CN117925646A - 甜瓜中抗旱相关基因CmPPR及其用途 - Google Patents
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Abstract
本发明公开了一个甜瓜中抗旱相关基因CmPPR及其用途，本发明通过利用苗期干旱复水存活率差异明显的甜瓜种质构建RIL遗传群体，针对抗旱性指标进行精细定位，发现一个五肽重复序列超家族蛋白(PPR)基因，该基因与甜瓜抗旱相关，其编码区在抗旱种质和不抗旱种质间存在一个非同义突变(Nonsynonymous SNP)，经验证该位点突变与甜瓜抗旱性密切相关。基于该SNP开发分子标记CmDSR标记和引物对，能用于甜瓜节水抗旱型的分子辅助育种。
Description
技术领域
本发明属于蔬菜品质性状分子标记开发和分子标记辅助育种技术领域，涉及甜瓜苗期抗旱性相关基因及辅助育种，具体涉及一个甜瓜中抗旱相关基因CmPPR及其用途。
背景技术
干旱严重胁迫植物的生长发育，是限制产量的主要环境因子。甜瓜作为葫芦科三大经济作物之一，广受世界范围内消费者的喜爱。露天栽培时的田间干旱以及设施栽培时由于灌水不足或灌溉不及导致的干旱会严重影响收获甜瓜的产量和品质。当前对于甜瓜干旱条件下生理生化特性的研究已较为全面与深入，但是从分子层面切入而进行的遗传基础上的研究还是以反向遗传学的思路为主，鲜有利用正向遗传学手段对甜瓜抗旱相关基因进行定位与功能验证的报道。挖掘定位甜瓜抗旱相关遗传变异位点并加以功能验证，开发对应的分子标记，对于提高育种效率、规避育种盲目性，促进节水抗旱型绿色农业的发展有着重要意义。
近年来，在对植株进行准确抗旱性评价的基础上，借助基因编辑等分子技术以及生物信息学手段，以模式植物拟南芥、水稻、番茄等为典型的部分抗旱相关基因及其机制被解析，其抗旱响应通路也越发明晰。围绕各抗旱指标，与干旱胁迫相关的脱落酸、细胞分裂素、生长素、赤霉素等植物内源激素，WRKY、MYB、NAC等转录因子以及水通道蛋白、过氧化物酶、胚胎晚期富集蛋白（LEA）等蛋白进行的基因组学、转录组学、蛋白组学以及代谢组学等多组学的研究使得我们对植物在干旱胁迫下的响应机制越发清楚，但仍有许多未知通路，还需更深入的研究。
21世纪初至如今，有许多关于甜瓜不同生育时期受到干旱胁迫时的外部形态、内部结构以及生理生化特性的研究。随着甜瓜基因组的解析完成（Argyris J.M., Ruiz-Herrera A., Madriz-Masis P., Sanseverino W., Morata J., Pujol M., Ramos-Onsins S.E., Garcia-Mas J. (2015) Use of targeted SNP selection for animproved anchoring of the melon (Cucumis melo L.) scaffold genome assembly.BMC Genomics, 16: 4），邢巧娟等人通过对PEG模拟干旱胁迫下编码应答非生物胁迫的脂氧合酶相关基因CmLOX01-18在甜瓜内的表达模式进行研究筛选出CmLOX08、CmLOX10和CmLOX13三个基因，并利用沉默与过表达对其功能进行验证，通过对其启动子进行分析发现存在脱落酸（ABA）、水杨酸（SA）以及茉莉酸甲酯（MeJA）响应元件，进一步揭示其干旱胁迫下的响应机制（邢巧娟. (2020). CmLOX10和CmLOX13在调控薄皮甜瓜耐旱及抗白粉病中的作用机制. 沈阳农业大学）。2017年土耳其研究团队对甜瓜干旱条件下LEA基因家族进行了全基因组以及转录组分析，进一步确认了LEA蛋白与甜瓜抗逆胁迫的关联性（Altunoglu, Y.C., Baloglu, M. C., Baloglu, P., Yer, E. N., Kara, S.. (2017). Genome-wideidentification and comparative expression analysis of lea genes in watermelonand melon genomes. Physiology and Molecular Biology of Plants, (1), 23）。上述研究对于甜瓜抗旱性的相关研究主要集中在筛选抗旱指标，研究植株生理生化变化，再利用反向遗传学手段筛选对应相关基因，但尚无通过正向遗传学手段对甜瓜抗旱基因进行定位的报道。故而亟待利用遗传图谱对甜瓜抗旱基因进行挖掘定位以开发切实可靠的分子标记。
