CN112457385B - 一种控制水稻生育期基因ljp1的应用 - Google Patents
一种控制水稻生育期基因ljp1的应用 Download PDFInfo
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- CN112457385B CN112457385B CN202011530070.XA CN202011530070A CN112457385B CN 112457385 B CN112457385 B CN 112457385B CN 202011530070 A CN202011530070 A CN 202011530070A CN 112457385 B CN112457385 B CN 112457385B
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Abstract
本发明属于植物基因工程技术领域,公开了一种控制水稻生育期基因LJP1的应用。具体涉及一种控制水稻生育期基因的克隆、功能验证和应用。所述的LJP1基因的核苷酸序列如序列表SEQ ID NO.1所示,其编码的氨基酸序列如序列表SEQ ID NO.2所示。LJP1决定水稻基本营养生长期与感光性,从而影响其产量的形成。该基因用于创制水稻育种的新种质,扩大水稻品种的种植区域与季节,提高水稻产量与品质,具有很好的应用前景。
Description
技术领域
本发明属于植物基因工程领域。具体的说,涉及一种利用基因定位结合基因组重测序技术克隆控制水稻生育期基因LJP1 (Long-Juvenile Phase),并利用转基因互补实验验证了该基因的功能。同时,还涉及利用该基因创制水稻育种新种质,扩大水稻品种的种植区域与季节,提高水稻产量与品质,具有很好的应用前景。
背景技术
水稻是我国乃至世界的主要粮食作物之一,水稻的产量与开花时间密切相关。抽穗期(即从播种到抽穗的天数)是作物最重要的农艺性状之一,它决定作物适宜的种植区域、种植季节及耕作方式。水稻的抽穗期主要由基本营养生长性和感光性决定。基本营养生长性强而感光性弱的水稻品种的抽穗期主要由基本营养生长期决定,在不同地区、不同季节种植抽穗期变化都不大,因而容易达到高产、稳产;而且,在温度条件许可的情况下,每年可种植2-3季。因此,选育基本营养生长期长、感光弱的品种已成为水稻育种的一个重要目标。
目前,水稻中共鉴别出50多个与抽穗期有关的基因(QTL),其中大多数与光周期响应(开花诱导、感光性)有关,涉及开花的诱导过程。但与基本营养生长性有关的基因(包括显性早熟基因和隐性迟熟基因)极少。可见,在水稻中,与光周期反应的研究相比,对基本营养生长的遗传研究还十分落后。其主要原因在于,开展基本营养生长期的遗传分析比较困难,它要求短光周期的生长条件来消除因光周期敏感座位的分离所带来的干扰。因目前对水稻基本营养生长性的分子遗传机理还知之甚少,已成为水稻生育期研究的重点方向。因此,深入研究水稻基本营养生长性的遗传基础,无论在理论上还是在应用上都具有十分重要的意义。而获得基本营养生长期长、感光弱或不感光的突变体,对开展水稻(及其它作物)的基本营养生长的遗传研究具有重要的促进作用。
我们在粳稻品种日本晴(营养生长期短、感光性强)的辐射诱变后代中发现了一个生育期明显延长、光钝感(无明显感光性)、产量显著增加的突变体。该突变性状是由单基因隐性突变所致,我们将该基因命名为LJP1。本发明利用基因定位结合基因组重测序的方法克隆了LJP1基因,该基因编码一个LUX/PCL1蛋白OsPCL1。在拟南芥等长日照植物中,LUX/ PCL1基因是开花抑制因子,其功能缺失表现为开花提早。OsPCL1是短日作物中克隆的第一个功能明确的LUX/PCL1基因,其作用与长日植物的LUX/PCL1基因的功能正好相反,是一个开花促进因子。LJP1基因突变引起水稻感光性丧失、基本营养生长期延长、开花延迟,符合当前水稻育种的目标要求。这为利用该基因,创制水稻生育期的新种质,以扩大水稻种植区域、延长种植季节,实现高产、稳产的育种目标奠定基础。
