高产稳产广适多抗小麦新品种，两个均为大面积推广的优良品种，综合性状良好。以这两个品种作诱变基础材料创制的优异突变体，更适宜于用作杂交亲本，以期快速地培育出高产优质小麦新品种。
<110> 山东省农业科学院作物研究所
<120> 小麦类胡萝卜素合成途径关键基因Lcye突变体及其应用
<130> GNCLN200630
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 536
<212> PRT
<213> Artificial sequence
<400> 1
Met Glu Ser Thr Gly Ala Ala Ile Ser Ala Pro Phe Gly Cys Arg Ala
1 5 10 15
Leu Arg Trp Ala Gly Gln Arg Pro Leu Arg Pro Ala Asp Gly Arg Arg
20 25 30
Arg Arg Val Gly Pro Gly Pro Gly Pro Glu Lys Trp Arg Ser Leu Lys
35 40 45
Ala Ser Cys Val Ala Thr Glu Lys Pro Asp Glu Lys Ala Ala Pro Gly
50 55 60
Leu Gly Val Glu Phe Ala Asp Glu Glu Asp Tyr Val Lys Gly Gly Gly
65 70 75 80
Gly Glu Leu Leu Tyr Val Gln Met Gln Ala Thr Lys Ala Met Glu Ser
85 90 95
Gln Ser Lys Ile Ala Ser Lys Leu Leu Pro Ile Ala Asp Glu Thr Ser
100 105 110
Val Leu Asp Leu Val Ile Ile Gly Cys Gly Pro Ala Gly Leu Ser Leu
115 120 125
Ala Ala Glu Ser Ala Lys Lys Gly Leu Thr Val Gly Leu Ile Gly Pro
130 135 140
Asp Leu Pro Phe Thr Asn Asn Tyr Gly Val Trp Glu Asp Glu Phe Lys
145 150 155 160
Asp Leu Gly Leu Glu Ser Cys Ile Glu His Val Trp Lys Asp Thr Val
165 170 175
Val Tyr Leu Asp Arg Asn Lys Pro Ile Met Ile Gly Arg Ala Tyr Gly
180 185 190
Arg Val Asp Arg Asp Leu Leu His Glu Glu Leu Leu Arg Arg Cys Asn
195 200 205
Glu Ala Gly Val Thr Tyr Leu Asn Ser Lys Val Glu Gln Ile Lys Glu
210 215 220
Ser Pro Asp Gly His Arg Val Val Tyr Cys Gly Arg Gly His Lys Ile
225 230 235 240
Leu Cys Arg Leu Ala Ile Val Ala Ser Gly Ala Ala Phe Gly Lys Leu
245 250 255
Leu Glu Tyr Glu Val Gly Gly Pro Arg Val Cys Val Gln Thr Ala Tyr
260 265 270
Gly Val Glu Val Glu Val Glu Arg Tyr Pro Tyr Asp Pro Ser Leu Met
275 280 285
Val Phe Met Asp Tyr Arg Asp Cys Phe Lys Glu Lys Phe Thr His Pro
290 295 300
Glu Glu Ala Asn Pro Thr Phe Leu Tyr Ala Met Ala Met Ser Ser Thr
305 310 315 320
Arg Val Phe Phe Glu Glu Thr Cys Leu Ala Ser Lys Asp Ala Met Pro
325 330 335
Phe Asp Leu Leu Lys Lys Arg Leu Met Ser Arg Leu Asp Ala Met Gly
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Val Arg Ile Ile Lys Val Tyr Glu Glu Glu Trp Ser Tyr Ile Pro Val
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Gly Gly Ser Leu Pro Asn Thr Asp Gln Lys Asn Leu Ala Phe Gly Ala
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Ala Ala Ser Met Val His Pro Ala Thr Gly Tyr Ser Val Val Arg Ser
385 390 395 400
Leu Ser Glu Ala Pro Arg Tyr Ala Ser Val Ile Ser Asp Ile Leu Arg
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Asn Arg Val Tyr Ser Gly Gln Tyr Leu Pro Gly Ser Ser Glu Met Ser
420 425 430
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Arg Met Asn Leu Val Arg His Leu Leu Ser Asp Pro Thr Gly Ser Ala
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Met Ile Arg Thr Tyr Leu Thr Leu
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<210> 2
<211> 3739
<212> DNA
<213> Artificial sequence
<400> 2
atggagtcca ccggcgccgc catctcggcg ccgttcggct gccgcgcgtt gcgctgggcc 60
gggcagcggc ctctccggcc ggccgatggc aggagaaggc gggtgggtcc gggccccggg 120
ccggagaagt ggaggagctt gaaggcgagc tgcgtggcca cggagaagcc cgacgagaag 180
gcggcgccgg ggctaggggt ggagtttgcc gacgaggagg actacgtcaa gggcggcggc 240
