CN114058600A - 一种具有草铵膦抗性的谷氨酰胺合成酶突变体及其应用 - Google Patents
一种具有草铵膦抗性的谷氨酰胺合成酶突变体及其应用 Download PDFInfo
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- CN114058600A CN114058600A CN202111352238.7A CN202111352238A CN114058600A CN 114058600 A CN114058600 A CN 114058600A CN 202111352238 A CN202111352238 A CN 202111352238A CN 114058600 A CN114058600 A CN 114058600A
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- glutamine synthetase
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Abstract
本发明公开了一种具有草铵膦抗性的谷氨酰胺合成酶突变体及其应用,涉及基因工程领域。通过对野生型谷氨酰胺合成酶的第n位进行突变从而获得具有草铵膦抗性的谷氨酰胺合成酶突变体,突变后为A、C、D、E、F、G、H、I、L、M、N、Q、R、S、T、V、W、或删除,该突变赋予谷氨酰胺合成酶草铵膦抗性。转化该谷氨酰胺合成酶突变体的植物不仅具有草铵膦抗性,也能够正常生长和发育。因此,该谷氨酰胺合成酶突变体可以用于培育草铵膦抗性的植物新品种、草铵膦抗性的重组菌以及重组细胞。
Description
技术领域
本发明涉及基因工程技术领域,具体而言,涉及一种具有草铵膦抗性的谷氨酰胺合成酶突变体及其应用。
背景技术
目前,培育草铵膦抗性品种主要是通过基因工程技术将外源的草铵膦抗性基因导入植物中。但是由于反转基因浪潮,转基因作物在全世界的接受程度仍然较低,即使在转基因作物种植面积最大的美洲,转基因也主要局限于玉米、大豆、棉花等几个作物。特别是bar基因和pat基因来源于微生物,而不是来源于农作物本身,更容易造成消费者的抵触心理。
bar基因和pat基因编码的草铵膦乙酰化酶可以使草铵膦乙酰化而失活,但是在草铵膦接触谷氨酰胺合成酶之前,草铵膦乙酰化酶很难使草铵膦彻底失活,由于很多谷氨酰胺合成酶分布在细胞膜上,部分未失活的草铵膦可以抑制细胞膜上谷氨酰胺合成酶的活性,从而干扰植物的氮代谢。因此草铵膦在转bar基因和pat基因农作物上应用时,会不同程度的干扰植物的氮代谢,同时影响植物正常的生长和发育。通过在植物中过量表达野生型谷氨酰胺合成酶虽然可以一定程度上降低转基因植物对草铵膦的敏感程度,但其对草铵膦的耐性程度远不足以商业化应用。
鉴于此,特提出本发明。
发明内容
本发明的目的在于提供一种具有草铵膦抗性的谷氨酰胺合成酶突变体及其应用以解决上述技术问题。
本发明是这样实现的:
本发明提供了一种具有草铵膦抗性的谷氨酰胺合成酶突变体,其如下(1)或(2)所示:
(1):其由来源于植物的野生型谷氨酰胺合成酶的第n位发生突变得到;第n位的位置通过如下方式确定:野生型谷氨酰胺合成酶与参考序列比对,野生型谷氨酰胺合成酶的第n位对应于参考序列的第158位,其中,参考序列的氨基酸序列如SEQ ID NO.1所示;
谷氨酰胺合成酶突变体的第n位的氨基酸为X,X包括A、C、D、E、F、G、H、I、L、M、N、Q、R、S、T、V、W、或删除;
(2):其与(1)所示的谷氨酰胺合成酶突变体至少具有85%以上的同一性、且与(1)所示的谷氨酰胺合成酶突变体在第n位的氨基酸相同、以及具有草铵膦抗性。
本发明提供的谷氨酰胺合成酶突变体原始来源于植物,通过突变后具有了草铵膦抗性,转化该谷氨酰胺合成酶突变体的植物不仅具有草铵膦抗性,也能够正常生长和发育。
发明人发现,将植物来源的野生型谷氨酰胺合成酶与参考序列进行比对,将其序列上对应于参考序列第158位的氨基酸位点即第n位进行突变,突变为A、C、D、E、F、G、H、I、L、M、N、Q、R、S、T、V、W、或删除,所得到的谷氨酰胺合成酶突变体具有草铵膦抗性,同时可以保持自身的生物酶催化活性,从而满足植物的正常氮代谢,维持植物的正常生长和发育。
此外,转化本发明提供的谷氨酰胺合成酶突变体的植物或重组菌也均能在草铵膦存在的条件下正常生长和发育,该植物谷氨酰胺合成酶突变体不仅用于转基因作物培育,也可应用于培育抗草铵膦非转基因植物或转基因植物例如水稻、烟草、大豆、玉米、小麦、油菜、棉花和高粱等,具有广阔的应用前景。
上述参考序列为水稻来源的野生型谷氨酰胺合成酶。
序列比对方法可使用Blast网站(https://blast.ncbi.nlm.nih.gov/Blast.cgi)进行Protein Blast比对;采用本领域熟知的其他序列比对方法或工具也可以得到相同的结果。
需要说明的是,野生型谷氨酰胺合成酶的第n位在其自身序列上可能是第158位(例如玉米、小麦、大豆、油菜等),但也可能不是第158位(例如花生对应为第159位),第n位的具体位置根据前述序列比对后确定,只要其通过与参考序列比对后,对应于参考序列第158位的位点即为本发明所述的第n位,也就是突变位点。
可选的,在本发明的一些实施方案中,上述植物包括不限于:小麦、水稻、大麦、燕麦、玉米、高粱、谷子、荞麦、黍稷、甘薯、马铃薯、棉花、油菜、芝麻、花生、向日葵、萝卜、胡萝卜、花椰菜、番茄、茄子、辣椒、韭菜、大葱、洋葱、韭葱、菠菜、芹菜、苋菜、莴苣、茼蒿、黄花菜、葡萄、草莓、甘蔗、烟草、芸薹属蔬菜、葫芦科植物、豆科植物、牧草、茶或木薯。
在一种可选的实施方式中,牧草包括不限于禾本科牧草或豆科牧草。禾本科牧草选自梯牧草、鸭茅、六月禾、细麦、羊茅、棕叶、狗尾草等;豆科牧草选自苜蓿、三叶草、三叶豆、巢菜、鸡眼草等。此外,在其他实施方式中,上述牧草也可选自草坪草。
在一种可选的实施方式中,芸薹属蔬菜包括不限于芜菁、白菜、芥菜、甘蓝、芥蓝、菜苔、苦芥、擎蓝、芸苔、青菜或甜菜。
在一种可选的实施方式中,葫芦科植物包括不限于黄瓜、西葫芦、南瓜、冬瓜、苦瓜、丝瓜、菜瓜、西瓜或甜瓜。
在一种可选的实施方式中,豆科植物包括不限于绿豆、蚕豆、豌豆、扁豆、大豆、菜豆、豇豆或毛豆。
所有植物的野生型谷氨酰胺合成酶都具有同源性,在植物体内具有基本相同的功能和结构域。因此,任意植物来源的野生型谷氨酰胺合成酶在第158位作上述突变后所得到的谷氨酰胺合成酶突变体都具有草铵膦抗性。因此,由任意植物来源的野生型谷氨酰胺合成酶作上述突变后得到的谷氨酰胺合成酶突变体均属于本发明的保护范围。
此外,本领域技术人员知晓并容易实现,在(1)所示的谷氨酰胺合成酶突变体上进行简单的氨基酸替换或删除或增加等操作并维持第n位为上述突变后的氨基酸,并使进一步突变得到的谷氨酰胺合成酶突变体与(1)所示的谷氨酰胺合成酶突变体具有至少具有85%(例如85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%)以上的同一性,且其功能包括酶催化活性和草铵膦抗性与(1)所示的谷氨酰胺合成酶突变体相当或略有下降或略有提高或大幅提高等。因此,此类谷氨酰胺合成酶也应属于本发明的保护范围。
本发明的研究还发现,针对不同的植物来源的谷氨酰胺合成酶,将其第n位突变为A、C、G或S之外,将其突变为其他的氨基酸也会使得谷氨酰胺合成酶具有草铵膦抗性。
例如,可选的,在本发明的一些实施方案中,当所述植物为水稻时,X=A、C、D、E、F、G、H、I、L、M、N、Q、R、S、T、V、W、或删除。
需要说明的是,X=删除,是指野生型谷氨酰胺合成酶第n位氨基酸被删除,即缺失突变。
当植物为大豆时,X=A、C、D、G、L、S或T;
当植物为玉米时,X=A、C、F、G、H、I、L、M、N、Q、S、T、V、W或删除;
当植物为小麦时,X=A、C、G、S、T或V;
当植物为油菜时,X=A、C、G、M或S。
可选的,在本发明的一些实施方案中,当所述植物为水稻时,水稻野生型谷氨酰胺合成酶为SEQ ID NO.1:
MASLTDLVNLNLSDTTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRKGNNILVMCDCYTPAGEPIPTNKRHNAAKIFSSPEVASEEPWYGIEQEYTLLQKDINWPLGWPVGGFPGPQGPYYCGIGADKSFGRDIVDSHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISAGDQVWVARYILERITEIAGVVVSFDPKPIPGDWNGAGAHTNYSTKSMRNDGGYEIIKSAIEKLKLRHKEHISAYGEGNERRLTGRHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYIVTSMIAETTIIWKP。
可选的,在本发明的一些实施方案中,当所述植物为玉米时,玉米野生型谷氨酰胺合成酶为SEQ ID NO.2:
MACLTDLVNLNLSDNTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRRGNNILVMCDCYTPAGEPIPTNKRYNAAKIFSSPEVAAEEPWYGIEQEYTLLQKDTNWPLGWPIGGFPGPQGPYYCGIGAEKSFGRDIVDAHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISSGDQVWVARYILERITEIAGVVVTFDPKPIPGDWNGAGAHTNYSTESMRKEGGYEVIKAAIEKLKLRHREHIAAYGEGNERRLTGRHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYVVTSMIAETTIIWKP。
可选的,在本发明的一些实施方案中,当所述植物为大豆时,大豆野生型谷氨酰胺合成酶为SEQ ID NO.3:
