CN115197952A - 水稻蜡质基因Wx的突变基因及其应用 - Google Patents
水稻蜡质基因Wx的突变基因及其应用 Download PDFInfo
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- CN115197952A CN115197952A CN202210541772.0A CN202210541772A CN115197952A CN 115197952 A CN115197952 A CN 115197952A CN 202210541772 A CN202210541772 A CN 202210541772A CN 115197952 A CN115197952 A CN 115197952A
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- rice
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- resistant starch
- starch content
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Abstract
本发明公开了一种水稻蜡质基因Wx的突变基因及其应用,所述突变基因的核苷酸序列如SEQIDNO.1所示。本发明发现水稻蜡质基因Wx第9个外显子第202位的T→C的碱基突变,会使水稻中抗性淀粉的含量提高,水稻蜡质基因Wx的突变基因是与高抗性淀粉含量密切相关的基因;根据该突变基因的突变位点设计引物对,再对水稻基因组DNA进行扩增,通过核酸电泳,即可有效地检测水稻品种是否为高抗性淀粉含量的水稻品种,并且能够对移栽前的水稻幼苗的基因型进行快速鉴定,从而可以有选择性地移栽,大大提高了高抗性淀粉含量的水稻品种的选择效率,加速育种进程。
Description
技术领域
本发明属于分子遗传学技术领域,更具体地说,本发明涉及一种水稻蜡质基因Wx的突变基因及其应用。
背景技术
水稻蜡质基因Wx编码结合态淀粉合成酶(Granule-Bound Starch Synthase,GBSS),该酶催化合成直链淀粉,影响水稻直链淀粉含量(Amylose content,AC),是决定直链淀粉含量的主效基因。
抗性淀粉(Resistant Starch,RS)最初由Englyst等(1982)提出,在人体的小肠中不被吸收,可降低餐后血糖水平,增加饱腹感,满足糖尿病人食用米饭的愿望,降低肥胖风险,提升2型糖尿病患者的胰岛素敏感性、防止慢性肾病(Kieffer et al.,2016;Bindelset al.,2017;Emilien et al.,2017)。食用大米中有近80%的成分都是淀粉,但大多数水稻品种的RS含量低于3%(Yang et al.,2006;Hu et al.,2004),高RS品种匮乏。因此,培育高RS水稻品种具有重要的应用价值。
研究表明,RS与直链淀粉的比例呈正相关(Cai et al.,2015;Lin et al.,2016;Chen et al.,2017;Xia et al.2018),水稻的蜡质基因Wx和RS含量间存在显著的相关性(Cai et al.,2015;Lin et al.,2016;Chen et al.,2017;Xia et al.2018),是调控RS含量的主效基因,也是决定不同水稻品种间RS含量的主效基因(Raben et al.,1994;Fitzgerald et al.,2011)。
Bao等(2017)通过全基因组关联分析,得到与水稻RS相关的4个候选基因都是与直链淀粉和支链淀粉合成有关的基因:Wx、SSⅡa、ISA1和AGPS1。水稻SBE3基因通过引入分支来促进支链淀粉的合成,研究发现,SBE3基因单碱基的突变,使得第599位的亮氨酸转变为脯氨酸,RS含量由0.41%提高至11.67%(Yang et al.,2012)。Zhou等(2016)发现SSⅢa基因突变后显著提高了水稻中RS的含量,其对RS的调控依赖于Wx基因的高表达。
Wxa基因是籼稻中最普遍的等位基因,这类品种具有较高的直链淀粉(成太辉和杨武,2017),而Wxa基因第10外显子的C/T位点决定了稻米胶稠度和软硬(Tran et al.,2011)。
高RS水稻品种的培育不可避免的要对大量中间材料进行RS含量的测定筛选,但目前RS含量主要是化学测定方法,测定步骤繁琐、效率低,测定成本高、重复性差。如果能够开发出跟目标性状紧密连锁的分子标记,应用于杂交后代中高RS含量单株的筛选,将极大地提高育种效率。
基于表型选择的常规育种方法存在选择效率低和育种周期长等缺点,迫切需要注入现代分子技术手段,辅之于高效率地基因型定向选择,才能快速高效地培育出优异水稻新品种。随着分子生物学和基因组学的迅速发展,分子标记技术的应用更加广泛。基于PCR的分子标记如微卫星或SSR(simple sequence repeat)等具有多态率高、相对稳定、检测方法简便快速及易于操作等特点而被广泛的应用。由于分子标记辅助选择不易受环境因素影响和性状显隐性干扰等,可以从分子水平定向选择目标性状基因,同时还有可能打破不利基因之间的连锁而高效率地聚合多个优良基因于一体。分子标记辅多基因聚合育种技术已经成为水稻、玉米等作物育种研究的一种发展趋势,关键在于能否获得与目标性状基因或主效QTL紧密连锁的实用分子标记。
目前鲜见与抗性淀粉含量紧密连锁的分子标记开发和利用的报道。仅在2008年,牟方贵等报道在杂交组合II-32B/RS 111中,位于第8染色体的RM72和RM547与抗性淀粉存在一定的连锁关系,而在宜香B/RS111的F2中,和Wx基因连锁的RM217和RM225与抗性淀粉存在一定的连锁关系。2009年王琳等利用BSA法在小麦中找到一个与高抗性淀粉含量密切相关的SSR标记Xbarc590。2012年朴钟泽等利用水稻淀粉分支酶SBE3基因第16个外显子的第105位处T→C的碱基突变设计了一个与高抗性淀粉含量密切相关的RFLP标记。
然而,SSR标记由于自身的特性在基因组的密度达不到理想要求,与目的基因的连锁关系不是非常密切,因此筛选精度往往不高;而基因本身的SNP标记往往开发出来必须以酶切为辅助手段,因此过程繁琐,而且费用也高。因此,挖掘更多的与高抗性淀粉含量密切相关的基因,以及开发与抗性淀粉含量紧密连锁的分子标记,利用开发的分子标记对育种材料进行早代选择,加快育种进程,改良水稻营养品质有着重要的意义。
