CN104404165A - 一种区分樱桃果实颜色的分子方法 - Google Patents
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Abstract
一种区分樱桃果实颜色的分子方法,该方法包括:A.取不同樱桃叶片,提取基因组DNA;B.采用PCR方法扩增基因LGS;C.电泳检测,在0.8%琼脂糖胶(EB)上电泳,紫外灯下观察扩增的DNA片段;D.对有条带的样品进行PCR产物测序;E.区分结果:没有扩增到LGS基因,该品种为黄色;能够扩增到完整LGS基因的为紫红色品种;能够扩增到LGS基因,但在1214bp位置上有一个碱基A发生缺失的,则为黄底红晕品种。该方法通过一个基因检测可以区分黄色、黄底红晕、紫红色樱桃三类品种,为樱桃育种服务,尤其为童期樱桃的快速选择提供技术支持。
Description
技术领域
本发明涉及一种区分樱桃果实颜色的方法,特别涉及一种区分樱桃果实颜色的分子方法。
背景技术
果实颜色是果实重要的外观品质,花青素是决定大多数果实颜色,也是果实成熟的标志。花青素又称花色素,是植物产生的次生代谢物质。花青素是一种水溶性色素,存在于植物中的特定组织和细胞中,如植物细胞的液泡内,花青素由外部和内部因素共同调控(Procissi等,Light-dependent spatial and temporalexpression of regulatory genes in developing maize seeds.The Plant Cell,1997,9:1547-1557;Cominelli E等,Expression analysis of anthocyanin regulatory genes inresponse to different light qualities in Arabidopsis thaliana.Journal of Plant Physiology,2008,165:886-894)。常见于花、果实的组织中及茎叶的表皮细胞与下表皮层,也是植物果实中的主要呈色物质(Allan等,MYB transcription factors that colour ourfruit.Trends in Plant Science,2008,13:99-102)。花青素对植物的生殖来说非常重要,由于鲜艳的花色可吸引昆虫授粉,鲜艳的果实也利于种子传播等(Jimenez-Garcia等,Vazquez-Cruz M A.Functional properties and qualitycharacteristics of bioactive compounds in berries:biochemistry,biotechnology,andgenomics.Food Research Internation,2013http://dx.doi.org/10.1016/i.foodres.2012. 11.004)。花青素也是果实成熟的标记(Allan等,MYB transcription factors thatcolour our fruit.Trends in Plant Science,2008,13:99-102;Jaakola,New insights intothe regulation of anthocyanin biosynthesis in fruits.Trends in Plant Science,2013,18:477-483)。
果实的颜色是自然选择和人工选择的结果(Chagné等,An ancient duplicationof apple MYB transcription factors is responsible for novel red fruit-flesh phenotypes.Plant Physiology,2013,161:225-239)。遗传决定了果实中的花青素种类,同时环境如光照、温度等影响花青素的积累。
樱桃(Prunus avium)是一种重要的果树,童期较长。另外,樱桃果实发育期又很短,从开花到果实成熟只需要1个多月。樱桃果皮颜色很丰富,有黄色、黄底红晕、紫红色等,在果实颜色的研究上基因(突变)是果实积累不同颜色的内在因素,基因如何区分不同果实颜色的樱桃关于这方面的研究还未见有相关报道。
发明内容
针对现有技术的上述问题,本发明提供了一种区分樱桃果实颜色的分子方法,通过一个基因检测可以区分黄色、黄底红晕、紫红色樱桃三类品种,为樱桃育种服务,尤其为童期樱桃的快速选择提供技术支持。
