CN116694799A - 水稻OsAUX5基因中与稻米必需氨基酸积累相关InDel的位点及应用 - Google Patents
水稻OsAUX5基因中与稻米必需氨基酸积累相关InDel的位点及应用 Download PDFInfo
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Abstract
本发明提供水稻OsAUX5基因中与稻米必需氨基酸积累相关的InDel位点及应用,位于水稻基因组第11条染色体第3344946位处,序列如SEQ ID NO.1所示,等位基因是缺失如SEQ ID NO.2所示的序列;本发明分离克隆了控制水稻胚乳必需氨基酸含量的OsAUX5蛋白,鉴定到OsAUX5基因启动子区域上与稻米必需氨基酸含量相关的InDel位点,还发现过表达OsAUX5基因能够显著增强稻米必需氨基酸含量,其转基因阳性植株的成熟稻米必需氨基酸含量显著高于野生型,还通过杂交方式将优异的OsAUX5等位基因型导入到常规水稻品种中,转基因植物或杂交后代可以在稻米中累积必需氨基酸。
Description
技术领域
本发明属于植物生物技术领域,具体涉及水稻OsAUX5基因中与稻米必需氨基酸积累相关的InDel位点及应用。
背景技术
必需氨基酸是人体自身不能合成或合成的速度不能满足机体需要,必需从食物中获得的一类氨基酸,是食物蛋白营养价值的关键成份。人们摄取必需氨基酸的主要途径是动物蛋白和植物蛋白,但过分依赖肉食品会带来高血压、高血脂、冠心病等伴随性疾病。通过改变植物蛋白的构成成分以提高必需氨基酸的含量,是解决这一问题的重要手段。
世界人口一半以上以稻米为主食,提高稻米必需氨基酸的含量,对强化人体营养与保健具有十分重要的意义。目前提高稻米必需氨基酸含量的方法通常是品种的选择或提高栽培技术,但是市面上选育出来的优质高必需氨基酸品种较少,从栽培技术和自然环境上提高稻米中必需氨基酸含量的过程较为复杂,而且受到人为和自然因素影响较大。
稻米必需氨基酸含量性状属于典型的数量遗传性状。全基因组关联分析方法(GWAS)是鉴定复杂数量性状的有效方法,近年来利用该方法已经发现了一些控制稻米必需氨基酸含量的数量性状基因位点(QTL)位点。但控制这些QTL位点的关键基因还未被克隆,并且不能确定这些QTL对稻米必需氨基酸含量影响的真实效应。插入/缺失(InDel)分子标记是指的是两种亲本在全基因组中的差异,相对于另一个亲本而言,其中一个亲本的基因组中有一定数量的核苷酸插入或缺失,而根据基因组中插入缺失位点,可以设计一些用于扩增这些插入缺失位点的引物,通过PCR扩增可以进行不同品种植株的辅助选育。因此,克隆控制稻米必需氨基酸含量的关键基因对于水稻营养品质改良具有巨大的应用前景。
发明内容
本发明的第一个目的提供一个水稻OsAUX5基因启动子区域上与稻米必需氨基酸积累显著相关的InDel位点,其位于水稻基因组第11条染色体第3344946位处,其核苷酸序列如SEQ ID NO.1所示,等位基因为缺失如SEQ ID NO.2所示的核苷酸序列。
本发明的第二个目的是提供一种所述的InDel位点检测试剂,检测试剂包含引物对,引物对用于检测所述的InDel位点的等位基因型为如SEQ ID NO.1所示;所述的引物对包括核苷酸序列如SEQ ID NO.3所示的上游引物和SEQ ID NO.4所示的下游引物。
本发明的第三个目的是提供一种鉴别高或低必需氨基酸含量水稻品种方法,包括如下步骤:
(1)提取待鉴别水稻的DNA;
(2)以提取的DNA为模板,用引物对进行PCR扩增;引物对包括如SEQ ID NO.3所示的上游引物和SEQ ID NO.4所示的下游引物;
(3)根据PCR扩增产物的琼脂糖凝胶电泳鉴定水稻OsAUX5基因启动子区域上的插入/缺失多态性,如扩增出条带,则该待鉴别水稻品种的稻米必需氨基酸低;如未扩增出条带,则该待鉴别水稻品种的稻米必需氨基酸高。