发明内容
基于此,本发明的目的之一在于提供一种水稻蜡质基因Wx的突变基因,该突变基因与水稻高抗性淀粉含量密切相关。
实现上述发明目的的具体技术方案包括如下:
一种水稻蜡质基因Wx的突变基因,所述突变基因的核苷酸序列如SEQ ID NO.1所示。
本发明还提供了上述水稻蜡质基因Wx的突变基因在筛选高抗性淀粉含量的水稻中的应用。
本发明还提供了一种水稻蜡质基因Wx的突变基因的编码蛋白,所述编码蛋白的氨基酸序列如SEQ ID NO.2所示。
本发明还提供了上述水稻蜡质基因Wx的突变基因的编码蛋白在筛选高抗性淀粉含量的水稻中的应用。
本发明还提供了一种筛选高抗性淀粉含量的水稻的引物对,所述引物对包括核苷酸序列如SEQ ID NO.5~SEQ ID NO.8所示的引物。
本发明还提供了一种筛选高抗性淀粉含量的水稻的引物对,所述引物对包括核苷酸序列如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.9和SEQ ID NO.10所示的引物。
本发明还提供了一种筛选高抗性淀粉含量的水稻的试剂盒,所述试剂盒包括SEQID NO.5~SEQ ID NO.8所示的引物对;和/或所述试剂盒包括SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.9和SEQ ID NO.10所示的引物对。
在其中一些实施例中,所述试剂盒还包括Taq DNA聚合酶、PCR缓冲体系和dNTP。
本发明还提供了一种筛选高抗性淀粉含量的水稻的方法,所述方法包括以下步骤:
(1)、以待筛选水稻的基因组DNA为模板,SEQ ID NO.5~SEQ ID NO.8或SEQ IDNO.5、SEQ ID NO.6、SEQ ID NO.9和SEQ ID NO.10为引物,进行PCR扩增;
(2)、核酸电泳检测扩增产物的谱带,鉴定水稻蜡质基因Wx第9个外显子第202位处是否具有T→C的碱基突变。
本发明还提供了一种筛选高抗性淀粉含量的水稻的方法,所述方法包括以下步骤:
(1)、以待筛选水稻的基因组DNA为模板,SEQ ID NO.3和SEQ ID NO.4为引物,进行PCR扩增;
(2)、对PCR扩增产物进行测序,检测水稻蜡质基因Wx第9个外显子第202位处是否具有T→C的碱基突变。
在其中一些实施例中,步骤(2)中还包括检测扩增产物中水稻蜡质基因Wx第10个外显子的第115位处是否具有C→T的碱基突变,或扩增产物的氨基酸序列第415位是否具有脯氨酸→丝氨酸的氨基酸突变,可以据此筛选出适口性较好的高抗性淀粉含量水稻品种。
与现有技术相比,本发明具有以下有益效果:
1、本申请的发明人通过大量的实验证明,水稻蜡质基因Wx第9个外显子第202位的T→C的碱基突变,会使水稻中抗性淀粉的含量提高,因此,水稻蜡质基因Wx的突变基因是与高抗性淀粉含量密切相关的基因;根据该突变基因的突变位点,以扩增受阻为原理设计引物对,再对水稻基因组DNA进行扩增,通过核酸电泳,即可有效地检测水稻品种是否为高抗性淀粉含量的水稻品种,并且能够对幼苗期水稻的基因型进行快速鉴定,从而可以有选择性地移栽,大大提高了高抗性淀粉含量的水稻品种的选择效率,加速育种进程。
2、本发明的筛选高抗性淀粉含量的水稻的方法,步骤简单快捷、稳定性可靠、准确率高、重复性好,适用于抗性淀粉分子标记辅助育种,可以省去复杂的抗性淀粉含量测定过程,节省育种成本。
附图说明
图1为本发明实施例1不同的水稻品种中水稻蜡质基因Wx第9个外显子处的核苷酸序列,其中黑色背景表示相同的核苷酸序列,灰色背景表示第202位处具有T→C的碱基突变。
图2为本发明实施例1水稻蜡质基因Wx第10个外显子处的核苷酸序列,其中黑色背景表示不同的水稻品种间相同的核苷酸序列,灰色背景表示第115位处具有C→T的碱基突变。
图3为本发明实施例2中使用引物组W9-IF1、W9-IR1、W9-OF1、W9-OR1对部分单株水稻基因组DNA进行扩增后的核酸电泳检测结果。
图4为本发明实施例2中使用引物组W9-IF1、W9-IR1、W9-OF2、W9-OR2对部分单株水稻基因组DNA进行扩增后的核酸电泳检测结果。
具体实施方式
为了便于理解本发明,下面将对本发明进行更全面的描述。本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明公开内容的理解更加透彻全面。
除非另有定义,本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不用于限制本发明。本发明所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
在本发明的第一个方面,提供了一种水稻蜡质基因Wx的突变基因,其可以编码水稻结合态淀粉合成酶,该突变基因的核苷酸序列如SEQ ID NO.1所示,其编码的氨基酸序列如SEQ ID NO.2所示。与水稻蜡质基因Wx相比,该突变基因在第9个外显子的第202位处具有T→C的碱基突变(同义突变,不造成氨基酸序列的改变),第10个外显子的第115位处具有C→T的碱基突变(造成脯氨酸突变为丝氨酸)。
SEQ ID NO.1(3039bp):涵盖了水稻蜡质基因Wx从第4个外显子至第14个外显子的编码区和非编码区,与水稻蜡质基因Wx相比,缺失4个碱基(“-”表示),第9个外显子的第202位处具有T→C的碱基突变,第10个外显子的第115位处具有C→T的碱基突变,另外还包括几处内含子的碱基突变。