为实现上述目的,本发明包括如下技术方案:
一种区分樱桃果实颜色的分子方法,包括如下步骤:
A.取不同樱桃叶片,提取基因组DNA;
B.采用PCR方法扩增基因LGS;
C.电泳检测,在0.8%琼脂糖胶(EB)上电泳,紫外灯下观察扩增的DNA片段;
D.对有条带的样品进行PCR产物测序;
E.区分结果:没有扩增到LGS基因,该品种为黄色;能够扩增到完整LGS基因的为紫红色品种;能够扩增到LGS基因,但在1214bp位置上有一个碱基A发生缺失的为黄底红晕品种。
如上所述的区分樱桃果实颜色的分子方法,优选地,所述步骤B采用PCR方法扩增基因LGS的具体方法如下:
上游引物为ATGGAGGGTATAACTTGGGTGTGAGAA;
下游引物为TAGTCCTTCTGAACATTGGTACACTGTC;
PCR反应体系:基因组DNA模板3.5μL(20ng-100ng),10PCR缓冲液2.5μL,MgCl22.5μL,dNTPS1μL(2.5mM),上游引物1μL(10pM),下游引物1μL(10pM),Taq酶0.2μL(2U),加水补足至20μL;
PCR反应程序:94℃5min,(94℃30s,55℃30s,72℃60s)35个循环,72℃10min。
果实颜色为黄底红晕的樱桃的LGS基因,该基因在LGS基因1214bp位置上有一个碱基A发生缺失,其基因序列如序列表中SEQID NO.4所示,其氨基酸序列如序列表中SEQID NO.6所示。
本发明的有益效果在于:使用本发明的方法,在樱桃生长的童期,取一片叶子就可以的区分黄色果实樱桃、黄底红晕樱桃以及红紫色樱桃。该方法操作简便、易于推广,非常有利于进行樱桃辅助育种。
附图说明
图1为成熟时樱桃大龙,雷尼和拉宾斯果皮和果肉的颜色。
图2为提取的樱桃大龙,雷尼和拉宾斯的基因组DNA。
图3为PCR扩增产物的凝胶检测结果,大龙没有扩增该基因,雷尼和拉宾斯扩增到该基因。
图4为樱桃雷尼和拉宾斯基因组LGS基因序列以及比对结果。
图5为26份樱桃PCR扩增LGS基因的电泳结果。M为DNA Marker,1-26分别为Royal Ann、Chelan、Critalina、Sam、B.TAR、佳红、红南阳、彩霞、先锋、红蜜、Linda、Sonata、BlackYork、巨红、伯兰特、Santina、早大果、早丹、Vega、Black Gold、S-106、彩虹、133、大龙,雷尼和拉宾斯。
图6为樱桃LGS基因关键序列的测序结果。
图7为樱桃LGS基因关键序列的比对结果。
具体实施方式
下面结合附图和实施例对本发明作进一步说明。
实施例1用本发明的方法检测大龙,雷尼和拉宾斯的颜色
选取大龙,雷尼和拉宾斯叶片作为实验材料。大龙成熟果皮颜色为黄色,果肉黄色;雷尼成熟果皮为黄底红晕,果肉浅黄色;拉宾斯成熟果皮颜色为紫红色,果肉红色(见图1)。具体操作如下:
使用Biomiga的Plant gDNA kit提取樱桃大龙,雷尼和拉宾斯三种材料的基因组DNA,方法参考试剂盒,在0.8%琼脂糖胶(EB)上电泳,紫外灯下观察提取的基因组DNA(见图2)。
采用PCR方法扩增基因LGS,PCR反应体系:分别对大龙,雷尼和拉宾斯三种材料进行扩增。
上游引物为ATGGAGGGTATAACTTGGGTGTGAGAA(序列表NO.1);
下游引物为TAGTCCTTCTGAACATTGGTACACTGTC(序列表NO.2);
基因组DNA模板3.5μL(20ng-100ng),10PCR缓冲液2.5μL,MgCl22.5μL,dNTPS 1μL(2.5mM),上游引物1μL(10pM),下游引物1μL(10pM),Taq酶0.2μL(2U),加水补足至20μL;PCR反应程序:94℃5min,(94℃30s,55℃30s,72℃60s)35个循环,72℃10min;
樱桃大龙,雷尼和拉宾斯的扩增产物在0.8%琼脂糖胶(EB)上电泳,紫外灯下观察扩增的DNA片段,发现黄色的大龙没有扩增到这个基因,而黄底红晕的雷尼和紫红色的拉宾斯可以扩增到约1600bp的片断(见图3);
对以上PCR产物送交生工公司测序,测序结果和比对结果如图4,发现雷尼材料在1214bp位置上有一个碱基A缺失(箭头所指)。
该实验证实了:黄色品种(大龙)没有扩增到LGS基因,紫红色品种(拉宾斯)能够扩增到完整LGS基因(基因序列见序列表NO.3,氨基酸序列见序列表NO.5);黄底红晕品种(雷尼)能够扩增到LGS基因(基因序列见序列表NO.4,氨基酸序列见序列表NO.6),但在1214bp位置上有一个碱基A发生缺失。