本发明的第四个目的是提供一种稻米必需氨基酸高的水稻植株,包括如下步骤:提取水稻的RNA并反转录为cDNA,采用引物对PCR扩增得到OsAUX5基因编码区,编码区的核苷酸序列为SEQ ID NO.7;引物对包括核苷酸序列如SEQ ID NO.5所示的上游引物和SEQ IDNO.6所示的下游引物,将OsAUX5基因编码区构建至表达载体,再将表达载体转化至稻米必需氨基酸含量低的水稻植株中,获得OsAUX5过表达植株即为稻米必需氨基酸高的水稻植株。
本发明的第五个目的是提供一种稻米必需氨基酸高的水稻植株,包括向稻米必需氨基酸低的水稻植株导入稻米必需氨基酸高的水稻植株的OsA UX5基因,得到含OsAUX5等位基因型的转基因水稻植株,稻米必需氨基酸高的水稻植株的OsAUX5基因核苷酸序列如SEQ ID NO.17所示。
本发明的第六个目的是提供一种必需氨基酸含量高的水稻植株,包括如下步骤:以水稻的第11号染色体第3344946位为靶标区域进行基因编辑,突变后OsAUX5基因为敲除如SEQ ID NO.2所示的核苷酸序列,从基因编辑植株的自交后代中获得纯合型植株;纯合型植株即为必需氨基酸高含量水稻植株。
本发明的第七个目的是提供一种基因芯片,用于检测所述的InDel位点,包含所述的检测试剂。
本发明的第八个目的是提供一种试剂盒,用于检测所述的InDel位点,试剂盒包含所述的检测试剂。进一步的,试剂盒应用在鉴定水稻品种稻米必需氨基酸含量高或低中。进一步的,试剂盒还包含另外一引物对,所述的引物对用于检测所述的InDel位点的等位基因型为T时。进一步的,试剂盒还包含:扩增缓冲液、dNTP混合物、DNA聚合酶以及ddH2O。
本发明的第九个目的是提供所述的InDel位点在分子育种中的应用。
本发明的第十个目的是提供所述的InDel位点在品种鉴定和保护中的应用。
本发明的第十一个目的是提供所述的InDel位点在杂交后代鉴定中的应用。
本发明的第十二个目的是提供所述的InDel位点在重要性状基因定位中的应用。
本发明利用多份水稻种质资源材料,通过GWAS方法分离克隆一个控制水稻胚乳必需氨基酸的氨基酸转运蛋白OsAUX5;同时鉴定到OsAUX5基因启动子区域上稻米必需氨基酸含量显著相关的InDel位点,并根据In Del位点设计扩增引物对。本发明还发现向受体水稻品种导入OsAUX5基因编码区核苷酸序列对增加稻米必需氨基酸含量具有重要意义,OsAUX5基因过表达能够显著增强稻米中必需氨基酸的含量,转基因阳性植株的稻米必需氨基酸含量显著高于野生型。本发明将稻米必需氨基酸含量高的等位基因型(T)转入到低含量的等位基因型(TCACATATACATTTGTCGA)植株,能够增强低基因型水稻品种的稻米必需氨基酸。
附图说明
图1是实施例1中195份不同水稻品种中必需氨基酸的含量图。
图2是实施例2中利用GWAS方法鉴定控制稻米必需氨基酸积累的基因OsAUX5图谱。
图3是实施例3中的OsAUX5的基因结构、InDel位置信息、粳稻和籼稻的基因型分析图。
图4是实施例4中的粳稻和籼稻的表达量检测。
图5是实施例5中的质粒图谱。
图6是实施例5中的OsAUX5过表达植株中OsAUX5的表达量检测。
图7是实施例6中的OsAUX5过表达植株稻米中氨基酸的含量测定。
图8是实例7中水稻OsAUX5启动子区域InDel标记的鉴定。
图9是实施例8中的质粒图谱。
图10是实施例9中导入高含量的OsAUX5等位基因型输掉植株稻米中必需氨基酸的含量测定。
具体实施方式
以下实施例中进一步定义本发明,根据以下的描述和这些实施例,本领域技术人员可以确定本发明的基本特征,并且在不偏离本发明本质和范围的情况下,可以对本发明做出各种改变和修改,以使其适用各种用途和条件。