ggtgagctgc tctacgtgca aatgcaggcc accaaggcca tggaaagcca gtccaagatc 300
gcctccaagg ttctctctct atccttttat gtactgctac cgctagtctg ctactgctag 360
tggtatcttg ttctttgttt tattccctta tttaattgct gatttctatg catgttttgt 420
tgatcttgaa tatactactc tagtacttaa gtgattcttt cccccaatta gcgttgtgtg 480
aacttccagc gttatggcga ataaaattaa aatccctaac attagtctgt ttctttcttg 540
cgatgtacag tagtaacatt agactgtttg aaagatgaag gattcccata aacaccacag 600
tagcaaaaat taatctgggt actattgcat actatactac tccctccgta tcaaaatata 660
agaccagaaa atacatagga gctcttgggt gctccacacc ctatatgaat attaaacgta 720
aaaaataccc aaaaaattaa aaaatctgaa atttggggat attaaacctg ggttcttaat 780
ctactcccgt gtgaaatttt gtgaaaaaat accagcacac gtatccgtgg cgaaggaaat 840
attgtccgaa caaaaatcca tccaaacagt tttttctata cataggaatt ttttgtcttt 900
tttgccacga atatgtttcc tggtattttt ttcacgaaat tttacacggg agtagatcgg 960
gaacccaggt ttgatatccc aaaatcttag ttttttttca aatttctcgg tattttttta 1020
aatttaatgt tcgtgtaggg gtgtggagca accgggtgct acaaatccgc ttccatccaa 1080
aggggtctta tatttggata tagagggagt agaacgcaag tggcattcaa atgcacgtca 1140
tgctagtact accatggcgt tgctcatttt tgacaaatca gggatagtct tgggtcgccc 1200
atgcttgtca ttcctgcctt cctggcagga gcagagtaca caatgtattt ggtcttgcgt 1260
taattaattg ctccttaggg acatgtcttt ggtcgtgcct ttgacttgct ttctttggaa 1320
acgccctttc actaagtata tcattcaata attcctacat agcaagacgc ggatcttagt 1380
gggttacgaa cgtcattgtg tacatgtttc aatccttctt ttactgatac tctgtacagt 1440
tctttttctt caaggttaat taattaagtt tcgaagttca tcgtcatttt gttaacaatt 1500
ttttgtctgg tagcatcctt caagtagttg attatgtctt ctgaatcttt ctttgcagct 1560
gttgcctata gctgatgaaa cttcagtgct tgatttggtt atcattggct gtggtccagc 1620
cggcctatct ctggctgcag aatcagcaaa gaaaggactc actgttggtc tcattggccc 1680
tgatcttcca ttcacaaaca attacggtgt atgggaggac gaattcaaag gtagccatta 1740
gtcgcagctg tgaaatgcag caccatgctt ggataacatc tttaccagca ttatcataaa 1800
gagtgatacg tttttatctt tttatttccc agatctcggc ctggagagct gtattgagca 1860
tgtatggaag gatactgtcg tgtaccttga ccgtaacaag ccgataatga ttggccgagc 1920
atatggccgg gtggaccgag acttgctgca cgaggagttg ctgagaaggt acattctatc 1980
ataacgatct tggaattcag tcgcgtcagg cagcatcata tatatgattg ttcctagtaa 2040
tgataattaa acaattggca gatgcaatga agctggtgtt acatacctca actcaaaggt 2100
tgaacagata aaagaatctc ctgatggaca tcgagtagta tattgtggaa ggggccacaa 2160
gatactttgc aggcttgcca ttgttgcatc tggagcagca tttggtaagc ttctagagta 2220
tgaggttgga ggaccacgtg tttgtgtgca gactgcatac ggtgtagaag tcgaggtaca 2280
caccaaccct gcacaaagtg ctccttctga ttcgtggttt atttcatgat ttgcatgtca 2340
cttcattcat gtcattcgag ctgagaaatt catatgttgt ttgtgcacca gttcaatttt 2400
tcagaatgat gtcaaaattt caggttgaaa gatatccgta cgatcccagc ttaatggttt 2460
tcatggacta cagagattgt ttcaaagaga agttcacaca ccctgaggaa gccaatccaa 2520
catttctcta tgccatggcc atgtcatcta cacgagtttt ctttgaggtc tacatgaatt 2580
tttttagcag tgtgtttctg caatggacac gtttccataa ggataggtca ctgaccaaga 2640
taatttatca taggaaacat gcttagcttc aaaagatgca atgccttttg atctccttaa 2700
gaagaggttg atgtctcggt tggatgcgat gggagttcgt atcataaaag tatacgagga 2760
ggtaacgagt ttggagttga tatccatact ggtttatctt gcacaggtgt gctgaatttc 2820
tgttgagtct tgatttcagg agtggtctta tattcctgtt ggaggatcct tacctaacac 2880
