CN117603986A - 大麦钙调蛋白结合蛋白基因HvCBP60b及其在调控大麦抗盐性中的应用 - Google Patents
大麦钙调蛋白结合蛋白基因HvCBP60b及其在调控大麦抗盐性中的应用 Download PDFInfo
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Abstract
本发明公开了一种大麦钙调蛋白结合蛋白基因HvCBP60b及其在调控大麦抗盐性中的应用。该基因的核苷酸序列为SEQ ID NO.1所示。本发明首次克隆和分析了大麦中响应盐胁迫的钙调蛋白结合蛋白基因HvCBP60b,公布了该基因的核苷酸序列、氨基酸序列和启动子序列,对阐明大麦耐盐调控的分子机理和耐盐材料的培育具有重要意义。
Description
技术领域
本发明属于生物基因工程技术领域,尤其涉及大麦钙调蛋白结合蛋白基因HvCBP60b及其在调控大麦抗盐性中的应用。
背景技术
土壤盐碱化是限制作物生长和产量的一种非生物逆境,严重制约粮食安全和农业可持续发展。目前,全球超过1/3的土地已受到盐碱化的影响。不同植物的耐盐性差异巨大,水稻、小麦等大多数作物属于甜土植物,对盐表现敏感,通常在100mM NaCl环境下生长就受到严重抑制,甚至死亡,而一些盐土植物,如海大麦、藜麦等,可在高于300mM NaCl环境下基本正常生长、发育。“转变育种观念,即由治理盐碱地适应作物向选育耐盐碱植物适应盐碱地转变”,为耐盐碱作物育种提出了任务并指明了方向。发掘耐盐基因并明确其功能是开展作物耐盐育种的基础与前提。
盐胁迫对植物的危害主要是由于高渗透和高浓度盐离子所致,并继而引发次生氧化胁迫影响植物的生理生化和代谢过程。通常,高浓度的盐离子(如Na+)改变土壤的基本属性,导致土壤孔隙度降低,从而减少土壤通气性和导水性,进而减弱植物吸收水分的能力,限制植物的生长。其次,植物细胞内Na+等离子的过度积累会抑制酶活性,破坏植物代谢,甚至导致植物死亡。细胞质是多种代谢的主要场所,包含许多参与初级代谢、卡尔文循环、苯丙烷途径、糖酵解以及多胺和淀粉合成的酶物质,而这些酶大多受到K+的调控。由于K+和Na+的化学性质相似,通常认为细胞内高浓度的Na+可在这些酶促反应中代替K+,从而降低代谢效率,进而产生毒害作用。
钙离子(Ca2+)是一种普遍存在的第二信使,在几乎所有生命形式的多种信号转导机制中发挥着关键作用。盐胁迫会在几分甚至几秒内引发细胞质Ca2+浓度的增加,表明Ca2+是响应盐胁迫的早期信号之一。有研究发现植物受盐胁迫时,整个根部内皮细胞中细胞质Ca2+浓度很快显著增加。另外,植物在盐环境中叶片中易产生系统性的钙离子波动。因此,盐胁迫过程中的钙信号是一种相当复杂的现象,钙离子信号在整个植物对盐胁迫的反应中具有核心作用。环境中的盐离子浓度,可以快速而强烈地调节细胞溶质内的Ca2+浓度。因此,适当调节细胞溶质Ca2+水平对细胞存活至关重要。在细胞中已发现了多种Ca2+感受器,其中含有EF-hand的蛋白分子可能在植物钙信号传导中起主要作用。通常,含EF-hand的蛋白质可主动结合Ca2+,即螯合细胞溶质中的钙,从而调节钙的稳态。钙调蛋白(CaM)即是含有EF-hand并能与Ca2+结合的一种感受器,它主要通过捕捉细胞内变化的Ca2+信号,形成Ca2+/CaM复合体,进而将这种信号传递给下游基因或反应。其中,一些转录因子可能被Ca2+/钙调蛋白直接激活,包括钙调蛋白结合转录因子(CAMTAs)、GT-元件结合样蛋白(GTLs)和MYBs,而这些转录因子反过来可调节各种基因的表达水平,并最终影响植物耐盐性。
大麦是全球第四大禾谷类作物,其用途且集粮食、饲料和啤酒原料于一体,并具有较强的耐盐性,是研究作物耐盐机制的理想材料。前期研究发现钙调蛋白HvCaM1具有负调控耐盐性的生物学功能,并推测其对耐盐性的调控是通过与HvCBP60b结合而实现的。钙调蛋白(CaM)结合蛋白60类(CBP60)蛋白是植物非典型转录因子,拟南芥基因组CBP60a-g和系统获得性抗性缺陷1(SARD1)共编码8个成员,CBP60g和SARD1在病原菌入侵过程中介导免疫基因的表达,在植物免疫中扮演重要角色。最新研究表明,CBP60b在病原菌诱导的防御中与CBP60g和SARD1一起发挥冗余作用,但CBP60b在基础防御上起关键作用,部分是通过直接调节CBP60g和SARD1来实现的。此外,有研究表明,CBP60g和SARD1也与非生物胁迫反应有关,这两种基因在寒冷胁迫下表达增强,而CBP60g过表达可提高植物的耐旱性,cbp60g敲除株系则表现对干旱胁迫更敏感。但是,迄今尚未有过CBP60响应盐胁迫的相关报道,亟待研究明确。