发明内容
本发明要解决的技术问题是提供一个甜瓜中抗旱相关基因CmPPR及其用途。
本发明的第一方面，提供了一个甜瓜中抗旱相关基因CmPPR，该基因与苗期干旱复水存活率相关，所述基因的核苷酸序列如SEQ ID NO.1所示。
本发明的第二方面，还同时提供了上述甜瓜中抗旱相关基因CmPPR编码的蛋白质，该蛋白质的氨基酸序列如SEQ ID NO.2所示。
本发明的第三方面，还同时提供了鉴定甜瓜中抗旱相关基因CmPPR的分子标记CmDSR(与甜瓜抗旱性CmPPR基因有关的分子标记CmDSR (drought survival rate))的引物对，包括：
Pa型正向引物：
5’-GAAGGTGACCAAGTTCATGCTCTGGCTAAAACAACGTAGCCAG-3’
Pb型正向引物：
5’-GAAGGTCGGAGTCAACGGATTCTGGCTAAAACAACGTAGCCAC-3’
反向引物：
5’-CAGGCTTGTAACCAATTGACTGCAT-3’
进一步地，引物(分子标记)可委托上海桑尼生物科技有限公司合成，在ABIVeriti96PCR仪上进行PCR扩增，并利用ABI公司Stepone荧光定量PCR仪检测荧光信号并分析基因分型。
本发明的第四方面，还同时提供了上述引物对的用途：用于甜瓜抗旱系或其后代辅助选择育种。具体为：
（1）提取待测甜瓜品种基因组DNA；
（2）利用所述的引物对对待测甜瓜基因组DNA进行PCR扩增；
（3）根据PCR产物荧光信号的差异，利用Kraken软件，检测PCR扩增结果的荧光信号判断待测甜瓜是否抗旱，其中，若PCR扩增结果荧光信号颜色与Pa型正向引物的荧光接头颜色一致，则待测甜瓜为纯合aa型基因型，表型为抗旱型，若PCR扩增结果荧光信号颜色与Pb型正向引物的荧光接头颜色一致，则待测甜瓜为纯合bb型基因型，表型为不抗旱；若PCR扩增结果荧光信号颜色均与Pa型正向引物、Pb型正向引物的荧光接头颜色不同，则为杂合ab基因型。
进一步地，所述步骤（2）中，PCR扩增时，PCR反应体系为：20～50ng/μl甜瓜基因组DNA 5.0μl，KASP Master Mix 5.0μl，KASP Assay Mix(Pa型正向引物： Pb型正向引物：反向引物＝2:2:5的摩尔浓度比)0.14μl，共10.14μl；
PCR程序为：94℃预变性，15分钟；94℃，20秒(变性) ；61℃(-0 .6℃/循环)退火60s， 10个循环；再94℃变性20s，55℃退火60s，26个循环。
进一步地，扩增结束后，利用BMG PHERAstar仪器检测荧光信号并查看分型情况。若分型不充分，则继续扩增，每3个循环查看分型情况，直至分型完全，从Kraken软件中导出实验结果。
按照本发明的设定，扩增26个循环，即能实现分型完全。
本发明的有益效果是：
发明人经过多年研究，发现甜瓜苗期干旱复水存活率与抗旱性高度相关。通过利用苗期干旱复水存活率差异的甜瓜种质构建RIL遗传群体，针对抗旱性指标进行精细定位，发现一个五肽重复序列超家族蛋白(PPR)基因，该基因与甜瓜抗旱相关，其编码区在抗旱种质和不抗旱种质间存在一个非同义突变(Nonsynonymous SNP)，经验证该位点突变与甜瓜抗旱性密切相关。基于该SNP开发分子标记CmDSR标记和引物对，能用于甜瓜节水抗旱型的分子辅助育种。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1为抗旱甜瓜Pa和不抗旱甜瓜Pb中抗旱基因CmPPR的部分基因组序列对比图。
图2是不同抗旱性甜瓜杂交RIL后代的CmDSR标记分型图；图中蓝色对应圆点代表纯合aa型基因型；红色对应圆点代表纯合bb型基因型；绿色对应圆点代表杂合ab基因型；黑色x标记代表无样品的对照；分型结果图形分区界限明显，说明该kasp标记分型可靠。