发明内容
本发明的目的在于提供一种控制水稻生育期的基因,该基因失活呈现光钝感、抽穗延迟、产量增加。本发明还提供利用该基因在改良水稻生育期,扩大种植区域、提高产量中的应用方法。为实现以上目的,采用以下技术方案:
本发明涉及一种LJP1基因在控制水稻生育期中的应用,所述LJP1基因的核苷酸序列如SEQ ID NO:1所示,所述LJP1基因编码的氨基酸序列如SEQ ID NO: 2所示。
具体方法包括以下步骤:将ljp1突变体分别与中花11、中花15中的任意一种水稻品种杂交,获得F1代;以中花11、中花15任意一种水稻品种作为轮回亲本,经5代以上回交,从中筛选出具有生育期长、光钝感、分蘖多、高产量等特性但其它性状与回交亲本一致的株系。
所述ljp1突变体为水稻LJP1基因的外显子上发生C→T的单核苷酸替换,引起其编码的精氨酸→色氨酸,所述ljp1突变体序列如SEQ ID No.3所示。
所述ljp1突变体由粳型水稻品种日本晴辐射诱变而来。
LJP1基因具有如SEQ ID No.1所示的DNA序列,也包括与SEQ ID No.1所示的DNA序列至少有90%同源性的基因序列。本发明中的SEQ ID No.2所编码的蛋白质是一种植物的LUX/PCL1蛋白,其中包括进行一个或几个氨基酸替换、***或缺失所获得的功能类似物。另外,也包括在SEQ ID No.1中添加、取代、***或缺失一个或多个核苷酸而生成的突变体、等位基因或衍生物,具有相同功能的序列也能达到本发明的目的。
本发明克隆的水稻LJP1基因的单碱基突变体表现为光钝感、抽穗延迟、产量增加。将正常功能的LJP1基因转化该突变体后,植株恢复正常表型。
本发明还提供一种用LJP1基因进行高效的植物转化的方法,具体地说,本发明提供了SEQ ID No.1所示的序列基因或该基因类似的部分功能片段的载体。该载体还有一种含有以上表达载体的宿主细胞。该宿主细胞包括大肠杆菌、农杆菌和植物细胞。
本发明利用传统的水稻杂交与回交技术,将突变等位基因替换正常的LJP1后,创制出长生育期、光钝感、高产量的水稻新种质,可用于培育水稻新品种。
SEQ ID NO. 1:
ATGGGCGAGGAGGCGCCGGAGGAGTACGAGCTGGGCGGCGGGGAGGACGAGCGGGTGATGGAGTGGGAGACGGGGCTGCCCGGCGCCGACGAGCTGACCCCGCTGTCGCAGCCGCTGGTGCCGGCGGGGCTGGCGGCGGCGTTCCGCATCCCGCCGGAGCCCGGGCGCACGCTGCTCGACGTGCACCGCGCGTCGGCGGCGACGGTGTCCCGGTTGCGGCGCGCGTCGTCGTCGTCGTCGAGCTCGTTCCCGGCGTTCGCGTCGAAGGGAGCGGGAACGGGAGCGGACGAGGCGGAGTCAGGGGGAGGCGCGGATGGGGGGAACGGGAACACCAACAACAGCAGCAGCAAGAGGGCGCGGCTGGTGTGGACGCCGCAGCTGCACAAGAGGTTCGTGGAGGTGGTGGCGCACCTGGGGATGAAGAACGCGGTGCCCAAGACGATCATGCAGCTGATGAACGTGGAGGGCCTCACCCGGGAGAACGTCGCCAGCCACCTCCAGAAGTATCGCCTCTACGTGAAGCGGATGCAGGGCCTCTCCAACGAGGGCCCTTCCCCCTCCGACCACATCTTCGCCTCCACCCCCGTCCCCCACGCCTCCCTCCACGACCAGGTTCCTTCTCCTTACCACCCCCACCCCCACCACCACTCCTACAACAACGCCGCCTATGCCGCCACCGTCTCCTCCTACCACCACTACCACCACGCCAACCACTGA;