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;所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中使用籼稻和粳稻是亚洲栽培稻的主要两个亚种,在长期栽培和驯化中经自然选择和人工选择逐渐分化而成,籼稻和粳稻遗传差异明显,其农艺形状和生理生化特性等方面存在着显著差异。
下述实施例涉及对OsAUX5的分离克隆和优异等位基因的鉴定,并对该片段的应用机理进行阐述。
实施例1、籼稻和粳稻品种稻米中的必需氨基酸含量的测定。
选取籼稻品种100个和粳稻品种95个,共计195个水稻品种种植在同一块大田中,得到的成熟稻米在60℃条件下烘干,再脱壳后粉碎成米粉,将米粉过筛后使用串联质谱系统(ABSCIEXQTRAP6500)进行氨基酸含量的测定。测定结果如图1所示,籼稻品种较粳稻品种表现出更高的必需氨基酸含量,具体包括缬氨酸,亮氨酸,苯丙氨酸和苏氨酸。
实施例2、调控稻米必需氨基酸含量基因OsAUX5的鉴定。
利用水稻RiceVarMap数据库(http://ricevarmap.ncpgr.cn/)中的水稻全基因组SNP信息,结合已经测定的稻米必需氨基酸含量,利用GWAS鉴定到控制稻米必需氨基酸含量的QTL位点。结果如图2所示,OsAUX5基因位点与必需氨基酸含量(缬氨酸,亮氨酸和苯丙氨酸)显著相关。
实施例3、籼稻和粳稻的OsAUX5基因启动子区域变异情况。
氨基酸转运蛋白OsAUX5(RAP-DB数据库中的登录号为Os11g0169200)。利用水稻RiceVarMap数据库中195份栽培稻基因组数据对OsAUX5启动子区域核苷酸序列变异情况进行分析。
通过序列分析可知:籼稻与粳稻的OsAUX5启动子区域存在1个InD el位点位于水稻11号染色体第3344946位,所述的InDel位点导致了籼粳稻亚种间序列的变异,其多态性为T/TCACATATACATTTGTCGA,如图3所示,籼稻的第11号染色体第3344946位为碱基T,粳稻的第11号染色体第3344946位为碱基序列TCACATATACATTTGTCGA。
实施例4、籼稻和粳稻品种中的OsAUX5基因表达量的检测。
提取50个水稻样品RNA,反转录为cDNA,籼稻粳稻各25个。分别以获得的cDNA为模板,采用如SEQ ID NO.11所示的上游引物和如SE Q ID NO.12所示的下游引物检测OsAUX5基因表达量,检测结果如图4所示,籼稻品种表达量高于粳稻品种,所以OsAUX5优异等位基因型可以用于高氨基酸含量积累水稻的分子标记开发。粳稻品种第11号染色体第3344946位为序列CACATATACATTTGTCGA,侧翼序列如SEQ ID NO.9所示。籼稻品种第11号染色体第3344946位为碱基T,侧翼序列如SEQ ID NO.10所示。
F:5'-TACCTCATCAGCGTGCTCTA-3'(SEQ ID NO.11)
R:5'-ACCACTGGATGACATGGTTG-3'(SEQ ID NO.12)
AATTTTTTGAGGTCACATATACATTTGTCGACAACTTTTCTTC(SE Q ID NO.9)
AATTTTTTGAGGTCAACTTTTCTTC(SEQ ID NO.10)
实施例5、OsAUX5过表达植株的创制及鉴定。
创制包括构建OsAUX5的过表达遗传转化载体,通过农杆菌介导的水稻遗传转化系统,获得OsAUX5的过表达水稻。具体是:提取粳稻品种的RNA,反转录为cDNA,获得的cDNA为模板,采用引物对扩增粳稻的OsAUX5编码区,如SEQ ID NO.7所示,所述的引物对包括核苷酸序列如SEQ ID NO.5所示的上游引物和SEQ ID NO.6所示的下游引物。使用KODFX(东洋纺)PCR酶进行扩增,退火温度为58℃,延伸时间为1min循环35次,将包含如SEQ ID NO.7所示序列的目的基因利用gateway体系连接至表达载体pJC034中,测序正确的图5中的表达载体通过电转法转入根癌农杆菌AH109中。编码蛋白的氨基酸序列如SEQ ID NO.8所示。实施例5通过水稻转基因技术将表达载体转化至粳稻品种ZH11中,获得OsAUX5过表达植株OsAUX5-OE1和OsAUX5-OE2。