图3是甜瓜RIL群体中不同基因型的苗期干旱复水存活率分布统计图；结果表明含有纯合aa型基因型的甜瓜苗期干旱复水存活率普遍高于含有bb型基因型的甜瓜。
图4是甜瓜RIL群体的苗期干旱复水存活率排序；结果表明供试甜瓜种质的苗期干旱复水存活率表型与CmDSR标记的基因分型存在极大关联性。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1、甜瓜中抗旱相关基因CmPPR获取
本发明的与甜瓜苗期干旱复水存活率有关的分子标记CmDSR，具体采用下述方法获得：
1)、以获取自中国作物种质资源信息库的抗旱甜瓜Pa(XLH)与不抗旱甜瓜Pb(HPSG)为亲本进行杂交得到的F1子代自交，随后F2子代运用单粒子传法进行繁种，构建可稳定遗传的永久性作图群体-重组自交系群体（Recombinant Inbred Lines, RIL），从而获得后代的抗旱/不抗旱单株；
2)、用CTAB(十六烷基三乙基溴化铵，Hexadecyl trimethyl ammonnium Bromide)法提取甜瓜亲本幼苗及杂交后代RIL群体幼苗基因组DNA；
3)、采用GBS（Genotyping by sequencing）测序技术对RIL群体进行重测序，并由测序得到的基因型，构建子代重组物理图谱。之后用WinQTLcart软件进行QTL定位；
4)、采用关联分析鉴定与抗旱性相关的基因；
5)、克隆到一个甜瓜中抗旱相关基因CmPPR。
6)、比较获取自中国作物种质资源信息库的抗旱甜瓜Pa和不抗旱甜瓜Pb中抗旱基因CmPPR的基因组序列，其中，抗旱甜瓜Pa中抗旱基因CmPPR的基因组序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO.2所示；不抗旱甜瓜Pb中抗旱基因CmPPR的基因组序列如SEQID NO.3所示，氨基酸序列如SEQ ID NO.4所示；鉴定到抗旱基因CmPPR的编码区在抗旱甜瓜Pa和不抗旱甜瓜Pb间存在一个非同义突变，即SNP变异；
上述SNP变异具体为：C（Pa）/ G（Pb），导致谷氨酰胺替换为组氨酸，如图1所示。
7)、基于抗/不抗旱甜瓜中CmPPR基因的SNP变异，在SNP附近区域设计一组引物(3条)，具体如下：
Pa型正向引物（SEQ ID NO.5）：
GAAGGTGACCAAGTTCATGCTCTGGCTAAAACAACGTAGCCAG
Pb型正向引物（SEQ ID NO.6）：
GAAGGTCGGAGTCAACGGATTCTGGCTAAAACAACGTAGCCAC
反向引物（SEQ ID NO.7）：
CAGGCTTGTAACCAATTGACTGCAT
其中，2个正向引物前分别设置各自的荧光接头(为不同颜色)，如果检测的材料是纯合基因型，扩增的时候只会选其中对应的一个引物扩增 (例如，纯合aa型只能与Pa型正向引物发生反应) ，根据荧光的差异，分辨出所测材料的基因型是纯合aa型还是纯合bb型，如果检测的材料是杂合型的，扩增时2个引物都会被用到，产生的荧光不同于纯合基因型的材料，从而实现区分杂合的基因型。
实施例2、用分子标记鉴定抗旱型甜瓜与非抗旱型甜瓜的序列差异：
1)、用CTAB法提取甜瓜亲本幼苗（抗旱甜瓜Pa和不抗旱甜瓜Pb）及70份RIL后代幼苗基因组DNA；
2)、采用PCR方法进行甜瓜抗旱CmPPR基因标记的筛选；
直接在ABI公司Stepone荧光定量PCR仪上进行PCR反应，所述PCR仪自带荧光分析软件，从而直接获得分析结果，即，软件自动将检测样品按照不同的基因型分成纯合aa型基因型，纯合bb型基因型和杂合ab基因型。
PCR反应体系为：20～50ng/μl甜瓜基因组DNA 5.0μl，KASP Master Mix 5.0μl，KASP Assay Mix(Pa型正向引物： Pb型正向引物：反向引物＝2:2:5的摩尔浓度比)0.14μl，共10.14μl；
PCR程序为：94℃预变性，15分钟；94℃，20秒(变性)；61℃(-0.6℃/循环)退火60s，10个循环；再94℃变性20s，55℃退火60s，26个循环。