SEQ ID NO. 2 :
MGEEAPEEYELGGGEDERVMEWETGLPGADELTPLSQPLVPAGLAAAFRIPPEPGRTLLDVHRASAATVSRLRRASSSSSSSFPAFASKGAGTGADEAESGGGADGGNGNTNNSSSKRARLVWTPQLHKRFVEVVAHLGMKNAVPKTIMQLMNVEGLTRENVASHLQKYRLYVKRMQGLSNEGPSPSDHIFASTPVPHASLHDQVPSPYHPHPHHHSYNNAAYAATVSSYHHYHHANH*。
SEQ ID No.3:
ATGGGCGAGGAGGCGCCGGAGGAGTACGAGCTGGGCGGCGGGGAGGACGAGCGGGTGATGGAGTGGGAGACGGGGCTGCCCGGCGCCGACGAGCTGACCCCGCTGTCGCAGCCGCTGGTGCCGGCGGGGCTGGCGGCGGCGTTCCGCATCCCGCCGGAGCCCGGGCGCACGCTGCTCGACGTGCACCGCGCGTCGGCGGCGACGGTGTCCCGGTTGCGGCGCGCGTCGTCGTCGTCGTCGAGCTCGTTCCCGGCGTTCGCGTCGAAGGGAGCGGGAACGGGAGCGGACGAGGCGGAGTCAGGGGGAGGCGCGGATGGGGGGAACGGGAACACCAACAACAGCAGCAGCAAGAGGGCGTGGCTGGTGTGGACGCCGCAGCTGCACAAGAGGTTCGTGGAGGTGGTGGCGCACCTGGGGATGAAGAACGCGGTGCCCAAGACGATCATGCAGCTGATGAACGTGGAGGGCCTCACCCGGGAGAACGTCGCCAGCCACCTCCAGAAGTATCGCCTCTACGTGAAGCGGATGCAGGGCCTCTCCAACGAGGGCCCTTCCCCCTCCGACCACATCTTCGCCTCCACCCCCGTCCCCCACGCCTCCCTCCACGACCAGGTTCCTTCTCCTTACCACCCCCACCCCCACCACCACTCCTACAACAACGCCGCCTATGCCGCCACCGTCTCCTCCTACCACCACTACCACCACGCCAACCACTGA;
实现本发明的具体技术步骤如下:
一.水稻ljp1突变体的分离和遗传分析:
利用辐射诱变水稻品种日本晴,本发明获得了一种如图1所示的长生育期、光钝感、高产量的突变体ljp1。该突变体长时间停留在营养生长阶段,植株明显增高、分蘖增多、单株产量提高1倍以上。遗传分析表明,该突变性状是由单基因隐性突变所致。
二、LJP1基因的克隆:
1.确定候选基因:为了分离LJP1基因,本发明采用基因定位结合基因组重测序的方法,首先创建了一个F2定位群体,由ljp1突变杂合体为母本、DZ60为父本杂交获得的F2中的ljp1突变体组成。利用水稻微卫星标记对LJP1基因进行定位,将LJP1基因精细定位于如图2A所示的水稻第1染色体的长臂端,与RM414连锁。进一步利用基因组重测序的方法确定候选基因。利用野生型日本晴与ljp1突变体杂交后代的F3代株系,分别构建野生型DNA池和突变型DNA池。将这两个DNA池进行全基因组重测序,并以日本晴基因组序列为参考,筛选两个DNA池的变异位点,对基因进行定位并确定候选基因。结果如图2B、2C所示。
2.LJP1基因的鉴定和功能分析:
构建了一个如图3所示的互补实验载体。本发明通过转基因技术将互补载体转入ljp1突变体后获得了如图4所示的表型恢复正常的转基因水稻,证明了本发明正确克隆了LJP1基因;氨基酸序列分析表明,LJP1编码一个MYB家族蛋白的转录因子,注释为OsPCL1。