MSLLSDLINLNLSDTTEKVIAEYIWIGGSGMDLRSKARTLPGPVSDPSKLPKWNYDGSSTGQAPGEDSEVIIYPQAIFRDPFRRGNNILVICDTYTPAGEPIPTNKRHDAAKVFSHPDVVAEETWYGIEQEYTLLQKDIQWPLGWPVGGFPGPQGPYYCGVGADKAFGRDIVDAHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISAGDEVWAARYILERITEIAGVVVSFDPKPIQGDWNGAGAHTNYSTKSMRNDGGYEVIKTAIEKLGKRHKEHIAAYGEGNERRLTGRHETADINTFLWGVANRGASVRVGRDTEKAGKGYFEDRRPASNMDPYVVTSMIADTTILWKP。
可选的,在本发明的一些实施方案中,当所述植物为小麦时,小麦野生型谷氨酰胺合成酶为SEQ ID NO.4:
MALLTDLLNLDLTDSTEKIIAEYIWIGGSGMDLRSKARTLPGPVTDPSKLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRKGNNILVMCDCYTPAGVPIPTNKRYNAAKIFSNPDVAKEEPWYGIEQEYTLLQKDINWPLGWPVGGFPGPQGPYYCSIGADKSFGRDIVDSHYKACLFAGVNISGINGEVMPGQWEFQVGPTVGISAGDQVWVARYLLERITEIAGVVVTFDPKPIPGDWNGAGAHTNYSTESMRKDGGFKVIVDAVEKLKLKHKEHIAAYGEGNERRLTGKHETADINTFSWGVANRGASVRVGRETEQNGKGYFEDRRPASNMDPYVVTSMIAETTILWKP。
可选的,在本发明的一些实施方案中,当所述植物为油菜时,油菜野生型谷氨酰胺合成酶为SEQ ID NO.5:
MSLLTDLVNLNLSETTDKIIAEYIWVGGSGMDMRSKARTLPGPVSDPSELPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRRGNNILVMCDAYTPAGEPIPTNKRHAAAKVFSHPDVVAEVPWYGIEQEYTLLQKDVNWPLGWPIGGFPGPQGPYYCSVGADKSFGRDIVDAHYKACLYAGINISGINGEVMPGQWEFQVGPAVGISAGDEIWVARFILERITEIAGVVVSFDPKPIPGDWNGAGAHCNYSTKSMREDGGYEIIKKAIDKLGLRHKEHIAAYGEGNERRLTGHHETADINTFLWGVANRGASIRVGRDTEKEGKGYFEDRRPASNMDPYIVTSMIAETTILWKP。
部分植物来源的野生型谷氨酰胺合成酶相互间的相似性(Similarity)和同一性(Identity)如下表所示,其序列比对的部分结果见图13,箭头所示为第158位氨基酸。
上述相似性(Similarity)和同一性(Identity)的比对方法为:将一个物种的氨基酸序列输入到Blast网站(https://blast.ncbi.nlm.nih.gov/Blast.cgi)进行ProteinBlast比对,从比对结果中查找此物种和其他需要比对的物种的相似性(Similarity)和同一性(Identity)。
另一方面,本发明提供一种分离的核酸分子,其编码如上任一项所述的具有草铵膦抗性的谷氨酰胺合成酶突变体。例如:重组DNA分子。
在本发明提供了上述氨基酸序列的情况下,本领域技术人员根据密码子的简并性容易获得编码上述谷氨酰胺合成酶突变体的核酸序列。例如,可以在编码野生型谷氨酰胺合成酶的核酸序列上作对应的核苷酸突变得到编码上述谷氨酰胺合成酶突变体的核酸序列。这对本领域技术人员来说是容易实现的。
例如,水稻野生型谷氨酰胺合成酶的编码核酸序列为SEQ ID NO.6:
atggcttctctcaccgatctcgtcaacctcaacctctccgacaccacggagaagatcatcgccgagtacatatggatcggtggatctggcatggatctcaggagcaaggctaggactctctccggccctgtgactgatcccagcaagctgcccaagtggaactacgatggctccagcaccggccaggcccccggcgaggacagtgaggtcatcctgtacccacaggctatcttcaaggacccattcaggaagggaaacaacatccttgtcatgtgcgattgctacacgccagccggagaaccgatccccaccaacaagaggcacaatgctgccaagatcttcagctcccctgaggttgcttctgaggagccctggtacggtattgagcaagagtacaccctcctccagaaggacatcaactggccccttggctggcctgttggtggcttccctggtcctcagggtccttactactgtggtatcggtgctgacaagtcttttgggcgtgatattgttgactcccactacaaggcttgcctctatgccggcatcaacatcagtggaatcaacggcgaggtcatgccaggacagtgggagttccaagttggcccgtctgtcggcatttctgccggtgatcaggtgtgggttgctcgctacattcttgagaggatcaccgagatcgccggagtcgtcgtctcatttgaccccaagcccatcccgggagactggaacggtgctggtgctcacaccaactacagcaccaagtcgatgaggaacgatggtggctacgagatcatcaagtccgccattgagaagctcaagctcaggcacaaggagcacatctccgcctacggcgagggcaacgagcgccggctcaccggcaggcacgagaccgccgacatcaacaccttcagctggggagttgccaaccgcggcgcctcggtccgcgtcggccgggagacggagcagaacggcaagggctacttcgaggatcgccggccggcgtccaacatggacccttacatcgtcacctccatgatcgccgagaccaccatcatctggaagccctga。
据此,在序列基础上,在对应于其编码氨基酸序列第158位的密码子进行对应的核苷酸突变,即可得到编码如上所述的水稻谷氨酰胺合成酶突变体。
玉米野生型谷氨酰胺合成酶的编码核酸序列为SEQ ID NO.7:
atggcctgcctcaccgacctcgtcaacctcaacctctcggacaacaccgagaagatcatcgcggaatacatatggatcggtggatctggcatggatctcaggagcaaagcaaggaccctctccggcccggtgaccgatcccagcaagctgcccaagtggaactacgacggctccagcacgggccaggcccccggcgaggacagcgaggtcatcctgtacccgcaggccatcttcaaggacccattcaggaggggcaacaacatccttgtgatgtgcgattgctacaccccagccggcgagccaatccccaccaacaagaggtacaacgccgccaagatcttcagcagccctgaggtcgccgccgaggagccgtggtatggtattgagcaggagtacaccctcctccagaaggacaccaactggccccttgggtggcccatcggtggcttccccggccctcagggtccttactactgtggaatcggcgccgaaaagtcgttcggccgcgacatcgtggacgcccactacaaggcctgcttgtatgcgggcatcaacatcagtggcatcaacggggaggtgatgccagggcagtgggagttccaagtcgggccttccgtgggtatatcttcaggcgaccaggtctgggtcgctcgctacattcttgagaggatcacggagatcgccggtgtggtggtgacgttcgacccgaagccgatcccgggcgactggaacggcgccggcgcgcacaccaactacagcacggagtcgatgaggaaggagggcgggtacgaggtgatcaaggcggccatcgagaagctgaagctgcggcacagggagcacatcgcggcatacggcgagggcaacgagcgccggctcaccggcaggcacgagaccgccgacatcaacacgttcagctggggcgtggccaaccgcggcgcgtcggtgcgcgtgggccgggagacggagcagaacggcaagggctacttcgaggaccgccgcccggcgtccaacatggacccctacgtggtcacctccatgatcgccgagaccaccatcatctggaagccctga。
大豆野生型谷氨酰胺合成酶的编码核酸序列为SEQ ID NO.8:
atgtcgctgctctcagatctcatcaaccttaacctctcagacactactgagaaggtgatcgcagagtacatatggatcggtggatcaggaatggacctgaggagcaaagcaaggactctcccaggaccagttagcgacccttcaaagcttcccaagtggaactatgatggttccagcacaggccaagctcctggagaagacagtgaagtgattatatacccacaagccattttcagggatccattcagaaggggcaacaatatcttggttatctgtgatacttacactccagctggagaacccattcccactaacaagaggcacgatgctgccaaggttttcagccatcctgatgttgttgctgaagagacatggtatggtattgagcaggaatacaccttgttgcagaaagatatccaatggcctcttgggtggcctgttggtggtttccctggaccacagggtccatactactgtggtgttggcgctgacaaggcttttggccgtgacattgttgacgcacattacaaagcctgtctttatgctggcatcaacatcagtggaattaatggagaagtgatgcccggtcagtgggaattccaagttggaccttcagttggaatctcagctggtgacgaggtgtgggcagctcgttacatcttggagaggatcactgagattgctggtgtggtggtttcctttgatcccaagccaattcagggtgattggaatggtgctggtgctcacacaaactacagcactaagtccatgagaaatgatggtggctatgaagtgatcaaaaccgccattgagaagttggggaagagacacaaggagcacattgctgcttatggagaaggcaacgagcgtcgtttaacagggcgccacgaaaccgctgacatcaacaccttcttatggggagttgcaaaccgtggagcttcagttagggttgggagggacacagagaaagcagggaagggatattttgaggacagaaggccagcttctaacatggacccatatgtggttacttccatgattgcagacacaaccattctgtggaagccatga。
小麦野生型谷氨酰胺合成酶的编码核酸序列为SEQ ID NO.9:
atggcgctcctcaccgatctcctcaacctcgacctcaccgactccacggagaagatcatcgccgagtacatatggatcggcggatctggcatggatctcaggagcaaagccaggaccctccccggcccggtcaccgaccccagcaagctgcccaagtggaactacgacggctccagcaccggccaggcccccggcgaggacagcgaggtcatcctgtacccacaggccatcttcaaggacccgttcaggaagggcaacaacatccttgtcatgtgcgattgctacaccccagctggagtgccaatccccaccaacaagagatacaacgctgccaagatctttagcaaccctgatgttgccaaggaggagccatggtacggtatcgagcaggagtacaccctcctacagaaggacatcaactggcctctcggctggcctgttggtggattccctggtcctcagggtccttactactgtagtattggtgctgacaagtcgtttgggcgtgacatagttgactcccactacaaggcctgcctctttgccggcgtcaacatcagtggcatcaacggcgaggtcatgcccggacagtgggagttccaagttggcccgactgtcggcatctctgctggtgaccaagtgtgggttgctcgctaccttcttgagaggatcactgagatcgccggagttgtcgtcacatttgaccccaagcccatcccaggcgactggaacggtgctggtgctcacacaaactacagtaccgagtcgatgaggaaggacggcgggttcaaggtcatcgtggacgctgtcgagaagctcaagctgaagcacaaggagcacatcgccgcctacggcgagggcaacgagcgccgtctcaccggcaagcacgaaaccgccgacatcaacaccttcagctggggtgtcgcgaaccgtggcgcgtcggtgcgcgtgggacgggagacggagcagaacggcaagggctacttcgaggaccgccggccggcgtccaacatggacccctacgtggtcacctccatgatcgccgagaccaccatcctgtggaagccctga。
油菜野生型谷氨酰胺合成酶的编码核酸序列为SEQ ID NO.10:
atgagtcttcttacagatctcgttaaccttaacctctcagagaccactgacaaaatcattgcggaatacatatgggttggaggttcaggaatggatatgagaagcaaagccaggactcttcctggaccagtgagtgacccttcggagctaccaaagtggaactatgatggctcaagcacaggccaagctcctggtgaagacagtgaagtcatcttataccctcaagccatattcaaagatcctttccgtagaggcaacaacattcttgtcatgtgcgatgcttacactccagcgggcgaaccgatcccaacaaacaaaagacacgctgcggctaaggtctttagccaccccgatgttgtagctgaagtgccatggtatggtattgagcaagagtatactttacttcagaaagatgtgaactggcctcttggttggcctattggcggcttccccggtcctcagggaccatactattgtagtgttggagcagataaatcttttggtagagacatcgttgatgctcactacaaggcctgcttatacgctggcatcaatattagtggcatcaacggagaagtcatgcctggtcagtgggagttccaagttggtccagctgttggtatctcggccggtgatgaaatttgggtcgcacgtttcattttggagaggatcacagagattgctggtgtggtggtatcttttgacccaaaaccgattcccggtgactggaatggtgctggtgctcactgcaactatagtaccaagtcaatgagggaagatggtggttacgagattattaagaaggcaatcgataaactgggactgagacacaaagaacacattgcagcttacggtgaaggcaatgagcgccgtctcacgggtcaccacgagactgctgacatcaacactttcctctggggtgttgcgaaccgtggagcatcaatccgtgtaggacgtgacacagagaaagaagggaaaggatactttgaggataggaggccagcttcgaacatggatccttacattgtgacttccatgattgcagagaccacaatcctctggaaaccttga。
本发明还提供了一种载体,其含有上述的核酸分子。
本发明还提供了一种重组菌或重组细胞,其含有上述的核酸分子或上述的载体。
重组菌可以选自农杆菌或大肠杆菌;重组细胞可以是感受态细胞。
本发明还提供了上述具有草铵膦抗性的谷氨酰胺合成酶突变体、核酸分子、载体或重组菌或重组细胞在培育具有草铵膦抗性的植物品种中的应用。
在本发明应用较佳的实施方式中,上述应用包括:将分离的核酸分子送入目的植物细胞,分离的核酸分子含有编码谷氨酰胺合成酶突变体的编码基因;
将载体转化目的植物,载体含有编码谷氨酰胺合成酶突变体的编码基因;
将重组菌或重组细胞导入目的植物,重组菌或重组细胞含有编码谷氨酰胺合成酶突变体的编码基因。
分离的核酸分子可以是质粒或DNA片段,在可选的实施方式中,可以通过基因枪法将分离的核酸分子送入目的植物细胞。
转化的方法包括不限于农杆菌介导基因转化法、基因枪转化法、花粉管通道法。
重组菌或重组细胞可通过侵染的方式导入目的植物体内。
在本发明应用较佳的实施方式中,上述应用包括:修饰目的植物的内源谷氨酰胺合成酶基因,使其编码谷氨酰胺合成酶突变体。
在本发明提供了谷氨酰胺合成酶突变体的基础上,本领域技术人员容易想到通过本领域常规的转基因技术、基因编辑技术(如通过锌指核酸内切酶(ZFN,zinc-fingernucleases)技术、类转录激活因子效应物核酸酶(TALEN,transcription activator-likeeffector nucleases)技术或CRISPR/Cas9)、诱变育种技术(如化学、辐射诱变等)等对目标植物进行改造,使其具有编码如上所述谷氨酰胺合成酶突变体的基因,进而获得草铵膦抗性并能够正常生长和发育,进行得到具有草铵膦抗性的植物新品种。因此,无论采用何种技术,只要其利用了本发明提供的谷氨酰胺合成酶突变体赋予植物草铵膦抗性,则属于本发明的保护范围。
在本发明应用较佳的实施方式中,上述应用包括:对植物细胞、组织、个体或群体进行诱变和筛选,使其编码谷氨酰胺合成酶突变体。
在一种可选的实施方式中,诱变为非致死剂量的理化诱变方式对植物进行诱变以获得植物材料。
上述非致死剂量是指将剂量控制在半致死剂量上下浮动20%的范围。
理化诱变方式包括以下物理诱变、化学诱变方式中的一种或多种的组合:物理诱变包括紫外线诱变、X射线诱变、γ射线诱变、β射线诱变、α射线诱变、高能粒子诱变、宇宙射线诱变、微重力诱变;化学诱变包括烷化剂诱变、叠氮化物诱变、碱基类似物诱变、氯化锂诱变、抗生素诱变、嵌入染料诱变;烷化剂诱变包括甲基环酸乙酯诱变、硫酸二乙酯诱变、乙烯亚胺诱变。
在一种可选的实施方式中,目的植物包括不限于小麦、水稻、大麦、燕麦、玉米、高粱、谷子、荞麦、黍稷、甘薯、马铃薯、棉花、油菜、芝麻、花生、向日葵、萝卜、胡萝卜、花椰菜、番茄、茄子、辣椒、韭菜、大葱、洋葱、韭葱、菠菜、芹菜、苋菜、莴苣、茼蒿、黄花菜、葡萄、草莓、甘蔗、烟草、芸薹属蔬菜、葫芦科植物、豆科植物、牧草、茶或木薯。
在一种可选的实施方式中,牧草包括不限于禾本科牧草或豆科牧草。
在一种可选的实施方式中,芸薹属蔬菜包括不限于芜菁、白菜、芥菜、甘蓝、芥蓝、菜苔、苦芥、擎蓝、芸苔、青菜或甜菜。
在一种可选的实施方式中,葫芦科植物包括不限于黄瓜、西葫芦、南瓜、冬瓜、苦瓜、丝瓜、菜瓜、西瓜或甜瓜。
在一种可选的实施方式中,豆科植物包括不限于绿豆、蚕豆、豌豆、扁豆、大豆、菜豆、豇豆或毛豆。
本发明具有以下有益效果:
本发明提供的具有草铵膦抗性的谷氨酰胺合成酶突变体,具有用于构建转化植物的表达载体、重组菌及培育抗草铵膦作物的应用潜力。本发明提供的谷氨酰胺合成酶突变体原始来源于植物,更容易被消费者接受。通过突变后具有了草铵膦抗性,转化该谷氨酰胺合成酶突变体的植物不仅具有适于商业化应用的草铵膦抗性,也能够保持谷氨酰胺合成酶正常的酶催化活性,可以满足植物正常的生长和发育。此外,具有上述突变的重组菌和重组细胞也均具有草铵膦抗性。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为本发明实施例1提供的水稻GS1突变体OY158A、OY158C、OY158D、OY158E、OY158F、OY158G、OY158H、OY158I、OY158L、OY158M、OY158N、OY158Q、OY158R、OY158S、OY158T、OY158V、OY158W和OY158X和野生型水稻OWT1的氨基酸序列部分比对结果;
图2为本发明实施例2提供的大豆GS1突变体GY158A、GY158D、GY158G、GY158L、GY158M、GY158N、GY158S和GY158T和野生型大豆GWT1的氨基酸序列部分比对结果;
图3为本发明实施例3提供的玉米GS1突变体ZY158A、ZY158C、ZY158F、ZY158G、ZY158H、ZY158I、ZY158L、ZY158M、ZY158N、ZY158Q、ZY158S、ZY158T、ZY158V、ZY158W和ZY158X和野生型玉米ZWT1的氨基酸序列部分比对结果;
图4为本发明实施例4提供的小麦GS1突变体TY158A、TY158C、TY158G、TY158S、TY158T和TY158V和野生型小麦TWT1的氨基酸序列部分比对结果;
图5为本发明实施例5提供的油菜GS1突变体BY158A、BY158C、BY158G、BY158M和BY158S和野生型油菜BWT1的氨基酸序列部分比对结果;
图6为本发明实验例1提供的pADV7载体的结构示意图;
图7为本发明实验例1提供的转化实施例1提供的水稻GS1突变体OY158A、OY158C、OY158D、OY158E、OY158F、OY158G、OY158H、OY158I、OY158L、OY158M、OY158N、OY158Q、OY158R、OY158S、OY158T、OY158V、OY158W和OY158X和野生型水稻OWT1的大肠杆菌在含不同浓度草铵膦的培养基上的生长结果;
图8为本发明实验例2提供的转化实施例2提供的大豆GS1突变体GY158A、GY158D、GY158G、GY158L、GY158M、GY158N、GY158S和GY158T和野生型大豆GWT1的大肠杆菌在含不同浓度草铵膦的培养基上的生长结果;
图9为本发明实验例3提供的转化实施例3提供的玉米GS1突变体ZY158A、ZY158C、ZY158F、ZY158G、ZY158H、ZY158I、ZY158L、ZY158M、ZY158N、ZY158Q、ZY158S、ZY158T、ZY158V、ZY158W和ZY158X和野生型玉米ZWT1的大肠杆菌在含不同浓度草铵膦的培养基上的生长结果;
图10为本发明实验例4提供的转化实施例4提供的小麦GS1突变体TY158A、TY158C、TY158G、TY158S、TY158T和TY158V和野生型小麦TWT1的大肠杆菌在含不同浓度草铵膦的培养基上的生长结果;
图11为本发明实验例5提供的转化实施例5提供的油菜GS1突变体BY158A、BY158C、BY158G、BY158M和BY158S和野生型油菜BWT1的大肠杆菌在含不同浓度草铵膦的培养基上的生长结果;
图12为本发明实验例6提供的水稻GS1突变体OY158A、大豆GS1突变体GY158A、玉米GS1突变体ZY158A、小麦GS1突变体TY158A、油菜GS1突变体BY158A、野生型水稻OWT1、野生型大豆GWT1、野生型玉米ZWT1、野生型小麦TWT1和野生型油菜BWT1的酶动力学参数和草铵膦抗性参数IC50;
图13为不同植物野生型谷氨酰胺合成酶的氨基酸序列比对结果;图中:TWT1:小麦野生型谷氨酰胺合成酶体;OWT1:水稻野生型谷氨酰胺合成酶体;ZWT1:玉米野生型谷氨酰胺合成酶体;GWT1:大豆野生型谷氨酰胺合成酶体;BWT1:油菜野生型谷氨酰胺合成酶体。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
除非另有定义,否则本文使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的含义相同的含义。尽管与本文描述的那些方法和材料类似或等同的任何方法和材料都可用于本文的制剂或单位剂量的实践或测试,但现在描述一些方法和材料。除非另有说明,否则本文采用或考虑的技术是标准方法。材料、方法和实例仅是说明性而非限制性的。
除非另外指明,否则实践本发明将采用植物生理学、植物分子遗传学、细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术,所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术,如《分子克隆:实验室手册(Molecular Cloning:A Laboratory Manual)》,第二版(Sambrook等人,1989);《寡核苷酸合成(Oligonucleotide Synthesis)》(M.J.Gait编,1984);《植物生理学》(苍晶等人,2017);《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press,Inc.);《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir和C.C.Blackwell编);《当代分子生物学方法(Current Protocols in Molecular Biology)》(F.M.Ausubel等人编,1987);《植物分子遗传学》(Monica A.Hughes等人著);《PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)》(Mullis等人编,1994),所述文献中的每个文献均通过引用明确并入本文中。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
本实施例提供的水稻(Oryza sativa)谷氨酰胺合成酶(GS1)突变体,其由野生型水稻谷氨酰胺合成酶自身(命名为OWT1,氨基酸序列如SEQ ID NO.1所示,编码核苷酸序列为SEQ ID NO.6)的第158位氨基酸残基Y突变为A、C、D、E、F、G、H、I、L、M、N、Q、R、S、T、V、W、或删除得到,得到的水稻GS1突变体分别命名为OY158A、OY158C、OY158D、OY158E、OY158F、OY158G、OY158H、OY158I、OY158L、OY158M、OY158N、OY158Q、OY158R、OY158S、OY158T、OY158V、OY158W和OY158X。
水稻GS1突变体OY158A、OY158C、OY158D、OY158E、OY158F、OY158G、OY158H、OY158I、OY158L、OY158M、OY158N、OY158Q、OY158R、OY158S、OY158T、OY158V、OY158W、OY158X和野生型水稻GS1的氨基酸序列比对如图1所示,图中:箭头所指示的位置为突变位点。
本实施例中,各水稻GS1突变体的编码序列在编码第158位氨基酸的位置上,对应氨基酸所用的密码子如下表所示,其余位置的核苷酸同相应的野生型编码序列。
本实施例提供的水稻GS1突变体OY158A、OY158C、OY158D、OY158E、OY158F、OY158G、OY158H、OY158I、OY158L、OY158M、OY158N、OY158Q、OY158R、OY158S、OY158T、OY158V、OY158W和OY158X和编码它们的核酸分子均可以通过化学合成的方法获得。
实施例2
本实施例提供的大豆(Glycine max)GS1突变体,其由野生型大豆GS1自身((命名为GWT1,氨基酸序列如SEQ ID NO.3所示,编码核苷酸序列为SEQ ID NO.8)的第158位(对应于参考序列(SEQ ID NO.1)的第158位)由氨基酸残基Y突变为A、C、D、G、L、S或T得到。得到的大豆GS1突变体分别命名为GY158A、GY158D、GY158G、GY158L、GY158M、GY158N、GY158S和GY158T。
大豆GS1突变体GY158A、GY158D、GY158G、GY158L、GY158M、GY158N、GY158S、GY158T和野生型大豆GS1的氨基酸序列比对如图2所示,图中:箭头所指示的位置为突变位点。
本实施例中,各大豆GS1突变体的编码序列在编码第158位氨基酸的位置上,对应氨基酸所用的密码子如下表所示,其余位置的核苷酸同相应的野生型编码序列。
本实施例提供的大豆GS1突变体GY158A、GY158D、GY158G、GY158L、GY158M、GY158N、GY158S和GY158T和编码它们的核酸分子均可以通过化学合成的方法获得。
实施例3
本实施例提供的玉米(Zea mays)GS1突变体,其由野生型玉米GS1自身(命名为ZWT1,氨基酸序列如SEQ ID NO.2所示,编码核苷酸序列为SEQ ID NO.7)的第158位(对应于参考序列(SEQ ID NO.1)的第158位)由氨基酸残基Y突变为A、C、F、G、H、I、L、M、N、Q、S、T、V、W或删除得到。得到的玉米GS1突变体分别命名为ZY158A、ZY158C、ZY158F、ZY158G、ZY158H、ZY158I、ZY158L、ZY158M、ZY158N、ZY158Q、ZY158S、ZY158T、ZY158V、ZY158W和ZY158X。
玉米GS1突变体ZY158A、ZY158C、ZY158F、ZY158G、ZY158H、ZY158I、ZY158L、ZY158M、ZY158N、ZY158Q、ZY158S、ZY158T、ZY158V、ZY158W、ZY158X和野生型玉米GS1的氨基酸序列比对如图3所示,图中:箭头所指示的位置为突变位点。
本实施例中,各玉米GS1突变体的编码序列在编码第158位氨基酸的位置上,对应氨基酸所用的密码子如下表所示,其余位置的核苷酸同相应的野生型编码序列。
氨基酸 | A | C | F | G | H |
密码子 | GCC | TGC | TTC | GGT | CAC |
氨基酸 | I | L | M | N | Q |
密码子 | ATC | TTG | ATG | AAC | CAG |