GCAGATCAAGGTTGCAGACAGGTACGAGAGGGTGAGGTTTTTCCATTGCTACAAGCGTGGAGTCGACCGTGTGTTCATCGACCATCCGTCATTCCTGGAGAAGGTGGAGTCATCATTAGTTTACCTTTTTTGTTTTTACTGAATTATTAACAGTGCATTTAGCAGTTGGACTGAGCTTAGCTTCCACTGGTGATTTCAGGTTTGGGGAAAGACCGGTGAGAAGATCTACGGACCTGACACTGGAGTTGATTACAAAGACAACCAGATGCGTTTCAGCCTTCTTTGCCAGGTCAGTGATTACTTCTATCTGATGATGGTTGGAAGCATCACGAGTTTACCATAGTATGTATGGATTCATAACTAATTCGTGTATTGATGCTAC-TGCAGGCAGCACTCGAGGCTCCTAGGATCCTAAACCTCAACAACAACCCATACTTCAAAGGAACTTATGGTGAGTTATAATTGATCTCAAGATCTTATAACTTTCTTCGAAGGAATCCATGATGATCAGACTAATTCCTTCCGGTTTGTTACTGACAACAGGTGAGGATGTTGTGTTCGTCTGCAACGACTGGCACACTGGCCCACTGGCGAGCTACCTGAAGAACAACTACCAGCCCAATGGCATCTACAGGAATGCAAAGGTCTATGCTTGTTCTTGCCATACCAACTCAAATCTGCATGCACACTGCATTCTGTTCAGAAACTGACTGTCTGAATCTTTTTCACTGCAGGTTGCTTTCTGCATCCACAACATCTCCTACCAGGGCCGTTTCGCTTTCGAGGATTACCCTGAGCTGAACCTCTCCGAGAGGTTCAGGTCATCCTTCGATTTCATCGACGGGTATGAGTAAGATTCTAAGAGTAACTTACTGTCAATTCGCCATATATCGATTCAATCCAAGATCCTTTTGAGCTGACAACCCTGCACTACTGTCCATCGTTCAAATCCGGTTAAATTTCAGGTATGACACGCCGGTGGAGGGCAGGAAGATCAACTGGATGAAGGCCGGAATCCTGGAAGCCGACAGGGTGCTCACCGTGAGCCCGTACTACGCCGAGGAGCTCATCTCCGGCATCGCCAGGGGATGCGAGCTCGACAACATCATGCGGCTCACCGGCATCACCGGCATCGTCAACGGCATGGACGTCAGCGAGTGGGATCCCAGCAAGGACAAGTACATCACCGCCAAGTACGACGCAACCACGGTAAGAACGAATGCATTCTTCACAAGATATGCAATCTGAATTTTCTTTGAAAAAGAAATTATCATCTGTCACTTCTTGATTGATTCTGACAAGGCAAGAATGAGTGACAAATTTCAGGCAATCGAGGCGAAGGCGCTGAACAAGGAGGCGTTGCAGGCGGAGGCGGGTCTTCCGGTCGACAGGAAAATCCCACTGATCGCGTTCATCGGCAGGCTGGAGGAACAGAAGGGCTCTGACGTCATGGCCGCCGCCATCCCGGAGCTCATGCAGGAGGACGTCCAGATCGTTCTTCTGGTATAATATAATACACTACAAGACACACTTGCACGATATGCCAAAAATTCAGAACAAATTCAGTGGCAAAAAAAAAACTCAAATATTAGGGAAGAACCTAAT---ATCAAATAATTAGAAGGGGTGAGGCTTTGAACCCAGGTCATCTAGCCCACCACCTTGTAGAGCTAGCCGGAAGAGCTCTGAGCATTTCTCGATTCAGTGGCAAATGATGTGTATAATTTTGATCCGTGTGTGTTTCAGGGTACTGGAAAGAAGAAGTTCGAGAAGCTGCTCAAGAGCATGGAGGAGAAGTATCCGGGCAAGGTGAGGGCCGTGGTGAAGTTCAACGCGCCGCTTGCTCATCTCATCATGGCCGGAGCCGACGTGCTCGCCGTCCCCAGCCGCTTCGAGCCCTGTGGACTCATCCAGCTGCAGGGGATGAGATACGGAACGGTATACAATTTCCATCTATCAATTCGATTGTTCGATTTCATCTTTGTGCAATGCAATGCAATTGCAAATGCAAATGCATGATGATTTTCCTTGTTGATTTCTCCAGCCCTGTGCTTGCGCGTCCACCGGTGGGCTCGTGGACACGGTCATCGAAGGCAAGACTGGTTTCCACATGGGCCGTCTCAGCGTCGACGTAAGCCTATACATTTACATAACAATCAGATATGACACATTCTAATACCGATAAGTCAGTACACTACTACACATTTACATGGTTGCTGGTTATATGGTTTTTTTGGCAGTGCAAGGTGGTGGAGCCAAGCGACGTGAAGAAGGTGGCGGCCACCCTGAAGCGCGCCATCAAGGTCGTCGGCACGCCGGCGTACGAGGAGATGGTCAGGAACTGCATGAACCAGGACCTCTCCTGGAAGGTATAAATTACGAAACAAATTTAACCCAAACATATACTATATACTCCCTCCGCTTCTAAATATTCAACGCCGTTGTCTTTTTAAAATATGTTTGACCGTTCGTCTTATTAAAAAAATTAAATAATTATAAATTATTTTCCTATCATTTGATTCATTGTTAAATATACTTATATGTATACATATAGTTTTACATATTTCATAAAAGTTTTTGAACAAGACGAACGGTCAAACATGTGCTAAAAAGTTAACGGTGTCGAATATTCAGAAACGGAGGGAGTATAAACGTCTTGTTCAGAAGTTCAGAGATTCACCTGTCTGATGCTGATGATGATTAATTGTTTGCAACATGGATTTCAGGGGCCTGCGAAGAACTGGGAGAATGTGCTCCTGGGCCTGGGCGTCGCCGGCAGCGCGCCGGGGATCGAAGGCGACGAGATCGCGCCGCTCGCCAAGGAGAACGTGGCTGCTCCTTGAAGAGCCTGAGATCTACATATGGAGTGATTAATTAATATAGCAGTATATGGATGAGAGACGAATGAACCAGTGGTTTGTTTGTTGTAGTGAATTTGTAGCTATAGCCAATTATATAGGCTAATAAGTTTGATGTTGTACTCTTCTGGGTGTGCTTAAGTATCTTATCGGACCCTGAATTTATGTGTGTGGCTTATTGCCA
在本发明的第二个方面,提供了一种水稻蜡质基因Wx的突变基因的编码蛋白,所述编码蛋白的氨基酸序列如SEQ ID NO.2所示。与水稻蜡质基因Wx的编码蛋白相比,该突变基因的编码蛋白在415位具有脯氨酸→丝氨酸的氨基酸突变。
SEQ ID NO.2