三、采用水稻杂交与回交技术,创制水稻不同生育期的材料:
采用传统的水稻杂交与回交技术,将突变等位基因取代水稻品种中具有正常功能的LJP1基因,创制出长生育期、光钝感、产量显著增加的水稻新种质,可用于培育适应不同种植区域、不同种植季节且高产、稳产的水稻品种(见实施例3)。
本发明的优点在于:
水稻的产量、品质与开花时间密切相关。抽穗期作为作物最重要的农艺性状,决定了作物适宜的种植区域、种植季节及耕作方式。水稻抽穗期主要由基本营养生长性和感光性决定。基本营养生长性长、感光性弱(或光钝感)的水稻品种对不同地域、不同季节的适应性强,且高产、稳产。目前,选育基本营养生长性长、光钝感的品种已成为水稻育种的一个重要目标。本发明获得的水稻ljp1突变体符合当前育种目标的水稻新种质,是十分宝贵的遗传资源。本发明通过基因定位结合基因组重测序方法确定了LJP1基因,并通过功能互补实验鉴定了该基因的功能。氨基酸序列分析表明,该基因编码一个MYB家族蛋白的转录因子。而将水稻品种中正常的LJP1基因替换为突变等位基因后,创制出长生育期、光钝感、产量高,可广泛用于培育适应性广、种植季节长且高产、稳产的水稻品种,以提高农业生产效益。因此,该基因具有非常重要的应用价值和广阔的应用前景。
附图说明
图1:水稻野生型与对应突变体ljp1的表型;A:野生型与ljp1突变体的植株表型;B:野生型与ljp1突变体的单株产量。
图2:LJP1基因的定位及候选基因确定图;A:LJP1基因与水稻RM414连锁分析图;B:生物信息学分析野生型DNA池和突变型DNA池中多态性位点上等位基因频率分布图;C:测序验证候选基因LOC_Os01g74020在突变体中的突变位点。
图3:互补实验载体的图谱。
图4:野生型、ljp1突变体与互补实验T0代植株的表型图。
具体实施方式
实施例1:水稻LJP1基因的克隆
1.水稻材料和定位群体:
如图1所示的水稻突变体ljp1为本发明者利用辐射诱变的方法获得,原始野生型亲本为粳型水稻品种日本晴。将ljp1突变体与野生型品种DZ60进行杂交,F1代自交,得到F2群体,在水稻抽穗期依据ljp1突变表型选出389个植株作为定位群体。
2.利用水稻微卫星标记定位LJP1基因:
采用水稻微量法快速提取水稻的总DNA。取大约0.3克水稻叶片,放入1.5ml离心管中经液氮冷冻后快速提取DNA,获得的DNA溶于100 ul超纯水中。利用DNA池的定位方法,初步定位LJP1基因。根据公布的水稻微卫星遗传图谱,均匀选取分布于各条染色体上的微卫星引物进行PCR扩增,于6%的非变性聚丙烯酰胺凝胶上分离和硝酸银染色,检测PCR产物的多态性。然后选取F2群体中的389个突变体进行连锁分析,将LJP1基因初步定位在水稻第1染色体短臂端,与水稻微卫星标记RM414连锁。扩增RM414标记的正向引物序列为:ATTGCAGTCATGCAGCAGTC,反向引物序列为:ATATCTCCAATGTGGCAGGG。利用RM414标记进行连锁分析的部分结果如图2A所示。
3.利用基因组重测序的方法确定候选基因:
以野生型日本晴和ljp1突变体为亲本进行杂交,从其F3代的不分离株系(纯合正常基因型)中随机挑选30个株系(5株/株系),取等量叶片混合、提取基因组DNA,构成野生型DNA池;从F3代的分离株系(杂合基因型)中随机挑选30个株系、每个株系选5个突变株,取叶片等量混合、提取基因组DNA,构成突变型DNA池。委托诺禾致源生物信息科技有限公司,采用Illumina PE测序方法对两个DNA池进行全基因组重测序:读段长度为300 bp,测序深度>30×。以日本晴基因组序列为参考,利用生物信息学方法,检测到如图2B所示的这两个DNA池中多态性位点上的等位基因频率分布,并将LJP1的候选基因定位在Chr.1的末端。经进一步的比对分析与测序验证,发现位于该区域中的基因LOC_Os01g74020,突变体在该基因的外显子上发生C→T的单核苷酸替换,引起其编码的精氨酸→色氨酸。突变体中,候选基因的突变位点的信息如图2C所示。从国家水稻数据中查找到,LOC_Os01g74020被注释为OsPCL1,编码一个MYB家族蛋白的转录因子。而在定位区域附近的其它基因,在突变体与野生型的基因组序列没有发生改变。因此,我们将LOC_Os01g74020确定为LJP1的候选基因。