氨基酸 | S | T | V | W | 删除 |
密码子 | TCC | ACC | GTT | TGG | 无 |
本实施例提供的玉米GS1突变体ZY158A、ZY158C、ZY158F、ZY158G、ZY158H、ZY158I、ZY158L、ZY158M、ZY158N、ZY158Q、ZY158S、ZY158T、ZY158V、ZY158W和ZY158X和编码它们的核酸分子均可以通过化学合成的方法获得。
实施例4
本实施例提供的小麦(Triticum aestivum)GS1突变体,其由野生型小麦GS1自身(命名为TWT1,氨基酸序列如SEQ ID NO.4所示,编码核苷酸序列为SEQ ID NO.9)的第158位(对应于参考序列(SEQ ID NO.1)的第158位)由氨基酸残基S突变为A、C、G、S、T或V得到。得到的小麦GS1突变体分别命名为TY158A、TY158C、TY158G、TY158S、TY158T和TY158V。
小麦GS1突变体TY158A、TY158C、TY158G、TY158S、TY158T、TY158V和野生型小麦GS1的氨基酸序列比对如图4所示,图中:箭头所指示的位置为突变位点。
本实施例中,各小麦GS1突变体的编码序列在编码第158位氨基酸的位置上,对应氨基酸所用的密码子如下表所示,其余位置的核苷酸同相应的野生型编码序列。
本实施例提供的小麦GS1突变体TY158A、TY158C、TY158G、TY158S、TY158T和TY158V和编码它们的核酸分子均可以通过化学合成的方法获得。
实施例5
本实施例提供的油菜(Brassica napus)GS1突变体,其由野生型油菜GS1自身(命名为BWT1,氨基酸序列如SEQ ID NO.5所示,编码核苷酸序列为SEQ ID NO.10)的第158位(对应于参考序列(SEQ ID NO.1)的第158位)由氨基酸残基S突变为A、C、G、M或S得到。得到的油菜GS1突变体分别命名为BY158A、BY158C、BY158G、BY158M和BY158S。
油菜GS1突变体BY158A、BY158C、BY158G、BY158M、BY158S和野生型油菜GS1的氨基酸序列比对如图5所示,图中:箭头所指示的位置为突变位点。
本实施例中,各油菜GS1突变体的编码序列在编码第158位氨基酸的位置上,对应氨基酸所用的密码子如下表所示,其余位置的核苷酸同相应的野生型编码序列。
氨基酸 | A | C | G | M | S |
密码子 | GCT | TGT | GGT | ATG | TCC |
本实施例提供的油菜GS1突变体BY158A、BY158C、BY158G、BY158M和BY158S和编码它们的核酸分子均可以通过化学合成的方法获得。
实验例1
检测实施例1提供的水稻GS1突变体OY158A、OY158C、OY158D、OY158E、OY158F、OY158G、OY158H、OY158I、OY158L、OY158M、OY158N、OY158Q、OY158R、OY158S、OY158T、OY158V、OY158W和OY158X的草铵膦抗性,方法如下:
根据实施例1提供的核酸分子的序列,采用化学合成的方法合成编码水稻GS1突变体OY158A、OY158C、OY158D、OY158E、OY158F、OY158G、OY158H、OY158I、OY158L、OY158M、OY158N、OY158Q、OY158R、OY158S、OY158T、OY158V、OY158W和OY158X的编码基因,两端引入酶切位点(Pac1和Sbf1),酶切后,在连接酶的作用下连接至经相同酶切处理后的表达载体(例如pADV7载体,其结构如图6所示)上,然后分别转化谷氨酰胺合成酶缺陷型大肠杆菌,经验证后,挑取阳性克隆,接种至含不同浓度草铵膦的M9培养基上生长,观察缺陷型大肠杆菌生长情况。以野生型水稻GS1突变体作为负对照,检测含有GS1突变体OY158A(Y158A,水稻GS1的第158位的氨基酸Y突变为A)、OY158C(Y158C)、OY158D(Y158D)、OY158E(Y158E)、OY158F(Y158F)、OY158G(Y158G)、OY158H(Y158H)、OY158I(Y158I)、OY158L(Y158L)、OY158M(Y158M)、OY158N(Y158N)、OY158Q(Y158Q)、OY158R(Y158R)、OY158S(Y158S)、OY158T(Y158T)、OY158V(Y158V)、OY158W(Y158W)和OY158X(Y158X,水稻GS1的第158位的氨基酸Y删除)的草铵膦抗性。结果如图7所示。
在含0mM草铵膦(KP0)的培养基上,转化编码野生型水稻GS1(OWT1)及水稻GS1突变体OY158A、OY158C、OY158D、OY158E、OY158F、OY158G、OY158H、OY158I、OY158L、OY158M、OY158N、OY158Q、OY158R、OY158S、OY158T、OY158V、OY158W和OY158X的编码基因的缺陷型菌株均能正常生长,表明由OY158A、OY158C、OY158D、OY158E、OY158F、OY158G、OY158H、OY158I、OY158L、OY158M、OY158N、OY158Q、OY158R、OY158S、OY158T、OY158V、OY158W和OY158X编码的GS1都具有正常GS1酶活力;
在含10mM草铵膦(KP10)的培养基上,转化野生型水稻GS1的大肠杆菌不能生长,但转化了水稻突变体OY158A、OY158C、OY158D、OY158E、OY158G、OY158H、OY158I、OY158L、OY158M、OY158N、OY158Q、OY158R、OY158S、OY158T、OY158V、OY158W和OY158X的大肠杆菌生长明显优于负对照,说明含OY158A、OY158C、OY158D、OY158E、OY158G、OY158H、OY158I、OY158L、OY158M、OY158N、OY158Q、OY158R、OY158S、OY158T、OY158V、OY158W和OY158X的单突变体抗草铵膦的能力明显优于野生型;在更好草铵膦浓度(20mM,KP20)的培养基上,转化水稻GS1突变体OY158A、OY158C、OY158D、OY158E、OY158G、OY158I、OY158L、OY158M、OY158N、OY158R、OY158S、OY158T、OY158V、OY158W和OY158X的大肠杆菌都还有明显生长。
这些结果说明OY158A、OY158C、OY158D、OY158E、OY158F、OY158G、OY158H、OY158I、OY158L、OY158M、OY158N、OY158Q、OY158R、OY158S、OY158T、OY158V、OY158W和OY158X的单突变体都具有抗草铵膦的能力。
实验例2
参考实验例1的检测方法,验证实施例2提供的大豆GS1突变体GY158A(Y158A,大豆GS1的第158位的氨基酸Y突变为A)、GY158D、GY158G、GY158L、GY158M、GY158N、GY158S和GY158T的草铵膦抗性。结果如图8所示。
根据图8的结果可看出:
在含0mM草铵膦(KP0)的培养基上,转化编码野生型大豆GS1(GWT1)及大豆GS1突变体GY158A、GY158D、GY158G、GY158L、GY158M、GY158N、GY158S和GY158T的编码基因的缺陷型菌株均能正常生长,表明由GY158A、GY158D、GY158G、GY158L、GY158M、GY158N、GY158S和GY158T编码的GS1都具有正常GS1酶活力;
在含2mM草铵膦(KP2)的培养基上,转化野生型大豆GS1的大肠杆菌基本上不能生长,但转化了大豆突变体GY158A、GY158D、GY158G、GY158L、GY158M、GY158N、GY158S和GY158T的大肠杆菌生长明显优于负对照,说明含GY158A、GY158D、GY158G、GY158L、GY158M、GY158N、GY158S和GY158T的单突变体抗草铵膦的能力明显优于野生型;在更高草铵膦浓度(20mM,KP20)的培养基上,转化大豆GS1突变体GY158G和GY158T的大肠杆菌都还有明显生长。
这些结果说明GY158A、GY158D、GY158G、GY158L、GY158M、GY158N、GY158S和GY158T的单突变体都具有抗草铵膦的能力,且大豆GS1突变体GY158G和GY158T的抗草铵膦能力更强。
实验例3
参考实验例1的检测方法,验证实施例3提供的玉米GS1突变体ZY158A(Y158A,玉米GS1的第158位的氨基酸Y突变为A)、ZY158C、ZY158F、ZY158G、ZY158H、ZY158I、ZY158L、ZY158M、ZY158N、ZY158Q、ZY158S、ZY158T、ZY158V、ZY158W和ZY158X(Y158X,玉米GS1的第68位的氨基酸S删除)的草铵膦抗性。结果如图9所示。
根据图9的结果可看出:
在含0mM草铵膦(KP0)的培养基上,转化编码野生型玉米GS1(ZWT1)及玉米GS1突变体ZY158A、ZY158C、ZY158D、ZY158E、ZY158F、ZY158G、ZY158H、ZY158I、ZY158K、ZY158L、ZY158M、ZY158N、ZY158P、ZY158Q、ZY158R、ZY158T、ZY158V、ZY158W、ZY158Y和ZY158X的编码基因的缺陷型菌株均能正常生长,表明由ZY158A、ZY158C、ZY158D、ZY158E、ZY158F、ZY158G、ZY158H、ZY158I、ZY158K、ZY158L、ZY158M、ZY158N、ZY158P、ZY158Q、ZY158R、ZY158T、ZY158V、ZY158W、ZY158Y和ZY158X编码的GS1都具有正常GS1酶活力;