agaccagaag aatcttgcat ttggtgctgc agcgagtatg gtccatcctg caactggtac 2940
atacaaatcc tcaactttaa ctcgtcattc tttattattt gacatgcata ttgacaatat 3000
tgtagaaaat tcataggata ttcggtggtc agatctttgt ctgaagctcc tagatatgct 3060
tctgtgatat ctgatatctt acgaaatcgt gtctattctg gacaatattt gcctggaagt 3120
tctgaaatgt ccagtccatc aatgcttggt acagatttct tcccttgttc actaaattca 3180
acagatagaa taaaatcact taaagatgtt ttgcagaata ggagaaatga gcagaagtat 3240
ctgctgctaa ccactgtctg atatttcagc atggggaaca ctatggcctc aagaacggaa 3300
acgtcagcgc tcattcttcc tctttggatt ggccttgata attcaactgg ataacgaagg 3360
cattcaaaca ttcttcgaga gctttttccg gttacccaaa tggtaattat gcccgctcga 3420
tgcatttact gcttgttttc aggccctgga ccaagtattt cctttttcgc gaatacaata 3480
cgcaaggatg cgtatctttc cattgacaga aaaagtttga acaagtaaat atttccttgc 3540
gcgtgacttt tgatggatcg atctcttgaa caggatgtgg cgaggattcc ttggttcgac 3600
gctttcgtca gcggatctca tgctgtttgc actctacatg tttgcaattg cgccaaacac 3660
tttgcgaatg aacctcgtca gacacctcct ctcggacccg actggttcgg caatgatcag 3720
gacctacctg accttgtaa 3739
Claims (10)
1.蛋白质，为如下任一：
(A1)将Lcye-D1蛋白的第253位的丝氨酸替换为苯丙氨酸后得到的；
(A2)将(A1)所限定的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同能力的由(A1)衍生的蛋白质；
(A3)与(A1)-(A2)中任一所限定的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白质；
(A4)在(A1)-(A3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
2.根据权利要求1所述的蛋白质，其特征在于：所述(A1)所示蛋白质为由SEQ ID No.1所示的氨基酸序列组成的蛋白质。
3.编码权利要求1或2所述蛋白质的核酸分子。
4.根据权利要求3所述的核酸分子，其特征在于：所述核酸分子为编码权利要求1或2所述蛋白质的基因，所述基因为如下任一所示的DNA分子：
(B1)SEQ ID No.2所示的DNA分子；
(B2)在严格条件下与(B1)限定的DNA分子杂交且编码权利要求1或2所述蛋白质的DNA分子；
(B3)与(B1)或(B2)限定的DNA序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且编码权利要求1或2所述蛋白质的DNA分子。
5.含有权利要求3或4所述核酸分子的重组载体、表达盒、转基因细胞系或重组菌。
6.权利要求1或2所述的蛋白质或权利要求3或4所述的核酸分子或权利要求5所述的重组载体、表达盒、转基因细胞系或重组菌在如下任一中的应用：
(C1)下调小麦Lcye基因总表达量；或制备用于下调小麦Lcye基因总表达量的产品；
(C2)下调小麦Lcye-B1基因和/或Lcye-D1基因表达量；或制备用于下调小麦Lcye-B1基因和/或Lcye-D1基因表达量的产品；
(C3)降低小麦籽粒黄色素含量；或制备用于降低小麦籽粒黄色素含量的产品。
7.一种降低小麦籽粒黄色素含量的方法，包括如下步骤：仅将受体小麦基因组中编码Lcye-D1蛋白的第253位的丝氨酸的密码子替换为编码苯丙氨酸的密码子。
8.根据权利要求7所述的方法，其特征在于：所述将受体小麦基因组中编码Lcye-D1蛋白的第253位的丝氨酸的密码子替换为编码苯丙氨酸的密码子为将所述受体小麦基因组中编码Lcye-D1蛋白的基因替换为编码由SEQ ID No.1所示的氨基酸序列组成的蛋白质的基因。
9.根据权利要求8所述的方法，其特征在于：所述编码由SEQ ID No.1所示的氨基酸序列组成的蛋白质的基因为SEQ ID No.2所示DNA分子。
10.如下任一应用：
(D1)权利要求1或2所述的蛋白质或权利要求3或4所述的核酸分子或权利要求5所述的重组载体、表达盒、转基因细胞系或重组菌或权利要求6所述应用或权利要求7-9中任一所述方法在小麦面粉或面制品颜色改良中的应用；
(D2)利用权利要求7-9中任一所述方法培育得到的籽粒黄色素含量降低的小麦品种在小麦育种中的应用。
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| CN119913161A (zh) * | 2024-10-31 | 2025-05-02 | 中国科学院分子植物科学卓越创新中心 | 调控玉米籽粒质地和类胡萝卜素合成的基因 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113801871A (zh) * | 2021-10-09 | 2021-12-17 | 中国农业科学院作物科学研究所 | SiLCYE调控玉米黄质等谷子类胡萝卜素合成代谢的功能及应用 |
| CN113801871B (zh) * | 2021-10-09 | 2022-09-27 | 中国农业科学院作物科学研究所 | SiLCYE调控玉米黄质等谷子类胡萝卜素合成代谢的功能及应用 |
| CN119913161A (zh) * | 2024-10-31 | 2025-05-02 | 中国科学院分子植物科学卓越创新中心 | 调控玉米籽粒质地和类胡萝卜素合成的基因 |
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| Publication number | Publication date |
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| CN111171129B (zh) | 2022-02-01 |
| US20210355477A1 (en) | 2021-11-18 |
| US11661594B2 (en) | 2023-05-30 |
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