实施例2 互补实验
1. 构建互补实验载体。
根据粳稻品种日本晴LJP1基因的序列,利用增加了酶切位点的pCAMBIA1300载体构建了如图3所示的侯选基因的互补实验载体。载体构建的具体过程是:在候选的LJP1基因区和启动子区设计一对带有酶切位点的引物,利用高保真酶进行高保真PCR扩增并测序,挑选序列完全正确的克隆,经酶切和连接,构建成如图3所示的载体,再将其转入农杆菌中。该互补载体的DNA序列为3420bp。扩增互补实验的DNA序列所用引物是:正向引物序列为:CGGAATTCATTTCTATGCGGTACAGTTTT,反向引物序列为:CGGAATTCGTATTTATTCGCTATTCAGTTCA。
2. 互补实验:
利用愈伤侵染法技术,转化ljp1突变体的愈伤组织。挑选生长迅速、颜色鲜黄、表面光滑、质地致密、直径大小为2-3mm的胚性愈伤组织颗粒作转化的受体。用含有双元质粒载体的农杆菌EHA105菌株浸染水稻愈伤,置25℃条件下暗培养3天;经30 mg/L 潮霉素的筛选培养基培养2次,每次15天;经50 mg/L 潮霉素的筛选培养基上培养7天;将经3次筛选获得的抗性愈伤转入分化培养基,于光照条件下进行分化培养;于幼苗长至2-3cm时,转至1/2MS培养基进行生根培养。对获得的25个再生植株进行鉴定和连续的观察,显示转基因水稻植株生长发育状况如图4所示,所有转基因植株的抽穗期与野生型比较没有明显区别,说明该侯选的OsPCL1基因就是LJP1基因。
实施例3 生育期长、光钝感、产量高的水稻种质的培育
将ljp1突变体分别与中花11、中花15杂交,获得F1代。以中花11、中花15作为轮回亲本,经多代(5代以上)回交,从中筛选出如表1所示,在海南省三亚市冬季自然短日照条件下(孕穗期约10 h日照/天)与福建省福州市夏季自然长日照条件下(孕穗期约14 h日照/天)种植,具有生育期长、光钝感、分蘖多、产量高等特性但其它性状与回交亲本一致的株系。同时,采用水稻微量法快速提取水稻的总DNA,利用PCR扩增出ljp1突变位点序列,对筛选出的株系予以测序验证。DNA扩增的正向引物序列为:ACGAGCGGGTGATGGAGTG,反向引物序列为:GCTTCACGTAGAGGCGATACT。这些生育期长、光钝感、产量高的水稻新种质,可用于培育适应不同种植区域、不同种植季节且高产、稳产的水稻品种。
表1 亲本与对应的回交株系的主要农艺性状差异
以上所述仅为本发明的若干个具体实施方式,应当指出,对与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。
SEQUENCE LISTING
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Claims (1)
1.一种突变体ljp1在控制水稻生育期中的应用,其特征在于,包括以下步骤:将来源于粳型水稻品种日本晴的突变体ljp1分别与粳稻品种中花11、中花15杂交,获得F1代;分别以中花11、中花15作为轮回亲本,经5代以上回交,从中筛选出具有生育期长、光钝感、分蘖多、高产量的特性但其它性状与回交亲本一致的株系;所述突变体ljp1为水稻LJP1基因的外显子上发生C→T的单核苷酸替换,引起其编码的精氨酸→色氨酸,所述突变体ljp1序列如SEQID No.3所示。
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