实施例2用本发明的方法检测26份不同颜色的樱桃
保持其它条件不变,对26份不同颜色的樱桃进行了盲测,也就是由分子检测结果推测果实颜色,再以成熟果实颜色证实推测结果。具体操作如下:
使用Biomiga的Plant gDNA kit提取樱桃Royal Ann、Chelan、Critalina、Sam、B.TAR、佳红、红南阳、彩霞、先锋、红蜜、Linda、Sonata、BlackYork、巨红、伯兰特、Santina、早大果、早丹、Vega、Black Gold、S-106、彩虹、133、大龙,雷尼和拉宾斯26份叶片材料的基因组DNA,方法参考试剂盒。
采用PCR方法扩增基因LGS,PCR反应体系:分别取26份基因组DNA作为模板进行扩增。
上游引物为ATGGAGGGTATAACTTGGGTGTGAGAA;
下游引物为TAGTCCTTCTGAACATTGGTACACTGTC;
模板DNA 3.5μL(20ng-100ng),10PCR缓冲液2.5μL,MgCl22.5μL,dNTPS1μL(2.5mM),上游引物1μL(10pM),下游引物1μL(10pM),Taq酶0.2μL(2U),加水补足至20μL;PCR反应程序94℃5min,(94℃30s,55℃30s,72℃60s)35个循环,72℃10min;
26份樱桃扩增产物在0.8%琼脂糖胶(EB)上电泳,紫外灯下观察扩增的DNA片段。发现没有扩增到这个基因的是黄色樱桃133和大龙,而其余材料均可扩增到约1600bp的片断(见图5);
对以上PCR产物送交生工公司测序,测序结果在1214bp上有一个碱基的差异,发现Royal Ann、佳红、红南阳、B.TAR、彩霞、红蜜、巨红、彩虹与雷尼材料在相同位置上有一个碱基A的缺失(箭头所指)(图6和图7),与比对结果一致(图7),而这些材料的果实颜色均为黄底红晕。测序完整的材料Chelan、Sam、Critalina、先锋、Linda、Sonata、BlackYork、伯兰特、Santina、早大果、早丹、Vega、Black Gold、S-106,其果实颜色为紫红色,与拉宾斯一致。
以上结果进一步证实本发明方法的可靠性和科学性。用一个基因LGS可以区分樱桃,没有扩增到LGS基因,该品种果实为黄色;能够完整扩增到该基因时为紫红色果实樱桃;能够扩增到该基因,但有一个碱基A发生缺失,则为黄底红晕樱桃。
Claims (3)
1.一种区分樱桃果实颜色的分子方法,其特征在于,该方法包括如下步骤:
A.取不同樱桃叶片,提取基因组DNA;
B.采用PCR方法扩增基因LGS;
C.电泳检测,在0.8%琼脂糖胶(EB)上电泳,紫外灯下观察扩增的DNA片段;
D.对有条带的样品进行PCR产物测序;
E.区分结果:没有扩增到LGS基因,该品种为黄色;能够扩增到完整LGS基因的为紫红色品种;能够扩增到LGS基因,但在1214bp位置上有一个碱基A发生缺失的为黄底红晕品种。
2.如权利要求1所述的区分樱桃果实颜色的分子方法,其特征在于,所述步骤B采用PCR方法扩增基因LGS的具体方法如下:
上游引物为ATGGAGGGTATAACTTGGGTGTGAGAA;
下游引物为TAGTCCTTCTGAACATTGGTACACTGTC;
PCR反应体系:基因组DNA模板3.5μL(20ng-100ng),10PCR缓冲液2.5μL,MgCl22.5μL,dNTPS1μL(2.5mM),上游引物1μL(10pM),下游引物1μL(10pM),Taq酶0.2μL(2U),加水补足至20μL;
PCR反应程序:94℃5min,(94℃30s,55℃30s,72℃60s)35个循环,72℃10min。
3.果实颜色为黄底红晕的樱桃的LGS基因,其特征在于,该基因在LGS基因1214bp位置上有一个碱基A发生缺失,其基因序列如序列表中SEQID NO.4所示,其氨基酸序列如序列表中SEQID NO.6所示。
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TWI699373B (zh) * | 2018-12-21 | 2020-07-21 | 國立屏東科技大學 | 用以鑑別蓮霧之果皮顏色的引子對及方法 |
CN111088388A (zh) * | 2020-01-18 | 2020-05-01 | 中国农业科学院郑州果树研究所 | 一种用于鉴定桃果肉红/非红性状的InDel标记、引物对及其应用 |
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