在含2mM草铵膦(KP2)的培养基上,转化野生型玉米GS1的大肠杆菌基本上不能生长,但转化了玉米突变体ZY158A、ZY158C、ZY158D、ZY158E、ZY158F、ZY158G、ZY158H、ZY158I、ZY158K、ZY158L、ZY158M、ZY158N、ZY158P、ZY158Q、ZY158R、ZY158T、ZY158V、ZY158W、ZY158Y和ZY158X的大肠杆菌生长明显优于负对照,说明含ZY158A、ZY158C、ZY158D、ZY158E、ZY158F、ZY158G、ZY158H、ZY158I、ZY158K、ZY158L、ZY158M、ZY158N、ZY158P、ZY158Q、ZY158R、ZY158T、ZY158V、ZY158W、ZY158Y和ZY158X的单突变体抗草铵膦的能力明显优于野生型;在更高草铵膦浓度(20mM,KP20)的培养基上,转化玉米GS1突变体ZY158A、ZY158C、ZY158D、ZY158E、ZY158G、ZY158I、ZY158K、ZY158L、ZY158N、ZY158P、ZY158R、ZY158T、ZY158V和ZY158Y的大肠杆菌都还有明显生长。
这些结果说明ZY158A、ZY158C、ZY158D、ZY158E、ZY158F、ZY158G、ZY158H、ZY158I、ZY158K、ZY158L、ZY158M、ZY158N、ZY158P、ZY158Q、ZY158R、ZY158T、ZY158V、ZY158W、ZY158Y和ZY158X的单突变体都具有抗草铵膦的能力。
实验例4
参考实验例1的检测方法,验证实施例4提供的小麦GS1突变体TY158A(Y158A,小麦GS1的第158位的氨基酸Y突变为A)、、TY158C、TY158G、TY158S、TY158T和TY158V的草铵膦抗性。结果如图10所示。
根据图10的结果可看出:
在含0mM草铵膦(KP0)的培养基上,转化编码野生型小麦GS1(TWT1)及小麦GS1突变体TY158A、TY158C、TY158G、TY158S、TY158T和TY158V的编码基因的缺陷型菌株均能正常生长,表明由TY158A、TY158C、TY158G、TY158S、TY158T和TY158V编码的GS1都具有正常GS1酶活力;
在含5mM草铵膦(KP5)的培养基上,转化野生型小麦GS1的大肠杆菌基本上不能生长,但转化了小麦突变体TY158A、TY158C、TY158G、TY158S、TY158T和TY158V的大肠杆菌生长明显优于负对照,说明含TY158A、TY158C、TY158G、TY158S、TY158T和TY158V的单突变体抗草铵膦的能力明显优于野生型;在更高草铵膦浓度(20mM,KP20)的培养基上,转化小麦GS1突变体TY158A、TY158C、TY158G、TY158S、TY158T和TY158V的大肠杆菌都还有明显生长。
这些结果说明TY158A、TY158C、TY158G、TY158S、TY158T和TY158V的单突变体都具有抗草铵膦的能力。
实验例5
参考实验例1的检测方法,验证实施例5提供的油菜GS1突变体BY158A(Y158A,油菜GS1的第158位的氨基酸Y突变为A)、BY158C、BY158G、BY158M和BY158S的草铵膦抗性。结果如图11所示。
根据图11的结果可看出:
在含0mM草铵膦(KP0)的培养基上,转化编码野生型油菜GS1(BWT1)及油菜GS1突变体BY158A、BY158C、BY158G、BY158M和BY158S的编码基因的缺陷型菌株均能正常生长,表明由BY158A、BY158C、BY158G、BY158M和BY158S编码的GS1都具有正常GS1酶活力;
在含2mM草铵膦(KP2)的培养基上,转化野生型油菜GS1的大肠杆菌基本上不能生长,但转化了油菜突变体BY158A、BY158C、BY158G、BY158M和BY158S的大肠杆菌生长明显优于负对照,说明含BY158A、BY158C、BY158G、BY158M和BY158S的单突变体抗草铵膦的能力明显优于野生型;在更高草铵膦浓度(20mM,KP20)的培养基上,转化油菜GS1突变体BY158A、BY158C、BY158G、BY158M和BY158S的大肠杆菌都还有明显生长。
这些结果说明BY158A、BY158C、BY158G、BY158M和BY158S的单突变体都具有抗草铵膦的能力。
实验例6
检测实施例1提供的OY158A、实施例2提供的GY158A、实施例3提供的ZY158A、实施例4提供的TY158A和实施例5提供的BY158A突变体的酶动力学参数和在有草铵膦时的酶动力学参数,以野生型水稻OWT1、野生型大豆GWT1、野生型玉米ZWT1、野生型小麦TWT1和野生型油菜BWT1为对照,方法如下:
载体构建:
将编码上述突变体的核酸序列克隆到原核表达载体pET32a中,测序验证克隆。
6His蛋白纯化:
通过6His和用标准方法纯化突变体酶蛋白,用Bradford法蛋白浓度测定试剂盒测定浓度,蛋白保存在蛋白贮存液中。
酶活测定:
1.仪器和试剂:酶标仪(德铁:HBS-1096A),草铵膦(利尔化学股份有限公司,http://www.lierchem.com/),底物L-谷氨酸钠(CAS:6106-04-3)。
2.操作步骤:
谷氨酰胺合成酶酶活测定反应液组分为:200mM Tris-HCl(pH7.5),1.67mM ATP,20mM L-谷氨酸钠,30mM氯化铵,20mM MgCl2,0.5uM的突变体蛋白液(BY158A为2.5uM),补水至30ul。30μl反应液(先不加突变体蛋白液)混匀后35℃预热5min后,加入突变体蛋白液开始反应,35℃反应30min后,加入100μl颜色反应D液(D=2A+B;A液:12%(W/V)抗坏血酸的1mol/L盐酸溶液,B液:2%(W/V)四水钼酸铵的水溶液)产生颜色,静置5min,再加入100μl反应终止F液(2%柠檬酸钠、2%乙酸的水溶液),静置15min,取200μl在660nm处测定光吸收值。
结果如图12所示。
根据图12的结果可以看出:
野生型对照OWT1、GWT1、ZWT1、TWT1和BWT1对草铵膦很敏感,IC50分别为17.82μM、6.37μM、16.57μM、11.14μM和14.38μM,突变体的IC50均明显高于野生型对照,OY158A、GY158A、ZY158A和BY158A的IC50远远高于野生型对照,表明突变体对草铵膦更不敏感。从突变体IC50和野生型IC50的倍数关系上也可以看出,OY158A、GY158A、ZY158A、TY158A和BY158A的IC50分别是对应野生型GS1 IC50的1379.12倍、230.39倍、347.13倍、37.54倍和145.96倍,这些数值也说明突变体的酶活性远远高于野生型对照。这些数据从酶动力学上说明了突变体的抗草铵膦机制。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 四川天豫兴禾生物科技有限公司
<120> 一种具有草铵膦抗性的谷氨酰胺合成酶突变体及其应用
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Glu Arg Arg Leu Thr Gly Lys His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300
Ser Trp Gly Val Ala Asn Arg Gly Ala Ser Val Arg Val Gly Arg Glu
305 310 315 320
Thr Glu Gln Asn Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser
325 330 335
Asn Met Asp Pro Tyr Val Val Thr Ser Met Ile Ala Glu Thr Thr Ile
340 345 350
Leu Trp Lys Pro
355
<210> 5
<211> 356
<212> PRT
<213> 人工序列
<400> 5
Met Ser Leu Leu Thr Asp Leu Val Asn Leu Asn Leu Ser Glu Thr Thr
1 5 10 15
Asp Lys Ile Ile Ala Glu Tyr Ile Trp Val Gly Gly Ser Gly Met Asp
20 25 30
Met Arg Ser Lys Ala Arg Thr Leu Pro Gly Pro Val Ser Asp Pro Ser
35 40 45
Glu Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro
50 55 60
Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp
65 70 75 80
Pro Phe Arg Arg Gly Asn Asn Ile Leu Val Met Cys Asp Ala Tyr Thr
85 90 95
Pro Ala Gly Glu Pro Ile Pro Thr Asn Lys Arg His Ala Ala Ala Lys
100 105 110
Val Phe Ser His Pro Asp Val Val Ala Glu Val Pro Trp Tyr Gly Ile
115 120 125
Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Val Asn Trp Pro Leu Gly
130 135 140
Trp Pro Ile Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Ser
145 150 155 160
Val Gly Ala Asp Lys Ser Phe Gly Arg Asp Ile Val Asp Ala His Tyr
165 170 175
Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn Gly Glu
180 185 190
Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ala Val Gly Ile
195 200 205
Ser Ala Gly Asp Glu Ile Trp Val Ala Arg Phe Ile Leu Glu Arg Ile
210 215 220
Thr Glu Ile Ala Gly Val Val Val Ser Phe Asp Pro Lys Pro Ile Pro