结果如图2和表1所示，当软件分析出现与抗旱型甜瓜Pa同样颜色（蓝色）的圆点时，表示圆点对应的材料基因型为纯合aa型；当出现同非抗旱型甜瓜Pb同样颜色（红色）的圆点时，表示圆点对应材料的为纯合bb型；当出现位置居中(绿色)圆点时，表示圆点对应材料的基因型是杂合型的。黑色x标记表示NTC，即为水对照，结果显示，对待测甜瓜的分型效果很好。
3)、在人工气候室（参数：光照16h、28℃/黑暗20℃、8h）中将消毒催芽后的甜瓜亲本及70份RIL后代种子每粒一穴播种于含基质的50穴育苗盘中，待甜瓜长至三叶一心期时对其进行自然干旱或用20%PEG600模拟干旱胁迫处理7天，随后复水（一次2.5L，隔3天浇一次）7天统计存活率，存活率越接近100%抗旱性越强，存活率越接近0%抗旱性越弱。
所得结果如表1及图3和图4所示，可以看出几乎所有纯合aa型基因型的甜瓜株系的苗期干旱复水存活率显著高于纯合bb型基因基因型的甜瓜株系(图3)。将所有甜瓜基因型按照苗期干旱复水存活率数据降序排列后，两类基因型呈现明显的两端分离(图4)，上述结果表明本发明的CmDSR分子标记与甜瓜苗期干旱复水存活率高度连锁，说明了CmDSR分子标记的可靠性和可用性。
表1、使用分子标记CmDSR检测的70份重组自交系的甜瓜所得结果
最后，还需要注意的是，以上列举的仅是本发明的若干个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。
Claims (8)
1.一个甜瓜中抗旱相关基因CmPPR，其特征在于，所述甜瓜中抗旱相关基因CmPPR的核苷酸序列如SEQ ID NO.1所示。
2.权利要求1所述的甜瓜中抗旱相关基因CmPPR编码的蛋白质，其特征在于，所述蛋白质的氨基酸序列如SEQ ID NO.2所示。
3.鉴定甜瓜中抗旱相关基因CmPPR的分子标记的引物对，其特征在于，所述引物对包括：
Pa型正向引物：
5’-GAAGGTGACCAAGTTCATGCTCTGGCTAAAACAACGTAGCCAG-3’
Pb型正向引物：
5’-GAAGGTCGGAGTCAACGGATTCTGGCTAAAACAACGTAGCCAC-3’
反向引物：5’-CAGGCTTGTAACCAATTGACTGCAT-3’。
4.权利要求3所述的引物对在甜瓜抗旱系或其后代辅助选择育种中的用途。
5.根据权利要求4所述的用途，其特征在于，具体为：
（1）提取待测甜瓜品种基因组DNA；
（2）利用权利要求3所述的引物对对待测甜瓜基因组DNA进行PCR扩增；
（3）检测PCR扩增结果的荧光信号判断待测甜瓜是否抗旱，其中，若PCR扩增结果荧光信号颜色与Pa型正向引物的荧光接头颜色一致，则待测甜瓜为纯合aa型基因型，表型为抗旱型，若PCR扩增结果荧光信号颜色与Pb型正向引物的荧光接头颜色一致，则待测甜瓜为纯合bb型基因型，表型为不抗旱型。
6.根据权利要求5所述的用途，其特征在于，所述步骤（2）中，PCR扩增时，PCR反应体系为：20~50 ng/μL甜瓜基因组DNA5.0μL，KASP Master Mix5.0μL，KASP Assay Mix 0.14μL，共10.14μL；KASP Assay Mix中，Pa型正向引物：Pb型正向引物：反向引物的摩尔浓度比为2:2:5；Pa型正向引物、Pb型正向引物前分别设置各自的不同颜色的荧光接头。
7.根据权利要求5所述的用途，其特征在于，所述步骤（2）中，PCR扩增时，PCR程序为：94℃预变性，15分钟；94℃变性，20秒；按照-0.6℃/循环的要求61℃退火60s，10个循环；再94℃变性20s，55℃退火60s，26个循环。
8.一种鉴定甜瓜抗旱系的方法，其特征在于，具体为：
（1）提取待测甜瓜品种基因组DNA；
（2）利用权利要求3所述的引物对对待测甜瓜基因组DNA进行PCR扩增；
（3）检测PCR扩增结果的荧光信号判断待测甜瓜是否抗旱，其中，若PCR扩增结果荧光信号颜色与Pa型正向引物的荧光接头颜色一致，则待测甜瓜为纯合aa型基因型，表型为抗旱型，若PCR扩增结果荧光信号颜色与Pb型正向引物的荧光接头颜色一致，则待测甜瓜为纯合bb型基因型，表型为不抗旱型。
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