MSALTTSQLATSATGFGIADRSAPSSLLRHGFQGLKPRSPAGGDATSLSVTTSARATPKQQRSVQRGSRRFPSVVVYATGAGMNVVFVGAEMAPWSKTGGLGDVLGGLPPAMAANGHRVMVISPRYDQYKDAWDTSVVAEIKVADRYERVRFFHCYKRGVDRVFIDHPSFLEKVWGKTGEKIYGPDTGVDYKDNQMRFSLLCQAALEAPRILNLNNNPYFKGTYGEDVVFVCNDWHTGPLASYLKNNYQPNGIYRNAKVAFCIHNISYQGRFAFEDYPELNLSERFRSSFDFIDGYDTPVEGRKINWMKAGILEADRVLTVSPYYAEELISGIARGCELDNIMRLTGITGIVNGMDVSEWDPSKDKYITAKYDATTAIEAKALNKEALQAEAGLPVDRKIPLIAFIGRLEEQKGSDVMAAAIPELMQEDVQIVLLGTGKKKFEKLLKSMEEKYPGKVRAVVKFNAPLAHLIMAGADVLAVPSRFEPCGLIQLQGMRYGTPCACASTGGLVDTVIEGKTGFHMGRLSVDCKVVEPSDVKKVAATLKRAIKVVGTPAYEEMVRNCMNQDLSWKGPAKNWENVLLGLGVAGSAPGIEGDEIAPLAKENVAAP
在本发明的第三方面,提供了上述水稻蜡质基因Wx的突变基因或其编码的蛋白在筛选高抗性淀粉含量的水稻中的应用。包含上述水稻蜡质基因Wx的突变基因或其编码的蛋白的水稻品种,其抗性淀粉含量显著高于不含上述水稻蜡质基因Wx的突变基因或其编码的蛋白的水稻品种。
在本发明的第四方面,提供了一种筛选高抗性淀粉含量的水稻的引物对,所述引物对包括两组:一组为SEQ ID NO.5~SEQ ID NO.8;另一组为SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.9和SEQ ID NO.10。所述引物对是以水稻蜡质基因Wx第9个外显子第202位的核酸序列为目标进行设计的。所述引物对可以作为筛选高抗性淀粉含量的水稻品种的分子标记,通过所述引物对能够准确筛选高抗性淀粉含量的水稻品种,从而加快高抗性淀粉含量水稻品种的选择进度,提高育种选择效率。
抗性淀粉含量是一个数量性状,它是由很多个基因控制的,数量性状的主效基因的贡献值如果能够达到30%就算非常高了,本发明中的发明人发现:水稻蜡质基因Wx的突变基因是高抗性淀粉含量的主效基因,应用筛选高抗性淀粉含量的水稻的引物对(即分子标记)对水稻品种进行筛选后,淘汰纯合阴性和杂合型的单株,最起码占总单株数量的3/4以上,剩下的纯合阳性单株理论上都是高抗性淀粉单株,由此,可以大大节约水稻品种筛选的工作量和成本。
本发明的第五方面,提供了一种用于筛选高抗性淀粉含量的水稻品种的试剂盒,所述试剂盒包括上述两组引物对的其中一组或两组,所述引物对为SEQ ID NO.5~SEQ IDNO.8;和/或SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.9和SEQ ID NO.10;所述试剂盒还包括Taq DNA聚合酶、PCR缓冲体系和dNTP。
在本发明的第六方面,提供了一种筛选高抗性淀粉含量的水稻的方法,所述方法包括以下步骤:
(1)、以待筛选水稻基因组DNA为模板,SEQ ID NO.5~SEQ ID NO.8为引物,进行PCR扩增;
(2)、核酸电泳检测扩增产物的谱带,若是190bp一条谱带,则是低抗性淀粉含量水稻品种;若是有350bp和215bp两条谱带,则是高抗性淀粉含量水稻品种的纯合基因型(水稻蜡质基因Wx第9个外显子第202位处双链具有T→C的碱基突变);若是有190bp和350bp两条谱带,则是高抗性淀粉含量水稻品种的杂合基因型(水稻蜡质基因Wx第9个外显子第202位处单链具有T→C的碱基突变)。
或所述方法包括以下步骤:
(1)、以待筛选水稻的基因组DNA为模板,SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.9和SEQ ID NO.10为引物,进行PCR扩增;
(2)、核酸电泳检测扩增产物的谱带,若是189bp一条谱带,则是低抗性淀粉含量水稻品种;若是有149bp一条谱带,则是高抗性淀粉含量水稻品种的纯合基因型(水稻蜡质基因Wx第9个外显子第202位处双链具有T→C的碱基突变);若是有189bp和149bp两条谱带,则是高抗性淀粉含量水稻品种的杂合基因型(水稻蜡质基因Wx第9个外显子第202位处单链具有T→C的碱基突变)。
W9-IF1:GCATGGACGTCAGCGAGTGGGATACT(SEQ ID NO.5)
W9-IR1:ACTTGGCGGTGATGTACTTGTCCTTGATG(SEQ ID NO.6)
W9-OF1:GGAGGGCAGGAAGATCAACTGGATGAA(SEQ ID NO.7)
W9-OR1:TTGCCTGAAATTTGTCACTCATTCTTGCC(SEQ ID NO.8)
W9-OF2:CCCGTACTACGCCGAGGAGCTCATCT(SEQ ID NO.9)
W9-OR2:TGCCTGAAATTTGTCACTCATTCTTGCCTT(SEQ ID NO.10)
本发明的第七方面,提供了一种筛选适口性好的高抗性淀粉含量的水稻的方法,所述方法包括以下步骤:
(1)、以待筛选水稻的基因组DNA为模板,SEQ ID NO.3和SEQ ID NO.4为引物,进行PCR扩增;
WL3:GCAGATCAAGGTTGCAGACA(SEQ ID NO.3)
WR:TGGCAATAAGCCACACACAT(SEQ ID NO.4)
(2)、通过测序,检测扩增产物中水稻蜡质基因Wx第10个外显子的第115位处是否具有C→T的碱基突变,或扩增产物的氨基酸序列第415位是否具有脯氨酸→丝氨酸的氨基酸突变,没有此碱基突变或氨基酸突变的品种为适口性好的高抗性淀粉含量的水稻品种。
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的药材原料、试剂材料等,如无特殊说明,均为市售购买产品。