F:5'-AAAAAGCAGGCTTAATGGCGTCGGAGAAGGT-3'(SEQ IDNO.5)
R:5'-AGAAAGCTGGGTAATGCAATTATGCAACAG-3'(SEQ ID NO.6)
鉴定OsAUX5转基因植株系的引物是如SEQ ID NO.11的上游引物和如SEQ IDNO.12的下游引物。
F:5'-TACCTCATCAGCGTGCTCTA-3'(SEQ ID NO.11)
R:5'-ACCACTGGATGACATGGTTG-3'(SEQ ID NO.12)
对其进行荧光实时定量PCR测定分析结果如图6所示,过表达OsAU X5的转基因家系OsAUX5-OE1和OsAUX5-OE2比OsAUX5的表达量明显增加。
实施例6、OsAUX5过表达转基因株系必需氨基酸含量的测定。
将实施例5中获得的OsAUX5过表达转基因株系成熟种子烘干去壳,再研磨至粉末,使用串联质谱系统(ABSCIEXQTRAP6500)进行必需氨基酸含量的测定,结果如图7所示:OsAUX5过表达转基因株系OsAUX5-OE1和OsAUX5-OE中稻米中的必需氨基酸含量与ZH11相比显著增加。
实施例7、稻米必需氨基酸积累基因OsAUX5相关的分子标记位点的应用。
以待检测水稻基因组DNA为模板,利用如SEQ ID NO.3所示上游引物和如SEQ IDNO.4所示下游引物进行PCR扩增水稻OsAUX5基因,再对PCR扩增片段进行琼脂糖凝胶电泳;根据琼脂糖凝胶电泳结果鉴定水稻OsAUX5基因上的插入/缺失多态性,结果如图8所示:扩增出条带则该待鉴别水稻品种的稻米必需氨基酸低;未扩增出条带则该待鉴别水稻品种的稻米必需氨基酸高。
F:5'-AGTTGTCGACAAATGTATATGTGAC-3'(SEQ ID NO.3)
R:5'-GACGCTCGTCGATTTGTACTG-3'(SEQ ID NO.4)
实施例8、将高含量的等位基因型(T)转入到低含量的基因型(TCACATATACATTTGTCGA)植株。
包括构建OsAUX5高含量等位基因型的遗传转化载体,通过农杆菌介导的水稻遗传转化系统,获得OsAUX5转高含量等位基因型的水稻株系。首先提取籼稻(珍汕97B品种)的DNA作为模板,再采用引物对如SEQ ID NO.17所示的籼稻的OsAUX5的基因进行PCR扩增。使用KODFX(东洋纺)PCR酶进行扩增,退火温度为58℃,延伸时间为3min循环35次,扩增采用如SEQ ID NO.13所示的上游引物和SEQ ID NO.14所示的下游引物。将扩增获得的片段利用gateway体系连接至表达载体pJE009,如图9所示。测序正确的表达载体通过电转法转入根癌农杆菌AH109中,通过水稻转基因技术将表达载体转化至粳稻品种ZH11中,获得OsAUX5转高含量等位基因型的水稻株系。对其进行载体插入鉴定,采用如SEQ ID NO.15所示的上游引物和SEQ ID NO.16所示的下游引物进行扩增,能扩增出条带为转基因阳性植株。
F:5'-AAAAAGCAGGCTTACTGGTACCAGTAGTGGAGACTCG-3'(SEQ ID NO.13)
R:5'-AGAAAGCTGGGTACTAGTGTCTTGGAGGGCACTGG-3'(SE Q ID NO.14)
F:5'-GAGAGAGATAGATTTGTAGAGAG-3'(SEQ ID NO.15)
R:5'-ATGGCGTCGGAGAAGGTGG-3'(SEQ ID NO.16)
本发明中通过杂交方式将优异的OsAUX5基因型导入到常规栽培品种中,转基因植物或杂交后代可以在水稻胚乳中累积必需氨基酸,从而提高稻米营养品质,满足人们对必需氨基酸的需求。
实施例9、转OsAUX5高含量等位基因型的水稻株系必需氨基酸含量的测定。
