CN116751887B - 棉花纤维强度性状关联的GhEIF5A基因的应用 - Google Patents
棉花纤维强度性状关联的GhEIF5A基因的应用 Download PDFInfo
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Abstract
本发明公开了一个棉花纤维强度性状关联的GhEIF5A基因的应用。所述的棉花纤维强度性状关联的基因GhEIF5A在四倍体陆地棉TM‑1中的cDNA序列为：SEQ ID NO.1，基因组序列为：SEQ ID NO.2；基因GhEIF5A受邻近SNP位点调控，该SNP位点碱基从T变为C；突变后的基因型纤维强度显著高于野生型。该基因在高效鉴定高纤维强度陆地棉品种、改良棉花品质性状和培育棉花高纤维强度新品种中具有重要的研究价值和应用前景。
Description
技术领域
本发明属于生物技术应用领域，涉及一个棉花纤维强度性状关联的基因GhEIF5A的应用。
背景技术
棉花(Gossypium spp.)是天然纺织纤维的主要来源，在国民经济中占有重要地位。实现棉纤维高产高品质是棉花育种的主要目标。随着棉纺织工业技术的进步、设备的更新换代以及消费市场对原棉纤维品质要求的不断提高，棉花纤维品质性状改良的要求日益迫切。棉花的纤维品质性状属数量性状，其表现型受基因型和环境共同影响，棉花纤维强度是重要的原棉品质指标之一，决定着成纱质量，挖掘棉花品种资源中高品质的优良基因对改良纤维强度、选育优质棉花品种具有重要的意义。
全基因组关联研究(GWAS:Genome-wide association studies)，也被称为连锁不平衡(LD)定位，利用一个物种内的表型和基因型变异来识别感兴趣的性状的遗传基础，提供了系统鉴定复杂性状的方法。目前在棉花的QTL和GWAS研究中鉴定到了大量控制纤维质量的QTL和GWAS位点：Fang(Fang,L.,Wang,Q.,Hu,Y.et al.Genomic analyses in cottonidentify signatures of selection and loci associated with fiber quality andyield traits.Nat Genet 49,1089–1098(2017))在258份陆地棉群体中鉴定到45个与纤维品质相关性状位点；Ma(Ma,Z.,He,S.,Wang,X.,Sun,J.,Zhang,Y.,Zhang,G.,et al.(2018).Resequencing a core collection of upland cotton identifies genomicvariation and loci influencing fiber quality and yield.Nat.Genet.50,803–813.doi:10.1038/s41588-018-0119-7)在419份陆地棉产量鉴定到6131个品质位点；Yuan(Yuan,Y.,Zhang,H.,Wang,L.,Xing,H.,Mao,L.,Tao,J.,et al.(2019).Candidatequantitative trait loci and genes for fiber quality in Gossypium hirsutuml.detected using single-and multi-locus association mapping.Indust.CropsProd.134,356–369)检测了196份棉花品种的41,815个SNPs，发现了40个SNPs、38个QTLs影响纤维性状；Wang(Wang,P.,He,S.,Sun,G.,Pan,Z.,Sun,J.,Geng,X.,et al.(2021).Favorable pleiotropic loci for fiber yield and quality in upland cotton(Gossypium hirsutum).Sci.Rep.11,15935)使用316份棉花的限制性位点DNA测序(Rad-seq)和特定位点扩增片段测序(SLAF-seq)，在27个基因组区域中，筛选231个SNP与纤维性状相关；Hu(Hu D,He S,Jia Y,Nazir MF,Sun G,Geng X,Pan Z,Wang L,Chen B,Li H,GeY,Pang B,Du X.Genome-wide association study for seedling biomass-relatedtraits in Gossypium arboreum L.BMC Plant Biol.2022Jan 27；22(1):54)收集215份棉种质，研究11个幼苗鲜重、干重、含水量和根冠比等生物量相关性状，利用142,5003个高质量SNPs的全基因组关联研究确定了83个显著关联和69个推定的候选基因；Li(Yiqian Li,Zhanfeng Si,Guoping Wang,Zhuolin Shi,Jinwen Chen,Guoan Qi,Shangkun Jin,ZegangHan,Wenhao Gao,Yue Tian,Yun Mao,Lei Fang,Yan Hu,Hong Chen,Xiefei Zhu,TianzhenZhang,Genomic Insights into the Genetic Basis of Cotton Breeding in China,Molecular Plant,2023,ISSN 1674-2052,https://doi.org/10.1016/j.molp.2023.01.012)选取16个在中国棉花育种中具有重要历史地位的陆地棉品种，构建MAGIC群体确定了54个与皮棉产量和纤维质量相关的基因组位点。