以下实施例中,抗性淀粉含量测定应用Megazyme公司提供的抗性淀粉含量测定试剂盒(Megazyme,Co.Wicklow,Ireland)进行测定,略有改进。具体步骤为:准确称取100mg米粉样品,小心放入带螺旋盖的塑料试管中,依次加入α-胰淀粉酶反应液和淀粉葡萄糖昔酶(AGM),37℃震荡孵育16小时,非抗性淀粉被溶解,水解成D-葡萄糖;孵育结束后加入等体积无水乙醇终止反应;离心上述溶液,弃上清,底部残留絮状团即为样品中的抗性淀粉,再用50%乙醇洗涤沉淀,洗涤后离心,再重复一次洗涤离心;倒置离心管,沉淀干燥后用2M KOH溶解沉淀,用醋酸盐缓冲液将这个溶液调至中性,并加入AGM,置于50℃水浴中孵育30分钟将抗性淀粉水解成葡萄糖,最后用葡糖氧化酶/过氧化物酶试剂(GOPOD)试剂测定葡萄糖含量,并计算抗性淀粉含量(以重量百分比计,简称RS(%))。
实施例1水稻蜡质基因Wx的突变基因及分子标记的获得
(一)、筛选高抗性淀粉含量水稻资源
从国内外广泛搜集各类水稻资源若干份,如糯稻资源、中低直链淀粉水稻资源、高直链淀粉水稻资源等,重点搜集直链淀粉含量高的水稻资源,如云南等地的“米粉稻”资源,印度、孟加拉、非洲等地的“手抓饭”资源。采用抗性淀粉含量测定试剂盒测定抗性淀粉含量,筛选出RS含量大于6%的水稻资源材料10份。14份不同水稻品种的直链淀粉含量(AC)和抗性淀粉(RS)含量如表1所示。
表1不同水稻品种直链淀粉含量和抗性淀粉含量
(二)、水稻蜡质基因Wx的克隆
(1)、用CTAB法提取上述14份不同RS含量水稻资源的基因组DNA
水稻小量DNA提取法,主要参考McCouch等(1988)的报道,方法简述如下:
1)、剪取一小片叶片4~5cm,加入700μl 1.5X CTAB(含1.5%CTAB、75mMTris-HCl、15mM EDTA、1.05M NaCl),充分研磨;
2)、将匀浆转入1.5ml的离心管,56℃水浴30min后冷却至室温;
3)、加入等体积的氯仿:异戊醇(24:1),摇匀;
4)、最高速度(12000rpm)离心10min;
5)、将上清液转入新的离心管,并加入0.6倍体积的预冷的异丙醇,静止20min后离心收集DNA;
6)、去上清,风干DNA,加50~100μl超纯水溶解,于紫外分光光度计中检测。稀释DNA,制备一套DNA工作溶液,其浓度为50~100ng/μL左右,4℃冰箱保存备用。
(2)、根据NCBI里的水稻Wx基因序列设计引物WL3(SEQ ID NO.3)和WR(SEQ IDNO.4),以14份不同RS含量水稻材料的基因组DNA为模板进行扩增,得到PCR产物;
WL3:GCAGATCAAGGTTGCAGACA(SEQ ID NO.3)
WR:TGGCAATAAGCCACACACAT(SEQ ID NO.4)
(3)、将PCR产物直接送生工生物工程(上海)有限公司测序,得到3043bp片段,测序结果与NCBI数据库中的序列进行比对,分析高抗性淀粉水稻材料与低抗性淀粉水稻材料之间的异同,通过比对分析,高抗性淀粉水稻材料相对于低抗性淀粉水稻材料的蜡质基因Wx,缺失4个碱基,且第9个外显子的第202位处都具有T→C的碱基突变;而第10个外显子的第115位处绝大部分具有C→T的碱基突变,而少数材料没有突变;另外还包括几处内含子的碱基突变。
(三)、高抗性淀粉分子标记的开发
针对水稻蜡质基因Wx第9个外显子的第202位处T→C的碱基突变,在线设计(http://primer1.soton.ac.uk/primer1.html)两组扩增受阻突变体系引物SEQ ID NO.5~SEQ ID NO.8、以及SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.9和SEQ ID NO.10,作为检测高抗性淀粉含量紧密相关的分子标记,引物对的序列分别如下:
W9-IF1:GCATGGACGTCAGCGAGTGGGATACT(SEQ ID NO.5)
W9-IR1:ACTTGGCGGTGATGTACTTGTCCTTGATG(SEQ ID NO.6)
W9-OF1:GGAGGGCAGGAAGATCAACTGGATGAA(SEQ ID NO.7)
W9-OR1:TTGCCTGAAATTTGTCACTCATTCTTGCC(SEQ ID NO.8)
W9-OF2:CCCGTACTACGCCGAGGAGCTCATCT(SEQ ID NO.9)
W9-OR2:TGCCTGAAATTTGTCACTCATTCTTGCCTT(SEQ ID NO.10)
实施例2实施例1中的分子标记的验证
1、材料
实施例1表1中的水稻品种的杂交F4代40个单株,包括:Lan/CN-5、Lan/CN-9、Lan/Sx、QR/Sx、QR/Ky43、Li/BR-50、QR/BR50杂交。
2、方法
(1)、采用实施例1的方法提取40个F4代单株样本DNA;
(2)、以步骤(1)的DNA作为模板,SEQ ID NO.5~SEQ ID NO.8或SEQ ID NO.5、SEQID NO.6、SEQ ID NO.9和SEQ ID NO.10作为引物,进行PCR扩增反应;反应体系为:2μ110XPCRbuffer(100mM Tris-HCl pH 8.0、15mM MgCl2、500mM KCl、1%TritonX-100、0.2mMdNTPs、上游引物各0.2μM、下游引物各0.2μM,50-100ng样本DNA、0.625U Taq酶;反应程序为:94℃预变性4min,循环(94℃30s,60℃30s,72℃1min)35次,最后72℃延伸10min。
(3)、PCR扩增产物经10%聚丙烯酰胺凝胶电泳分析检测多态性,后代与低抗性淀粉亲本基因型一致的标记为“-”,与高抗性淀粉亲本基因型一致的标记为“+”,杂合型的标记为H。
a、当利用SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8所示的引物对,对水稻基因组DNA进行PCR扩增后;核酸电泳检测,若是有190bp一条谱带,则是低抗性淀粉含量水稻品种;若是有350bp和215bp两条谱带,则是高抗性淀粉含量水稻品种的纯合基因型;若是有190bp和350bp两条谱带,则是高抗性淀粉含量水稻品种的杂合基因型。
b、当利用SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.9、SEQ ID NO.10所示的引物对,对水稻基因组DNA进行PCR扩增后;核酸电泳检测,若是有189bp一条谱带,则是低抗性淀粉含量水稻品种;若是有149bp一条谱带,则是高抗性淀粉含量水稻品种的纯合基因型;若是有189bp和149bp两条谱带,则是高抗性淀粉含量水稻品种的杂合基因型。