225 230 235 240
Gly Asp Trp Asn Gly Ala Gly Ala His Cys Asn Tyr Ser Thr Lys Ser
245 250 255
Met Arg Glu Asp Gly Gly Tyr Glu Ile Ile Lys Lys Ala Ile Asp Lys
260 265 270
Leu Gly Leu Arg His Lys Glu His Ile Ala Ala Tyr Gly Glu Gly Asn
275 280 285
Glu Arg Arg Leu Thr Gly His His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300
Leu Trp Gly Val Ala Asn Arg Gly Ala Ser Ile Arg Val Gly Arg Asp
305 310 315 320
Thr Glu Lys Glu Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser
325 330 335
Asn Met Asp Pro Tyr Ile Val Thr Ser Met Ile Ala Glu Thr Thr Ile
340 345 350
Leu Trp Lys Pro
355
<210> 6
<211> 1071
<212> DNA
<213> 人工序列
<400> 6
atggcttctc tcaccgatct cgtcaacctc aacctctccg acaccacgga gaagatcatc 60
gccgagtaca tatggatcgg tggatctggc atggatctca ggagcaaggc taggactctc 120
tccggccctg tgactgatcc cagcaagctg cccaagtgga actacgatgg ctccagcacc 180
ggccaggccc ccggcgagga cagtgaggtc atcctgtacc cacaggctat cttcaaggac 240
ccattcagga agggaaacaa catccttgtc atgtgcgatt gctacacgcc agccggagaa 300
ccgatcccca ccaacaagag gcacaatgct gccaagatct tcagctcccc tgaggttgct 360
tctgaggagc cctggtacgg tattgagcaa gagtacaccc tcctccagaa ggacatcaac 420
tggccccttg gctggcctgt tggtggcttc cctggtcctc agggtcctta ctactgtggt 480
atcggtgctg acaagtcttt tgggcgtgat attgttgact cccactacaa ggcttgcctc 540
tatgccggca tcaacatcag tggaatcaac ggcgaggtca tgccaggaca gtgggagttc 600
caagttggcc cgtctgtcgg catttctgcc ggtgatcagg tgtgggttgc tcgctacatt 660
cttgagagga tcaccgagat cgccggagtc gtcgtctcat ttgaccccaa gcccatcccg 720
ggagactgga acggtgctgg tgctcacacc aactacagca ccaagtcgat gaggaacgat 780
ggtggctacg agatcatcaa gtccgccatt gagaagctca agctcaggca caaggagcac 840
atctccgcct acggcgaggg caacgagcgc cggctcaccg gcaggcacga gaccgccgac 900
atcaacacct tcagctgggg agttgccaac cgcggcgcct cggtccgcgt cggccgggag 960
acggagcaga acggcaaggg ctacttcgag gatcgccggc cggcgtccaa catggaccct 1020
tacatcgtca cctccatgat cgccgagacc accatcatct ggaagccctg a 1071
<210> 7
<211> 1071
<212> DNA
<213> 人工序列
<400> 7
atggcctgcc tcaccgacct cgtcaacctc aacctctcgg acaacaccga gaagatcatc 60
gcggaataca tatggatcgg tggatctggc atggatctca ggagcaaagc aaggaccctc 120
tccggcccgg tgaccgatcc cagcaagctg cccaagtgga actacgacgg ctccagcacg 180
ggccaggccc ccggcgagga cagcgaggtc atcctgtacc cgcaggccat cttcaaggac 240
ccattcagga ggggcaacaa catccttgtg atgtgcgatt gctacacccc agccggcgag 300
ccaatcccca ccaacaagag gtacaacgcc gccaagatct tcagcagccc tgaggtcgcc 360
gccgaggagc cgtggtatgg tattgagcag gagtacaccc tcctccagaa ggacaccaac 420
tggccccttg ggtggcccat cggtggcttc cccggccctc agggtcctta ctactgtgga 480
atcggcgccg aaaagtcgtt cggccgcgac atcgtggacg cccactacaa ggcctgcttg 540
tatgcgggca tcaacatcag tggcatcaac ggggaggtga tgccagggca gtgggagttc 600
caagtcgggc cttccgtggg tatatcttca ggcgaccagg tctgggtcgc tcgctacatt 660
cttgagagga tcacggagat cgccggtgtg gtggtgacgt tcgacccgaa gccgatcccg 720
ggcgactgga acggcgccgg cgcgcacacc aactacagca cggagtcgat gaggaaggag 780
ggcgggtacg aggtgatcaa ggcggccatc gagaagctga agctgcggca cagggagcac 840
atcgcggcat acggcgaggg caacgagcgc cggctcaccg gcaggcacga gaccgccgac 900
atcaacacgt tcagctgggg cgtggccaac cgcggcgcgt cggtgcgcgt gggccgggag 960
acggagcaga acggcaaggg ctacttcgag gaccgccgcc cggcgtccaa catggacccc 1020
tacgtggtca cctccatgat cgccgagacc accatcatct ggaagccctg a 1071
<210> 8
<211> 1071
<212> DNA
<213> 人工序列
<400> 8
atgtcgctgc tctcagatct catcaacctt aacctctcag acactactga gaaggtgatc 60
gcagagtaca tatggatcgg tggatcagga atggacctga ggagcaaagc aaggactctc 120
ccaggaccag ttagcgaccc ttcaaagctt cccaagtgga actatgatgg ttccagcaca 180
ggccaagctc ctggagaaga cagtgaagtg attatatacc cacaagccat tttcagggat 240
ccattcagaa ggggcaacaa tatcttggtt atctgtgata cttacactcc agctggagaa 300
cccattccca ctaacaagag gcacgatgct gccaaggttt tcagccatcc tgatgttgtt 360
gctgaagaga catggtatgg tattgagcag gaatacacct tgttgcagaa agatatccaa 420
tggcctcttg ggtggcctgt tggtggtttc cctggaccac agggtccata ctactgtggt 480
gttggcgctg acaaggcttt tggccgtgac attgttgacg cacattacaa agcctgtctt 540
tatgctggca tcaacatcag tggaattaat ggagaagtga tgcccggtca gtgggaattc 600
caagttggac cttcagttgg aatctcagct ggtgacgagg tgtgggcagc tcgttacatc 660
ttggagagga tcactgagat tgctggtgtg gtggtttcct ttgatcccaa gccaattcag 720
ggtgattgga atggtgctgg tgctcacaca aactacagca ctaagtccat gagaaatgat 780
ggtggctatg aagtgatcaa aaccgccatt gagaagttgg ggaagagaca caaggagcac 840
attgctgctt atggagaagg caacgagcgt cgtttaacag ggcgccacga aaccgctgac 900
atcaacacct tcttatgggg agttgcaaac cgtggagctt cagttagggt tgggagggac 960
acagagaaag cagggaaggg atattttgag gacagaaggc cagcttctaa catggaccca 1020