遗传变异位点通过引起基因表达变异进而引起表型的变异，这些鉴定到的大量控制纤维质量的QTL和GWAS位点通常位于在染色体较大的连锁不平衡区段中。而通过基因表达数量位点关联研究(eQTL:Expression quantitative trait loci)将基因的表达和基因组进行关联，建立遗传变异和基因表达的相关性，可以把表型变异定位到单基因。
EIF5A属于真核生物起始因子5A(Eukaryotic translation initiation factor5A)基因家族，是唯一一个被认知的细胞内含有翻译后修饰形成氨基酸衍生物hypusine[Ne-(4-氨基丁基-2-羟丁基)赖氨酸]的蛋白，对细胞的持续性增殖起着关键的作用，一直受到广泛关注。EIF5A以蛋白质合成起始功能为基础，通过促进一些特异mRNA的转移及相关特异基因的表达来促进细胞的增值或者细胞的衰老、死亡，参与植物产生环境胁迫的应答和抵抗能力。
Kemper(1976)从兔子内质网膜上首次分离出EIF5A，酵母缺失EIF5A会导致致死。而植物EIF5A的研究起步较晚，Thompson(2004)从模式植物拟南芥中分离出3个AtEIF5A基因，AtEIF5A1参与次生木质部的形成，超量表达AtEIF5A1的转基因拟南芥木质部增大。AtEIF5A2主要在受机械损伤的组织中高水平表达，AtEIF5A3主要在细胞分裂很活跃的种子中表达，超量表达AtEIF5A3的转基因拟南芥叶子变为并生叶、种子变大、同时能够响应高盐、干旱以及渗透等胁迫信号。
发明内容
本发明的目的是针对现有技术的不足，提供一个与棉花纤维强度性状相关联的基因，全基因组关联分析和基因表达数量位点(eQTL)共定位分析表明该基因是棉花纤维强度的因果基因。
本发明的另一目的是提供该基因的应用。
本发明的目的可通过如下技术方案实现：
所述的棉花纤维强度性状相关联的真核生物起始因子5A基因GhEIF5A在四倍体陆地棉TM-1中的cDNA序列为：SEQ ID NO.1，基因组序列为：SEQ ID NO.2；该基因表达由SNP(A03:65556479)控制，该SNP位点碱基从T变为C，突变后基因型的棉纤维强度增加。
本发明所述的棉花纤维强度性状关联的GhEIF5A基因在鉴定高纤维强度陆地棉品种中的应用。
检测棉花基因组中控制GhEIF5A基因的SNP位点A03:65556479的碱基类型，其中碱基为C的棉花即为高纤维强度棉花品种。
本发明所述的棉花纤维强度性状关联的GhEIF5A基因在改良棉花品质性状中的应用。
本发明所述的棉花纤维强度性状关联的GhEIF5A基因在通过基因工程手段培育棉花高纤维强度新品种中的应用。具体地，可以通过基因工程的手段将含有高产单倍型GhEIF5A(C)的基因转入棉花品种中提高棉花产量，也可以将低产单倍型GhEIF5A(T)中的SNP位点进行定点突变，改造成高纤维强度单倍型从而培育高质棉花新品种。
一种用于检测所述的SNP位点的引物对，上游引物为：SEQ ID NO.3，下游引物为：SEQ ID NO.4。
所述的引物对在筛选高纤维强度棉花品种中的应用。
一种鉴定高纤维强度棉花品种的方法，检测棉花基因组中控制GhEIF5A基因的SNP位点A03:65556479的碱基类型，其中碱基为C的棉花即为高纤维强度棉花品种。
本发明的有益效果在于：
本发明利用棉花品种群体基因组重测序和棉花开花后20天的棉纤维基因表达数据，通过全基因组关联分析和表达数量标签位点共定位，挖掘了一个与棉花品质性状纤维强度相关联的GhEIF5A基因。GhEIF5A由SNP位点(A03:65556479)调控，本发明提供的包含检测SNP的基因组序列由PCR技术获得，该技术具有起始模板量小，试验步骤简单易行而且灵敏度高的优点。
GhEIF5A在相对高纤维强度和低纤维强度品种群体中的SNP基因型通过PCR技术进行了验证(表1)，较易操作、灵敏度高和准确性好。
根据调控GhEIF5A的不同SNP基因型可以将品种群体分为两大类，统计学分析方法发现，这两类群体之间的纤维强度性状存在显著性差异(图3)，进一步证明该基因与棉花纤维强度性状之间的相关性，可用于鉴定、改造获得高纤维强度棉花品种。
附图说明
图1 2008年安阳，棉花纤维强度性状与全基因组测序数据关联分析结果；
其中，横坐标表示染色体上的位置，纵坐标表示SNP位点关联的显著性，用-log10(Pvalue)表示。染色体A3位置有连续SNP位点与棉花纤维强度性状关联显著性较高。
图2GhEIF5A相对表达水平与纤维强度性状共定位结果；