3、结果
对F4代群体的40个单株的分子标记进行验证,部分单株的核苷酸电泳检测结果见图3和图4。结果表明,分离群体单株出现3种带型,即分别与低抗性淀粉亲本、高抗性淀粉亲本和杂合型一致。带型均与RS抗性淀粉含量测定结果相对应。具体的结果统计如表1所示。
表1 40个单株的基因型和对应的抗性淀粉含量测定结果
从表1结果可知,40个F4代单株中,有23个单株基因型与低抗性淀粉亲本一致(不含有水稻蜡质基因Wx的突变基因),其抗性淀粉含量以重量百分比计为0.41%~1.99%,平均值为0.88%;8个单株的基因型与高抗性淀粉亲本基因型一致(含有水稻蜡质基因Wx的突变基因,且是纯合型),其抗性淀粉含量为3.10%~11.10%,平均值为5.91%;9株基因型为杂合型(一条链含有水稻蜡质基因Wx的突变基因,一条链不含有水稻蜡质基因Wx的突变基因),其抗性淀粉含量为1.42%~4.00%,平均值为3.00%。
本实施例的结果说明:利用本发明实施例1提供的分子标记能够准确筛选出高抗性淀粉含量的纯合单株。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 重庆市农业科学院
<120> 水稻蜡质基因Wx的突变基因及其应用
<130> 1
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3039
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gcagatcaag gttgcagaca ggtacgagag ggtgaggttt ttccattgct acaagcgtgg 60
agtcgaccgt gtgttcatcg accatccgtc attcctggag aaggtggagt catcattagt 120
ttaccttttt tgtttttact gaattattaa cagtgcattt agcagttgga ctgagcttag 180
cttccactgg tgatttcagg tttggggaaa gaccggtgag aagatctacg gacctgacac 240
tggagttgat tacaaagaca accagatgcg tttcagcctt ctttgccagg tcagtgatta 300
cttctatctg atgatggttg gaagcatcac gagtttacca tagtatgtat ggattcataa 360
ctaattcgtg tattgatgct actgcaggca gcactcgagg ctcctaggat cctaaacctc 420
aacaacaacc catacttcaa aggaacttat ggtgagttat aattgatctc aagatcttat 480
aactttcttc gaaggaatcc atgatgatca gactaattcc ttccggtttg ttactgacaa 540
caggtgagga tgttgtgttc gtctgcaacg actggcacac tggcccactg gcgagctacc 600
tgaagaacaa ctaccagccc aatggcatct acaggaatgc aaaggtctat gcttgttctt 660
gccataccaa ctcaaatctg catgcacact gcattctgtt cagaaactga ctgtctgaat 720
ctttttcact gcaggttgct ttctgcatcc acaacatctc ctaccagggc cgtttcgctt 780
tcgaggatta ccctgagctg aacctctccg agaggttcag gtcatccttc gatttcatcg 840
acgggtatga gtaagattct aagagtaact tactgtcaat tcgccatata tcgattcaat 900
ccaagatcct tttgagctga caaccctgca ctactgtcca tcgttcaaat ccggttaaat 960
ttcaggtatg acacgccggt ggagggcagg aagatcaact ggatgaaggc cggaatcctg 1020
gaagccgaca gggtgctcac cgtgagcccg tactacgccg aggagctcat ctccggcatc 1080
gccaggggat gcgagctcga caacatcatg cggctcaccg gcatcaccgg catcgtcaac 1140
ggcatggacg tcagcgagtg ggatcccagc aaggacaagt acatcaccgc caagtacgac 1200
gcaaccacgg taagaacgaa tgcattcttc acaagatatg caatctgaat tttctttgaa 1260
aaagaaatta tcatctgtca cttcttgatt gattctgaca aggcaagaat gagtgacaaa 1320
tttcaggcaa tcgaggcgaa ggcgctgaac aaggaggcgt tgcaggcgga ggcgggtctt 1380
ccggtcgaca ggaaaatccc actgatcgcg ttcatcggca ggctggagga acagaagggc 1440
tctgacgtca tggccgccgc catcccggag ctcatgcagg aggacgtcca gatcgttctt 1500
ctggtataat ataatacact acaagacaca cttgcacgat atgccaaaaa ttcagaacaa 1560
attcagtggc aaaaaaaaaa ctcaaatatt agggaagaac ctaatatcaa ataattagaa 1620
ggggtgaggc tttgaaccca ggtcatctag cccaccacct tgtagagcta gccggaagag 1680
ctctgagcat ttctcgattc agtggcaaat gatgtgtata attttgatcc gtgtgtgttt 1740
cagggtactg gaaagaagaa gttcgagaag ctgctcaaga gcatggagga gaagtatccg 1800