tatgtggtta cttccatgat tgcagacaca accattctgt ggaagccatg a 1071
<210> 9
<211> 1071
<212> DNA
<213> 人工序列
<400> 9
atggcgctcc tcaccgatct cctcaacctc gacctcaccg actccacgga gaagatcatc 60
gccgagtaca tatggatcgg cggatctggc atggatctca ggagcaaagc caggaccctc 120
cccggcccgg tcaccgaccc cagcaagctg cccaagtgga actacgacgg ctccagcacc 180
ggccaggccc ccggcgagga cagcgaggtc atcctgtacc cacaggccat cttcaaggac 240
ccgttcagga agggcaacaa catccttgtc atgtgcgatt gctacacccc agctggagtg 300
ccaatcccca ccaacaagag atacaacgct gccaagatct ttagcaaccc tgatgttgcc 360
aaggaggagc catggtacgg tatcgagcag gagtacaccc tcctacagaa ggacatcaac 420
tggcctctcg gctggcctgt tggtggattc cctggtcctc agggtcctta ctactgtagt 480
attggtgctg acaagtcgtt tgggcgtgac atagttgact cccactacaa ggcctgcctc 540
tttgccggcg tcaacatcag tggcatcaac ggcgaggtca tgcccggaca gtgggagttc 600
caagttggcc cgactgtcgg catctctgct ggtgaccaag tgtgggttgc tcgctacctt 660
cttgagagga tcactgagat cgccggagtt gtcgtcacat ttgaccccaa gcccatccca 720
ggcgactgga acggtgctgg tgctcacaca aactacagta ccgagtcgat gaggaaggac 780
ggcgggttca aggtcatcgt ggacgctgtc gagaagctca agctgaagca caaggagcac 840
atcgccgcct acggcgaggg caacgagcgc cgtctcaccg gcaagcacga aaccgccgac 900
atcaacacct tcagctgggg tgtcgcgaac cgtggcgcgt cggtgcgcgt gggacgggag 960
acggagcaga acggcaaggg ctacttcgag gaccgccggc cggcgtccaa catggacccc 1020
tacgtggtca cctccatgat cgccgagacc accatcctgt ggaagccctg a 1071
<210> 10
<211> 1071
<212> DNA
<213> 人工序列
<400> 10
atgagtcttc ttacagatct cgttaacctt aacctctcag agaccactga caaaatcatt 60
gcggaataca tatgggttgg aggttcagga atggatatga gaagcaaagc caggactctt 120
cctggaccag tgagtgaccc ttcggagcta ccaaagtgga actatgatgg ctcaagcaca 180
ggccaagctc ctggtgaaga cagtgaagtc atcttatacc ctcaagccat attcaaagat 240
cctttccgta gaggcaacaa cattcttgtc atgtgcgatg cttacactcc agcgggcgaa 300
ccgatcccaa caaacaaaag acacgctgcg gctaaggtct ttagccaccc cgatgttgta 360
gctgaagtgc catggtatgg tattgagcaa gagtatactt tacttcagaa agatgtgaac 420
tggcctcttg gttggcctat tggcggcttc cccggtcctc agggaccata ctattgtagt 480
gttggagcag ataaatcttt tggtagagac atcgttgatg ctcactacaa ggcctgctta 540
tacgctggca tcaatattag tggcatcaac ggagaagtca tgcctggtca gtgggagttc 600
caagttggtc cagctgttgg tatctcggcc ggtgatgaaa tttgggtcgc acgtttcatt 660
ttggagagga tcacagagat tgctggtgtg gtggtatctt ttgacccaaa accgattccc 720
ggtgactgga atggtgctgg tgctcactgc aactatagta ccaagtcaat gagggaagat 780
ggtggttacg agattattaa gaaggcaatc gataaactgg gactgagaca caaagaacac 840
attgcagctt acggtgaagg caatgagcgc cgtctcacgg gtcaccacga gactgctgac 900
atcaacactt tcctctgggg tgttgcgaac cgtggagcat caatccgtgt aggacgtgac 960
acagagaaag aagggaaagg atactttgag gataggaggc cagcttcgaa catggatcct 1020
tacattgtga cttccatgat tgcagagacc acaatcctct ggaaaccttg a 1071
Claims (10)
1.一种具有草铵膦抗性的谷氨酰胺合成酶突变体,其特征在于,其如下(1)或(2)所示:
(1):其由来源于植物的野生型谷氨酰胺合成酶的第n位发生突变得到;所述第n位的位置通过如下方式确定:所述野生型谷氨酰胺合成酶与参考序列比对,所述野生型谷氨酰胺合成酶的所述第n位对应于所述参考序列的第158位,其中,所述参考序列的氨基酸序列如SEQ ID NO.1所示;
所述谷氨酰胺合成酶突变体的所述第n位的氨基酸为X,X包括A、C、D、E、F、G、H、I、L、M、N、Q、R、S、T、V、W、或删除;
(2):其与(1)所示的谷氨酰胺合成酶突变体至少具有85%以上的同一性、且与(1)所示的谷氨酰胺合成酶突变体在第n位的氨基酸相同、以及具有草铵膦抗性。
2.根据权利要求1所述的具有草铵膦抗性的谷氨酰胺合成酶突变体,其特征在于,所述植物选自小麦、水稻、大麦、燕麦、玉米、高粱、谷子、荞麦、黍稷、甘薯、马铃薯、棉花、油菜、芝麻、花生、向日葵、萝卜、胡萝卜、花椰菜、番茄、茄子、辣椒、韭菜、大葱、洋葱、韭葱、菠菜、芹菜、苋菜、莴苣、茼蒿、黄花菜、葡萄、草莓、甘蔗、烟草、芸薹属蔬菜、葫芦科植物、豆科植物、牧草、茶或木薯;
优选地,所述牧草选自禾本科牧草或豆科牧草;
优选地,所述芸薹属蔬菜选自芜菁、白菜、芥菜、甘蓝、芥蓝、菜苔、苦芥、擎蓝、芸苔、青菜或甜菜;
优选地,所述葫芦科植物选自黄瓜、西葫芦、南瓜、冬瓜、苦瓜、丝瓜、菜瓜、西瓜或甜瓜;
优选地,所述豆科植物选自绿豆、蚕豆、豌豆、扁豆、大豆、菜豆、豇豆或毛豆。
3.根据权利要求1或2所述的具有草铵膦抗性的谷氨酰胺合成酶突变体,其特征在于,当所述植物为水稻时,X=A、C、D、E、F、G、H、I、L、M、N、Q、R、S、T、V、W、或删除;
当所述植物为大豆时,X=A、C、D、G、L、S或T;
当所述植物为玉米时,X=A、C、F、G、H、I、L、M、N、Q、S、T、V、W或删除;
当所述植物为小麦时,X=A、C、G、S、T或V;
当所述植物为油菜时,X=A、C、G、M或S。
4.一种分离的核酸分子,其特征在于,其编码权利要求1-3任一项所述的具有草铵膦抗性的谷氨酰胺合成酶突变体。
5.一种载体,其特征在于,其含有权利要求4所述的核酸分子。
6.一种重组菌或重组细胞,其特征在于,其含有权利要求4所述的核酸分子或权利要求5所述的载体。
7.权利要求1-3任一项所述的具有草铵膦抗性的谷氨酰胺合成酶突变体、权利要求4所述的核酸分子、权利要求5所述的载体或权利要求6所述的重组菌或重组细胞在培育具有草铵膦抗性的植物品种中的应用。
8.根据权利要求7所述的应用,其特征在于,其包括:将分离的核酸分子送入目的植物细胞,所述分离的核酸分子含有编码所述谷氨酰胺合成酶突变体的编码基因;
将所述载体转化目的植物,所述载体含有编码所述谷氨酰胺合成酶突变体的编码基因;
将所述重组菌或重组细胞导入目的植物,所述重组菌或重组细胞含有编码所述谷氨酰胺合成酶突变体的编码基因。
9.根据权利要求7所述的应用,其特征在于,其包括:修饰目的植物的内源谷氨酰胺合成酶基因,使其编码所述谷氨酰胺合成酶突变体。
10.根据权利要求8或9所述的应用,其特征在于,其包括:对植物细胞、组织、个体或群体进行诱变和筛选,使其编码所述谷氨酰胺合成酶突变体;
优选的,所述目的植物选自小麦、水稻、大麦、燕麦、玉米、高粱、谷子、荞麦、黍稷、甘薯、马铃薯、棉花、油菜、芝麻、花生、向日葵、萝卜、胡萝卜、花椰菜、番茄、茄子、辣椒、韭菜、大葱、洋葱、韭葱、菠菜、芹菜、苋菜、莴苣、茼蒿、黄花菜、葡萄、草莓、甘蔗、烟草、芸薹属蔬菜、葫芦科植物、豆科植物、牧草、茶或木薯;
优选地,所述牧草选自禾本科牧草或豆科牧草;
优选地,所述芸薹属蔬菜选自芜菁、白菜、芥菜、甘蓝、芥蓝、菜苔、苦芥、擎蓝、芸苔、青菜或甜菜;
优选地,所述葫芦科植物选自黄瓜、西葫芦、南瓜、冬瓜、苦瓜、丝瓜、菜瓜、西瓜或甜瓜;
优选地,所述豆科植物选自绿豆、蚕豆、豌豆、扁豆、大豆、菜豆、豇豆或毛豆。
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