其中，横坐标表示染色体A3上的位置(Mb)，纵坐标表示SNP位点关联的显著性，用-log10(P value)表示。上图为SNP位点与纤维强度性状关联的显著性，下图为SNP位点与GhEIF5A相对表达水平关联的显著性。在65Mb之后有连续SNP位点与GhEIF5A相对表达水平显著性较高，同时附近位点与棉花纤维强度性状关联。
图3GhEIF5A的不同单倍型之间基因表达量、纤维强度比较分析。
箱线图分别代表品种群体的GhEIF5A表达量、纤维强度的分布情况。表达量为20DPA纤维中GhEIF5A的FPKM值(经正态中心化后)，表示GhEIF5A的相对表达水平。含有CC和TT两种单倍型的品种分别是6和102个。方框内的横线代表性状分布的中值。方框内的横线代表性状分布的中值。SNP位点碱基从T变为C；突变后的基因型纤维强度显著高于野生型；基因GhEIF5A相对表达水平提高。
具体实施方式
实施例1棉花强度性状关联的基因GhEIF5A的挖掘：
针对258份现代品种或者品系，从2007到2009年，分别在河南安阳、江苏南京和新疆库车进行了棉花相关性状的详细调查。同时，对这258份棉花品种进行了全基因组测序。将测序结果比对到棉花陆地棉基因组序列上，最后和表型进行全基因组关联分析。发现了A03染色体上的一个SNP信号关联位点(A03:65556479)，如图1和图2所示，可以关联棉花纤维强度性状，同时该SNP关联GWAS信号区内的真核生物起始因子5A基因GhEIF5A，GhEIF5A在四倍体陆地棉TM-1中的cDNA序列为：SEQ ID NO.1，基因组序列为：SEQ ID NO.2。该基因属于真核生物起始因子5A基因家族，是唯一一个被认知的细胞内含有翻译后修饰形成氨基酸衍生物hypusine[Ne-(4-氨基丁基-2-羟丁基)赖氨酸]的蛋白质(GH_A03G1210)。从陆地棉基因组序列中获取了GhEIF5A(GH_A03G1210)的cDNA序列和基因组序列，根据cDNA两端设计基因全长引物，进行PCR扩增，引物序列为F1：SEQ ID NO.6和R1：SEQ ID NO.7。PCR反应程序如下：95℃预变性5min；95℃变性15sec，58℃退火15sec，72℃延伸3min，34个循环；最后72℃延伸5min。PCR扩增产物进行测序，与cDNA进一步比对确定序列的准确性，即可获得GhEIF5A基因。
实施例2鉴定高纤维强度棉花品种和改良品质性状中的应用：
基于SNP位点(A03:65556479)在染色体A03上的位置及其附近的序列(SEQ IDNO.5)，在其两端设计基因组扩增引物，引物序列为F2：SEQ ID NO.3和R2：SEQ ID NO.4。利用这对引物，在258个品种的DNA中进行PCR扩增并测序。PCR反应程序如下：94℃预变性5min；94℃变性30sec，58℃退火1min，72℃延伸45sec，30个循环；最后72℃延伸7min。根据测序结果分析该SNP位点上每个品种群体的基因型。发现该SNP位点碱基从T变为C，根据两个SNP位点碱基信息，将低纤维强度品种材料标记为GhEIF5A(T)，将高纤维强度品种材料标记为GhEIF5A(C)。根据SNP基因型，鉴定出单倍型GhEIF5A(C)材料6个，单倍型GhEIF5A(T)材料102个(图3和表1)。利用t-test检测方法，计算了两组单倍型之间纤维强度性状的相关性和GhEIF5A基因表达水平的相关性(图3)。结果显出，相比GhEIF5A(T)，GhEIF5A(C)材料纤维强度增加了10.91％(P<0.001)，GhEIF5A基因表达水平更高。
通过以上结果可以看出，基因GhEIF5A在改良棉花品质性状和培育棉花高纤维强度新品种中具有重要的研究价值。一方面可以根据基因GhEIF5A的调控SNP位点的两种单倍型设计分子标记，可以有效的鉴定棉花品质性状，在高纤维强度棉花品种选育研究中有很好的应用价值。另一方面，可以通过基因工程的手段将含有高产单倍型GhEIF5A(C)的基因转入棉花品种中提高棉花产量，也可以将低产单倍型GhEIF5A(T)中的SNP位点进行定点突变，改造成高纤维强度单倍型从而培育高质棉花新品种。
表1高纤维强度和低纤维强度单倍型在群体品种材料中的鉴定
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其他不同形式的变化或变动。这里无需也无法把所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明的保护范围。
Claims (2)
1.检测SNP位点的试剂在鉴定高纤维强度陆地棉品种中的应用，所述SNP位点位于陆地棉基因组A03:65556479，该SNP位点碱基从T突变为C，突变后基因型的棉纤维强度增加；其特征在于，所述应用具体为：
利用检测SNP位点的试剂检测棉花基因组中控制GhEIF5A基因的SNP位点A03:65556479的碱基类型，其中碱基为C的棉花即为高纤维强度棉花品种。
2.根据权利要求1所述的应用，其特征在于，检测SNP位点的试剂包括引物对：如SEQ IDNO.3所示的上游引物和如SEQ ID NO.4所示的下游引物。
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