ggcaaggtga gggccgtggt gaagttcaac gcgccgcttg ctcatctcat catggccgga 1860
gccgacgtgc tcgccgtccc cagccgcttc gagccctgtg gactcatcca gctgcagggg 1920
atgagatacg gaacggtata caatttccat ctatcaattc gattgttcga tttcatcttt 1980
gtgcaatgca atgcaattgc aaatgcaaat gcatgatgat tttccttgtt gatttctcca 2040
gccctgtgct tgcgcgtcca ccggtgggct cgtggacacg gtcatcgaag gcaagactgg 2100
tttccacatg ggccgtctca gcgtcgacgt aagcctatac atttacataa caatcagata 2160
tgacacattc taataccgat aagtcagtac actactacac atttacatgg ttgctggtta 2220
tatggttttt ttggcagtgc aaggtggtgg agccaagcga cgtgaagaag gtggcggcca 2280
ccctgaagcg cgccatcaag gtcgtcggca cgccggcgta cgaggagatg gtcaggaact 2340
gcatgaacca ggacctctcc tggaaggtat aaattacgaa acaaatttaa cccaaacata 2400
tactatatac tccctccgct tctaaatatt caacgccgtt gtctttttaa aatatgtttg 2460
accgttcgtc ttattaaaaa aattaaataa ttataaatta ttttcctatc atttgattca 2520
ttgttaaata tacttatatg tatacatata gttttacata tttcataaaa gtttttgaac 2580
aagacgaacg gtcaaacatg tgctaaaaag ttaacggtgt cgaatattca gaaacggagg 2640
gagtataaac gtcttgttca gaagttcaga gattcacctg tctgatgctg atgatgatta 2700
attgtttgca acatggattt caggggcctg cgaagaactg ggagaatgtg ctcctgggcc 2760
tgggcgtcgc cggcagcgcg ccggggatcg aaggcgacga gatcgcgccg ctcgccaagg 2820
agaacgtggc tgctccttga agagcctgag atctacatat ggagtgatta attaatatag 2880
cagtatatgg atgagagacg aatgaaccag tggtttgttt gttgtagtga atttgtagct 2940
atagccaatt atataggcta ataagtttga tgttgtactc ttctgggtgt gcttaagtat 3000
cttatcggac cctgaattta tgtgtgtggc ttattgcca 3039
<210> 2
<211> 609
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ser Ala Leu Thr Thr Ser Gln Leu Ala Thr Ser Ala Thr Gly Phe
1 5 10 15
Gly Ile Ala Asp Arg Ser Ala Pro Ser Ser Leu Leu Arg His Gly Phe
20 25 30
Gln Gly Leu Lys Pro Arg Ser Pro Ala Gly Gly Asp Ala Thr Ser Leu
35 40 45
Ser Val Thr Thr Ser Ala Arg Ala Thr Pro Lys Gln Gln Arg Ser Val
50 55 60
Gln Arg Gly Ser Arg Arg Phe Pro Ser Val Val Val Tyr Ala Thr Gly
65 70 75 80
Ala Gly Met Asn Val Val Phe Val Gly Ala Glu Met Ala Pro Trp Ser
85 90 95
Lys Thr Gly Gly Leu Gly Asp Val Leu Gly Gly Leu Pro Pro Ala Met
100 105 110
Ala Ala Asn Gly His Arg Val Met Val Ile Ser Pro Arg Tyr Asp Gln
115 120 125
Tyr Lys Asp Ala Trp Asp Thr Ser Val Val Ala Glu Ile Lys Val Ala
130 135 140
Asp Arg Tyr Glu Arg Val Arg Phe Phe His Cys Tyr Lys Arg Gly Val
145 150 155 160
Asp Arg Val Phe Ile Asp His Pro Ser Phe Leu Glu Lys Val Trp Gly
165 170 175
Lys Thr Gly Glu Lys Ile Tyr Gly Pro Asp Thr Gly Val Asp Tyr Lys
180 185 190
Asp Asn Gln Met Arg Phe Ser Leu Leu Cys Gln Ala Ala Leu Glu Ala
195 200 205
Pro Arg Ile Leu Asn Leu Asn Asn Asn Pro Tyr Phe Lys Gly Thr Tyr
210 215 220
Gly Glu Asp Val Val Phe Val Cys Asn Asp Trp His Thr Gly Pro Leu
225 230 235 240
Ala Ser Tyr Leu Lys Asn Asn Tyr Gln Pro Asn Gly Ile Tyr Arg Asn