将实施例8中获得的OsAUX5转高含量等位基因型的水稻株系成熟种子烘干去壳,再研磨至粉末,使用串联质谱系统(ABSCIEXQTRAP6500)进行必需氨基酸含量的测定,结果如图10所示:OsAUX5转高含量等位基因型的水稻株系稻米中的必需氨基酸含量与粳稻ZH11相比显著增加。
实施例10、以水稻的第11号染色体第3344946位为靶标区域进行基因编辑,突变后OsAUX5基因为敲除所述的SEQ ID NO.2序列,从基因编辑植株的自交后代中获得纯合型基因编辑植株,纯合型基因编辑植株即为必需氨基酸高含量水稻植株。
通过上述实施例可知:OsAUX5是水稻中重要的氨基酸转运基因,Os AUX5基因过表达能够显著增加稻米中必需氨基酸的含量;将籼稻品种Os AUX5基因型导入到粳稻品种中,可以增加粳稻品种的必需氨基酸含量。所以通过种植过表达OsAUX5或其优异等位基因的水稻品种,满足人体对必需氨基酸的需求,进而提高稻米的营养品质。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,所述的实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对所述的实施例进行变化、修改、替换和变型。
Claims (10)
1.水稻OsAUX5基因中与稻米必需氨基酸积累相关的InDel位点,其位于水稻基因组第11条染色体第3344946位处,其核苷酸序列如SEQ ID NO.1所示,等位基因为缺失如SEQ IDNO.2所示的核苷酸序列。
2.一种检测试剂,其特征在于:所述的检测试剂包含引物对,所述的引物对用于检测所述的InDel位点的等位基因型为如SEQ ID NO.1所示;所述的引物对包括核苷酸序列如SEQID NO.3所示的上游引物和SEQ ID NO.4所示的下游引物。
3.一种鉴别高或低必需氨基酸含量水稻品种方法,其特征在于,包括如下步骤:
(1)提取待鉴别水稻的DNA;
(2)以提取的DNA为模板,用引物对进行PCR扩增;引物对包括如SEQ ID NO.3所示的上游引物和SEQ ID NO.4所示的下游引物;
(3)根据PCR扩增产物的琼脂糖凝胶电泳鉴定水稻OsAUX5基因启动子区域上的插入/缺失多态性,如扩增出条带,则该待鉴别水稻品种的稻米必需氨基酸低;如未扩增出条带,则该待鉴别水稻品种的稻米必需氨基酸高。
4.一种稻米必需氨基酸高的水稻植株,其特征在于,包括如下步骤:包括向受体水稻品种导入的如SEQ ID NO.7所示的OsAUX5基因编码区序列,使OsAUX5基因在受体中过表达得到转基因水稻植株即为稻米必需氨基酸高的水稻植株。
5.一种稻米必需氨基酸高的水稻植株,其特征在于,包括如下步骤:包括向稻米必需氨基酸低的水稻植株中导入稻米必需氨基酸高的水稻植株OsA UX5基因,其核苷酸序列如SEQID NO.17所示,得到含OsAUX5优异等位基因型的转基因水稻植株。
6.一种必需氨基酸含量高的水稻植株,其特征在于,包括如下步骤:以水稻的第11号染色体第3344946位为靶标区域进行基因编辑,突变后OsAUX5基因为敲除如SEQ ID NO.2所示的序列,从基因编辑植株的自交后代中获得纯合型基因编辑植株;纯合型基因编辑植株即为必需氨基酸高含量水稻植株。
7.如权利要求1所述的InDel位点在以下一种或几种的应用:
(1)在水稻分子育种中的应用;
(2)在水稻品种鉴定和保护中的应用;
(3)在水稻杂交后代鉴定中的应用;
(4)在水稻重要性状基因定位中的应用。
8.一种基因芯片,其特征在于:包含如权利要求2所述的检测试剂。
9.一种试剂盒,其特征在于:包含如权利要求2所述的检测试剂。
10.一种试剂盒,其特征在于:还包含另外一引物对,所述的引物对用于检测所述的InDel位点的等位基因型为T时。
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