245 250 255
Ala Lys Val Ala Phe Cys Ile His Asn Ile Ser Tyr Gln Gly Arg Phe
260 265 270
Ala Phe Glu Asp Tyr Pro Glu Leu Asn Leu Ser Glu Arg Phe Arg Ser
275 280 285
Ser Phe Asp Phe Ile Asp Gly Tyr Asp Thr Pro Val Glu Gly Arg Lys
290 295 300
Ile Asn Trp Met Lys Ala Gly Ile Leu Glu Ala Asp Arg Val Leu Thr
305 310 315 320
Val Ser Pro Tyr Tyr Ala Glu Glu Leu Ile Ser Gly Ile Ala Arg Gly
325 330 335
Cys Glu Leu Asp Asn Ile Met Arg Leu Thr Gly Ile Thr Gly Ile Val
340 345 350
Asn Gly Met Asp Val Ser Glu Trp Asp Pro Ser Lys Asp Lys Tyr Ile
355 360 365
Thr Ala Lys Tyr Asp Ala Thr Thr Ala Ile Glu Ala Lys Ala Leu Asn
370 375 380
Lys Glu Ala Leu Gln Ala Glu Ala Gly Leu Pro Val Asp Arg Lys Ile
385 390 395 400
Pro Leu Ile Ala Phe Ile Gly Arg Leu Glu Glu Gln Lys Gly Ser Asp
405 410 415
Val Met Ala Ala Ala Ile Pro Glu Leu Met Gln Glu Asp Val Gln Ile
420 425 430
Val Leu Leu Gly Thr Gly Lys Lys Lys Phe Glu Lys Leu Leu Lys Ser
435 440 445
Met Glu Glu Lys Tyr Pro Gly Lys Val Arg Ala Val Val Lys Phe Asn
450 455 460
Ala Pro Leu Ala His Leu Ile Met Ala Gly Ala Asp Val Leu Ala Val
465 470 475 480
Pro Ser Arg Phe Glu Pro Cys Gly Leu Ile Gln Leu Gln Gly Met Arg
485 490 495
Tyr Gly Thr Pro Cys Ala Cys Ala Ser Thr Gly Gly Leu Val Asp Thr
500 505 510
Val Ile Glu Gly Lys Thr Gly Phe His Met Gly Arg Leu Ser Val Asp
515 520 525
Cys Lys Val Val Glu Pro Ser Asp Val Lys Lys Val Ala Ala Thr Leu
530 535 540
Lys Arg Ala Ile Lys Val Val Gly Thr Pro Ala Tyr Glu Glu Met Val
545 550 555 560
Arg Asn Cys Met Asn Gln Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn
565 570 575
Trp Glu Asn Val Leu Leu Gly Leu Gly Val Ala Gly Ser Ala Pro Gly
580 585 590
Ile Glu Gly Asp Glu Ile Ala Pro Leu Ala Lys Glu Asn Val Ala Ala
595 600 605
Pro
Claims (10)
1.一种水稻蜡质基因Wx的突变基因,其特征在于,所述突变基因的核苷酸序列如SEQID NO.1所示。
2.一种水稻蜡质基因Wx的突变基因的编码蛋白,其特征在于,所述编码蛋白的氨基酸序列如SEQ ID NO.2所示。
3.权利要求1所述的突变基因或权利要求2所述的突变基因的编码蛋白在筛选高抗性淀粉含量的水稻中的应用。
4.一种筛选高抗性淀粉含量的水稻的引物对,其特征在于,所述引物对包括核苷酸序列如SEQ ID NO.5~SEQ ID NO.8所示的引物。
5.一种筛选高抗性淀粉含量的水稻的引物对,其特征在于,所述引物对包括核苷酸序列如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.9和SEQ ID NO.10所示的引物。
6.一种筛选高抗性淀粉含量的水稻的试剂盒,其特征在于,所述试剂盒包括权利要求4所示的引物对,或所述试剂盒包括权利要求5所示的引物对。
7.根据权利要求6所述的筛选高抗性淀粉含量的水稻的试剂盒,其特征在于,所述试剂盒还包括Taq DNA聚合酶、PCR缓冲体系和dNTP。
8.一种筛选高抗性淀粉含量的水稻的方法,其特征在于,所述方法包括以下步骤:
(1)、以待筛选水稻的基因组DNA为模板,SEQ ID NO.5~SEQ ID NO.8或SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.9和SEQ ID NO.10为引物,进行PCR扩增;
(2)、核酸电泳检测扩增产物的谱带,鉴定水稻蜡质基因Wx第9个外显子第202位处是否具有T→C的碱基突变。
9.一种筛选高抗性淀粉含量的水稻的方法,其特征在于,所述方法包括以下步骤:
(1)、以待筛选水稻的基因组DNA为模板,SEQ ID NO.3和SEQ ID NO.4为引物,进行PCR扩增;
(2)、对PCR扩增产物进行测序,检测水稻蜡质基因Wx第9个外显子第202位处是否具有T→C的碱基突变。
10.根据权利要求9所述的筛选高抗性淀粉含量的水稻的方法,其特征在于,步骤(2)中还包括检测扩增产物中水稻蜡质基因Wx第10个外显子的第115位处是否具有C→T的碱基突变,或检测扩增产物中水稻蜡质基因Wx编码的氨基酸序列第415位是否具有脯氨酸→丝氨酸的氨基酸突变。
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