发明内容
本发明提供了一种大麦钙调蛋白结合蛋白基因HvCBP60b及其在调控大麦抗盐性中的新用途。
具体技术方案如下:
本发明提供了一种钙调蛋白结合蛋白基因HvCBP60b,其核苷酸序列为SEQ IDNO.1所示。本发明首先克隆得到一个大麦的钙调蛋白结合蛋白编码基因,命名为HvCBP60b,其转录本cDNA全长核苷酸序列为SEQ ID NO.2所示。
SEQ ID NO.1:ATGAACGAGAAGCGCGGGCTGGAAGCGGCCGCCGCCGGCGACGG CCGCCCCGAGGCTAAGCGGTCGCGCCCCCCGGCTCTTGCCAGGTCAGGAATTCGTCGAGTTTCGGTACCCCTTTTAAGTGCGTGCGTGATGTGATTTTTGTGTGTCCGTGCCGATTCAGCCGCGCGAGTTCTAGCCAGTGTAGCTGCTTGGGTTCGTATAGTGGCAAAGGCTTTCGTGATTTAGCGCCGGCTTACGTATTTATAACTGAGATTTGGGAATTCATCTTCAGATTAATTGCTCGCGATTTGGTGCCGTCTAGCTGAGAATCTGAAGATGTATTTTTTTTTTATTGACAAAGGGTCGTCAGGTTGTTATTAGCGGGGTGATGATTAGTCTCCTAGTTATGGTTTTGATGACCATGGGCGTTTTGAAGTTGCCTTTTGGTGTTATGTACTCCTTAGTATTAGAAACAATTTGAGCTGGAACTGGTGTGACCTGCTGGGAAATGAGTGAATTATTGGTAACTTGGTGGTTAACTGTTGAGAGAAAGGACAAGATCCGTTATAAGTAGTTTCAGGAGCACACTCGTGACCACGTTATAAGCATCAAGGACGTACACTCAAGGACGCCTATGCTTACCACTGAAAACAATTTAAGAAAAATGTATACATCATCCATCACATATTAGGACAAAATCAAATGCCAGGCTTGTTCTGTAGCTTTAACTCCAGGCTTGTCATCCACTCTATTCTGTTGTTAGTATAATATTTTAACCTCATGCACATAAGTTAAGCAAATACATAAGATTACCACTGTGGCCTTGTTAAAATTATCTCATTGAACTTGTCCAACAAGAATCTCCCTAGTTAATGGCAAGAACTATCTCTAAAGCGCTATCTTCTGGGCGTTATGTTTATAGGCATAATCTCCATACTATTCTCCTATTTATTTGAATAGCCAAAGCTTAAACATTGTTTAGTGTGACCATGTGAACATATATAAGCAGTTCCAGTGCAATTGGCACATGGCATATTCTTGGTTGTGGTCTGTGTGAACATGCGGTGTTGGATACTTAGATGTGCTACAGACTACATAAATCATTGTAGGACAGTTAAGAAGTACTCCCTCCGTCCCAAAATAACTGTCTCGACTTTGTACTAGCTCTAATATAAAGTTATACTAAGCTTAAGACAGTTATTTTGGGACGGACGGAGTATTATTTTACCATCTTTCAGTAACCAGTGTGTCTCTTTTGCTTTCATGTATCTGTACTTGTAATTCTGTTTCGTAATGCTAGCAGAAGACTGTCAAGTGCTGTAACATTATGATGTTGAGCTGTGGCCGGTTGAATATTAGCTAAGTATTATCTTAAATGGTTTTAGTGAACATTTTTTCTGTTGGGTTTACCTTTTAATTGGAACAACGTTTAATCATGGCCTTTATACTTATTCATTGTACTTGTACAGTGTTATTGTTGAAGCACTAAAGGTGGATAGTCTGCAGAGGCTTTGCTCATCATTGGAACCAATTCTCCGTAGAGTGGTAAGAACAAAGCAAGTATTAAGCAGGAATGCAGTTATGACATATCTTTGTGGAGTTGCAAAATGTCTATTGACATTTTGTGTTTCTTTCTGTTGGTTGTAATATGATTAATTTCGTATTTTGCTTGTAGGTTAGTGAAGAAGTTGAGCGCGCCTTGGGAAAACTTGGTCCTGCTGCAATCACTGGGAGGTATGACATCTAACCAACAATGAAGCAAATATATTAGTTTCTTTGTTAAAAAGTCGATATGTTACTTTCCATTTAAGATGCACCAAACCGTTTGTTTGGTGCTTTCTCCATGTTGTGTTATGTACCTGACACGGATATGCCTCATGTGGTATGTATCAGATTATTTTTGAAATATTTGTACACACTGTACATGGCTATGCAGCCCTCTGCCAGAAAAGAAAGCTCATCAAACAACCTAAGCATGACCTCTAACCCTACCTTGACATAGGAGTATGCGAGTAGCCAAGCTATAGCTTCCCTCTCCTATTGACACCCACATACATTCCTCCCCCATCTACAGGTCCATCTCTTGGCGTAAATGGGGCACAATCGGTTCCGTTCCGTTTATAATTTTTTCTCTGTTTCAATCTATAGACTCATGTTACACCTGACTTCCGGACTGCAATAAGGAAGTGGGATATTTCTTATGGTTGATTTTGCACAGAAATCGAAGATATTTTATGTGGCTTATTATGTTTTACTTGGAGACCCCATATTGTCAGTGTCAATTCTAGACTAGAGGAACTACACTATTGGATACTTCATAAGTTTTCAAATGTATATGTAGCGTTGTCAGTTTGTCAAGGAAAAAACATGTATCACCATAGCCTACTTTTTCATTATACCTTTTAGCTGTGCACTTCATTCGTTTCAAAATAGATGTCCATCTAGGGTTAGGCATAGAAATGAAGAGTGAGTGAAATGTCAATATTGCCCTTCACTCATTTCTGTCTCCATATACTTTTCCTCACCAAGGCATTAAATGATACCTCTTTTTCCTCCCCCAAGCAACACCTGCAAGCAAAAGATGGGTTATTGGGAATTGCAAACATGAGGAAGAGTTAATTATCTCTTATCTTCTACTACGTTGTTGACGGGAAAAAAAGAGTAAAGTGACTAAAATGTATTGGAGGTGCAGGAGGGCATTATTTTGAAATGGAAGGAGTATGGCACGTAACACCCTTTCCTTACTGAATTGTAGAATATATTAATATGGTGCAAGTAACACATGATGGTATTGCCAGTAACTTGATGTTTGTGGTGTGCAATGTAATTTGGCTTCACGCGTCATGCTATATCCTCGCAAAGTATGTAAACTTTGAGGCCTAAAAGATGAAATGGCCTTCTTTGGGAGCCACATTGATACTAATTATCTGCTACTGATTGGAATAATGCAGGTCTTCTCCAAAGCGAATTGAAGGTCCTGGTGGAAGAAATCTGCAACTCCAATTCAGGACAAGATTGTCCCTTCCCCTTTTTACCGGGGGAAAAGTTGAAGGGGAGCAGGGGGCTGCAATTCATGTTGTTTTGCTTGATACCAGCAATGGCTGTGTTGTATCATCTGGGCCAGAGTCGTCTGCCAAGCTTGATATTGTTGTTCTAGAAGGCGATTTCAACAATGAAGATGAGGAAGGCTGGACAGAGGAGGAATTTGACAGTCATGTAGTGAAGGAGCGTGAGGGAAAACGACCTATTATAACTGGCGATATACAAGTCACACTAAAAGACGGTGTTGGCACAATTGGGGAGTTCACGTTCACAGATAACTCTAGCTGGATAAGGAGTAGGAAATTCAGACTTGGTTTGAAAATTGCCTCAGGGTTTTGTCAAGGTGTTCGCATCCGTGAGGCAAAAACTGAAGCTTTCATGGTTAAGGACCATAGAGGAGAATGTAAGTTTTCTGTAATGGTAATCATGTGCTTCCGCTTGAGATATGTCTGAACAATTTGAATTTCACTTCTTTAGTGTACAAGAAGCATTATCCACCTGCATTGAAGGATGAGGTCTGGAGGTTAGAGAAAATAGGAAAAGATGGGTCGTTCCATAAGAGGCTGAATAAATCTGGAATTTTAACTGTTGAAGATTTTCTCAGGCTTGTTGTTCGGGATCCACAGAAGTTGCGTACTGTAAGTTCGTTTCTTTCCATGGCCTTTGATCTATTGCTGTTGTAGTTCTAATTTGTATACTATTCTGTTGTTTTAGATAGTTCGTTTCTTTCCACGGCCTTTGATCTATTGCTGTTGTAGTTCTAATTTGTATACTGTTCTGTTGTTTTAGATCCTCGGAAGTGGCATGTCCAACAAGATGTGGGATTCTCTCGTTGAACATGCAAAAACCTGCGTCTTAAGTGGAAAATATTACATATACTATTCTGATGAGCACAGAACTGCTGGTGCTATTTTCAACGACCTCTATGCATTCTGTGGGTTGATTTCTGGCGAGCAATTCTATTCATCTGAAAGTCTCGATGATGGCCAAAAGGTTTGTGCTATGGGCATATATTTGGGGGCATTTGATATCTAGTGTTATGTTTTATTGAATTTAAAGGTTGTTTCCATTCATTTTAACATGCACCCATCCTAGATAATGAAACTAAATTTGTATTGTTTTGGAGTTTTGAAATTGGTATGCTCTCTGTTCCTGATTCTAGGGTGCTTTAGTTTGGGCGTCAATATTAAGGAGTGTGGGCATGGACATGATTAGTTGAGAGTTGGTCGATTTTGCTCTCACTCTTGTCTTAGAAGTTCATCTCATGCAGAGTCTGCATATCTTGCTTGCCCTGATCTCTGCTCACCAATAGTCTGCCTACTATTGAACATGCTACATGGAAGGAGATGTTCCAGAGATTTTGTTTCCAGTATATGGTCGGGTCGTTTAGATTTTGGAAGTTCAGGGATCTTTAGCGTTTTTACTATGGTAGGGGAACAGGAACGTCATGTATGCAGGAGTATTATCAGGAGAAAGGAGGAGAAAAACCTCCAGGGCCCTAGATCTGGGAAAAGACGACCAAGGAACTGTGATTGAGGGAGTAATATGTAGATGTCTGACTTCATCCTATATCACATTGGTCTTGATCTGTCCACACCCTTTATATTCCAAGAATGGTGGCCATGGATAGTGTGTATTAACTATTAATTTCTGGAACCGTTATATGTTTTTAACGCACTAAATGCTCATTAGGTACGTTCTCTTTGCTGATGGGCTGGTGAAGAAAGCATATGATAATTGGATGTATGTTATTGAATATGATGGTAAAGCTCTCTTGAACCCTAAACCGAAGAAAAAGGCTGCATTAACTAGTCAACCTGAGGCTCGTGCTCCTGCTGCATATGTACAGCGCATTTCTTCAACGAGTATGCCAGGACCATCTACAGCAGGTAAGTAGCACACAAGTGCTTAGTTACAGAATTCCAAGTATTTTTACGATTTTACTTCAGAAGTAGGAAAATACGACTTAATGCGTTGGAAAAACTGAAACATTGATTTTGTATTTAAGGCTGGCCATAGTGGTGGTATCTTAGCCGATATCATGCACTCGGGAATAGCAAACGTGCTGATGTGTCAGAGAATTAAAGAAGAGAGAGATGGGTAGAGTAACATAGGTTGATACCGTATCATGTTAAATGCTATGCTACTTTGTGTCATGCATGACAATAAATAAGATCATATATGATATTACTCTGATACTATGCATTATAAAGGTACCATCATACACTAGTATAATATGCATGATATTAGTATATGATACTTCCCACTATGAGCAGCCTAAATATATACTCCCTCCATTCCATAATATAAGATCTTATTACATCTTATATGCGAGGATATTGGATGTAATAAGGTCTTATATTATTGGTCGGAGGGAGTACAAATTAAGCAAATGGTGTATGAAATATTTTATGATGTCACTGTGGAATAGCAGTTTCTGAAAACCATATGTTTTGTTTCATCTATTGCAGGCTCAAATGGTTCTATAGGGTATGATGGCAACCAGACAGCAACACAGTCTGTTCAGCTTCAGAGTTCATCTGCTAATGTGCCGTTGCCATATGATGAGGCCTTTTCATTTTTGCCGCCTACCATGTTGATGGGTTCTAATCAAGGAACAGCAAGTGATGCCATGGGTCTGGAACTCAGCCAATTGCATCACACAATTTCTCAAGCCCATCCAATTCAACCAGCAAATGTCAGCTACGATAATTGGCATCACAACCGTGATAGTCAGTATGCTGATGATTTCACCGAAGACATTCGCATGAAAAGCCACCAACTGCTCGAGGGTGAAGACATGCAGCAGCTGCTCAGGGTCTTCAATATGGGTGGAGCTTCTCCTGGTTTGCCAGACGAGACGTTTTCCTACCAGACCTACATGCCATCTCCATTGCCAACCCCAGGCTTCGAAGGTGAGCCCAGCCGTCCATCGGGCAAAGCCGTTGTTGGGTGGCTCAAGATAAAGGCCGCTATGAGGTGGGGAATATTTGTCAGGAAGAAAGCTGCGGAGAGAAGGGCACAGATTGTTGAGCTGGATGATTAA。
SEQ ID NO.2:ATGAACGAGAAGCGCGGGCTGGAAGCGGCCGCCGCCGGCGACGGCCGCCCCGAGGCTAAGCGGTCGCGCCCCCCGGCTCTTGCCAGTGTTATTGTTGAAGCACTAAAGGTGGATAGTCTGCAGAGGCTTTGCTCATCATTGGAACCAATTCTCCGTAGAGTGGTTAGTGAAGAAGTTGAGCGCGCCTTGGGAAAACTTGGTCCTGCTGCAATCACTGGGAGGTCTTCTCCAAAGCGAATTGAAGGTCCTGGTGGAAGAAATCTGCAACTCCAATTCAGGACAAGATTGTCCCTTCCCCTTTTTACCGGGGGAAAAGTTGAAGGGGAGCAGGGGGCTGCAATTCATGTTGTTTTGCTTGATACCAGCAATGGCTGTGTTGTATCATCTGGGCCAGAGTCGTCTGCCAAGCTTGATATTGTTGTTCTAGAAGGCGATTTCAACAATGAAGATGAGGAAGGCTGGACAGAGGAGGAATTTGACAGTCATGTAGTGAAGGAGCGTGAGGGAAAACGACCTATTATAACTGGCGATATACAAGTCACACTAAAAGACGGTGTTGGCACAATTGGGGAGTTCACGTTCACAGATAACTCTAGCTGGATAAGGAGTAGGAAATTCAGACTTGGTTTGAAAATTGCCTCAGGGTTTTGTCAAGGTGTTCGCATCCGTGAGGCAAAAACTGAAGCTTTCATGGTTAAGGACCATAGAGGAGAATTGTACAAGAAGCATTATCCACCTGCATTGAAGGATGAGGTCTGGAGGTTAGAGAAAATAGGAAAAGATGGGTCGTTCCATAAGAGGCTGAATAAATCTGGAATTTTAACTGTTGAAGATTTTCTCAGGCTTGTTGTTCGGGATCCACAGAAGTTGCGTACTATCCTCGGAAGTGGCATGTCCAACAAGATGTGGGATTCTCTTGTTGAACATGCAAAAACCTGCGTCTTAAGTGGAAAATATTACATATACTATTCTGATGAGCACAGAACTGCTGGTGCTATTTTCAACGACCTCTATGCATTCTGTGGGTTGATTTCTGGCGAGCAATTCTATTCATCTGAAAGTCTCGATGATGGCCAAAAGCTCTTTGCTGATGGGCTGGTGAAGAAAGCATATGATAATTGGATGTATGTTATTGAATATGATGGTAAAGCTCTCTTGAACCCTAAACCGAAGAAAAAGGCTGCATTAACTAGTCAACCTGAGGCTCGTGCTCCTGCTGCATATGTACAGCGCATTTCTTCAACGAGTATGCCAGGACCATCTACAGCAGGCTCAAATGGTTCTATAGGGTATGATGGCAACCAGACAGCAACACAGTCTGTTCAGCTTCAGAGTTCATCTGCTAATGTGCCGTTGCCATATGATGAGGCCTTTTCATTTTTGCCGCCTACCATGTTGATGGGTTCTAATCAAGGAACAGCAAGTGATGCCATGGGTCTGGAACTCAGCCAATTGCATCACACAATTTCTCAAGCCCATCCAATTCAACCAGCAAATGTCAGCTACGATAATTGGCATCACAACCGTGATAGTCAGTATGCTGATGATTTCACCGAAGACATTCGCATGAAAAGCCACCAACTGCTCGAGGGTGAAGACATGCAGCAGCTGCTCAGGGTCTTCAATATGGGTGGAGCTTCTCCTGGTTTGCCAGACGAGACGTTTTCCTACCAGACCTACATGCCATCTCCATTGCCAACCCCAGGCTTCGAAGGTGAGCCCAGCCGTCCATCGGGCAAAGCCGTTGTTGGGTGGCTCAAGATAAAGGCCGCTATGAGGTGGGGAATATTTGTCAGGAAGAAAGCTGCGGAGAGAAGGGCACAGATTGTTGAGCTGGATGATTAA。
本发明还提供了HvCBP60b基因启动子,该启动子的核苷酸序列如SEQ ID No:4所示。该启动子为3kb,用于引导该基因在植物细胞中大量表达,以满足快速合成钙调蛋白结合蛋白和响应各种刺激或胁迫所需。
SEQ ID NO.4:AAGATGAGAGGGAAGGGGTGCGCCCCTGACACGCGTTTTCAAATA CCGAATCACGTTCGACGAATAACCGAAGACGACGCAGCTACGGTCGACCGTTCGGGTACGAGACGGACTCCGATTGCGATGAAATTTGGCAAGCGGCCTAGCTACAACTAATTAAGACCGCACGCCAAGTTTCAACTCAATCAGAGAATATTTTACACACACTTTTAAAACAGGGTTTAATCGATGCCGCGGGCGCGTATGTGTGCGGTCGGGCTCAGAACGGACAACGACGAGAACCGGCAACTAATAACGGACACAAGTTTTGAAAACTAGCGGCAACGGGATGCCGATGCAATGCGGATGATGCGACAAGTAAAAATAGACACACGACGAAAACGGAATAAAGGGGGGAATCTTCTGGAACGTCGGTCTCGGGCTGTCACAGTCGGTTGGCCTCGTTCTGGTGGCGGTGACGGGCTATGTTGCGGCGGCACATCATCCCCCTTCAAATTGGCGCCAGCGTCGAGGGCCTGGTGGATTGCTCGGCGTCATCTGATCAGATATACTATGGCCGATGCAGCCGACAGTGGCGGTCATCTCCTCCGTATAGGTGGTCGACCTGAGGCTGGGTGGTTGGATCTCGCGATCCATCATCCAGTCCTGGCTGTGAGTCGGGAAGACATAGTTGTCGGTGAAAACCGAGCTGATGGCGGCGTTCCGCGTCGTTACCTTGATGAAGGCATCATCGTGTAACTACTGTTGACCCACTCATGATGCTTAGGGGGAAACCCTAAGATCTGATCGTCAGATCGGACGATGGCGGCACTACGGTGTCTTGCCTCTTTTGGGAGCATCGTTTCTGGAGCAGCGCTGGATGATAGAGGAAGGCGATGGAGCGACTTCGTCTTGATCGGAGCTTTGGTGGAGATGTCAAGTCATGCCTGGGCGACAGGTGCTACGTTGTGTCATGCCTGCTCGACAGGTGCTACGCACGACAGATCTTTCAGAGCCTTCATGTCAGGATGGACGAGCGCAGGACGGTGGCGCCGTTGGGCGTCGTGGTGGTGTCGACGGATGGACGAACTGGCAAGGGTGATACAGATCTCTCTCCTGAAAATGGGACAGTGGTTCGATGATGATGGCGGCTTCTAAAACGTGTGCAAGTGGTATACGATTTAAGTTTGCTGCACCGTTTGTTTTTTCGATACGTTATAGGGATGGATAGGTGATGACAACGGTTTTTGATATGGAGAGTGAGAGCATTCAATATTATCATGTTTGTAGGTGTGAGTGATGGCTTCGGATGGCTTAATGTATGATCTTGTCAGACCTTTGTGTAATAATTAATAAAAATGGTGTATGCATCAATTGATGTAGATGCCGAGGGTTGAACCTTCTTTTCTAAAACAAAGTAAGAGTTACCGAAAACCAAACATATTTCCTTGCATTTTTCTGGTGAAAAATACATACTTCCTCTCATAAAGAGCTAGCATTTAGTTCATCACCAAAATGGACCCTCGCGAACCAGTCTTCATTGACTTACTCCCACTTTCTCTTGGTTACTTGGGTACAAAAGCAGTACTCCCTTTTTCAAAATAAGTGTCATAATTTAAATTAAAATTAGTTTAGAACTGCAACACTTAGGGGGTGTTTGTTTCCACGGACTTTTTGGTGCAGGGACTAAAAAAAGTCCCTTTTAGTCCCATGTGAAACAAACAGGAGGGACTTTTAGGGACTAAAAGGGGGTATTTGGGACTAAAGGAAGAAGTCCCTATGGGAGGGTCTTTTTGGGACTTTTTGACACTTTTTCCAATATTGCCCCTACTTTTAGCATGTTATTTAATAACTACTACAACATTACTAGGGGTAACATGGTCTTTTTGCATGTTATTTAATGACCTCTAATCTCTGTTTAGTCCATGAAAACAAACGGGTAGGGACTAGGGACTTTTTAGTTGGGACTTAAAAAAGTCCTGGGATTTCTGAAACAAACAAGGCCTTATTATGGATCTGAGGGAGTACTAATTACTCCCTCCGTTCCTAAATATAAGACCTTTTAAAGATTGTACTATAAACTACATACGGATGTACATAGACATATTTTAGAGTATAAATTCACTCATTTTGCTTCGTATGTAGTCTTCTAATAAAATACCTAAAAGGTCTTATATTTTAAGACGGAGGGAGTATTATCGTATCTACTTCAATAGACTGCTAAATCTTGCCCTAATATGAAAATGCGGAGGTCGCTACTACAAGTAGAAACTGAAAAAGATAATAATAAGAGAAAGGGTTTTGAGCACAACAAACCTGCGCGAGCGCTGCCGGTGCCGACACCGACGCCCAGGAGGAAGCCACCGTGCGACATGACTCCGACGACGGCTATGGGGAGCAGTCGGAGCCGGGGACGAGGAGCAGCTATCACTTCCTCGTGGGCCTCGCGTGGATCACATGGGTTTGATTGGGTGGGGCCTGATCACAAGTTGGGTTGAGGTCTGGGCCCACTTTAAACTATTCTACGCGGGCCGCCGTTCCACGTGTGCTAATCATCGTCCTAACCATGCCCCAGCGAAAAGATCGCGCCGACCGACCCACACCGCGCCACCACTTGCCACCACGGGCGTATCGAGCTGGGCTTTCGGATAGAATATAGGAGTATCTACGCTGCGCCTGGGCCCAGCCGCGGGCACGTCACCCACCGGGCGGCGACGTCACACTGCCGCCGTGGGCCCCGCGCGCTTTTCGTCAGTGGATGGTACAATCTCTATCTGTCTCGCCAAAAGGATCTCTGACCCATCCGCCCCGCCGACGCCGACGCGCCGTTGCCTTCTCTCCCCTCACCTCCCGACCTCATCGCCGCGATCCCCCTCCCTCCGCCCGGCCCCGAATCCAGCGCCCGGCCCAGGCGACCGCGGCCGATCGATCTCCTCCGGCGCGCGGAACCGGTGAAGGCGGCGGCGGCGGCGGTCGCGCGCTCGATTCGGTGCGGGTGCCGGCTAGGGTTCGCTGCCGGCG。
本发明还提供了钙调蛋白结合蛋白基因HvCBP60b的编码蛋白,编码蛋白的氨基酸序列如SEQ ID NO.3所示。
SEQ ID NO.3:MNEKRGLEAAAAGDGRPEAKRSRPPALASVIVEALKVDSLQRLCSSL EPILRRVVSEEVERALGKLGPAAITGRSSPKRIEGPGGRNLQLQFRTRLSLPLFTGGKVEGEQGAAIHVVLLDTSNGCVVSSGPESSAKLDIVVLEGDFNNEDEEGWTEEEFDSHVVKEREGKRPIITGDIQVTLKDGVGTIGEFTFTDNSSWIRSRKFRLGLKIASGFCQGVRIREAKTEAFMVKDHRGELYKKHYPPALKDEVWRLEKIGKDGSFHKRLNKSGILTVEDFLRLVVRDPQKLRTILGSGMSNKMWDSLVEHAKTCVLSGKYYIYYSDEHRTAGAIFNDLYAFCGLISGEQFYSSESLDDGQKLFADGLVKKAYDNWMYVIEYDGKALLNPKPKKKAALTSQPEARAPAAYVQRISSTSMPGPSTAGSNGSIGYDGNQTATQSVQLQSSSANVPLPYDEAFSFLPPTMLMGSNQGTASDAMGLELSQLHHTISQAHPIQPANVSYDNWHHNRDSQYADDFTEDIRMKSHQLLEGEDMQQLLRVFNMGGASPGLPDETFSYQTYMPSPLPTPGFEGEPSRPSGKAVVGWLKIKAAMRWGIFVRKKAAERRAQIVELDD。
本发明还提供了包含权利要求1所述的钙调蛋白结合蛋白基因HvCBP60b的CRISPR敲除重组载体,所述CRISPR敲除重组载体由编码基因HvCBP60b编码区域的19bp基因片段连入pUB-Cas9-TaU6-sgRNA载体构建得到。
本发明还提供了所述的钙调蛋白结合蛋白基因HvCBP60b作为负调控因子在提高大麦耐盐性中的应用。
本发明还提供了一种基因工程菌,所述基因工程菌中包含有编辑HvCBP60b钙调蛋白结合蛋白基因的CRISPR/Cas9载体;所述HvCBP60b钙调蛋白结合蛋白基因的核苷酸序列为SEQ ID NO.1所示。
本发明还提供了所述的基因工程菌在调控大麦耐盐性中的应用。
钙调蛋白结合蛋白基因HvCBP60b参与大麦耐盐性的调控,抑制该基因的表达可提高大麦的耐盐性。作为优选,所述大麦品种为大麦栽培品种Golden Promise。
进一步地,所述应用的途径为通过沉默HvCBP60b基因、敲除HvCBP60b基因或抑制HvCBP60b基因的表达获得突变体,从而提高大麦的耐盐性。
进一步地,敲除的手段为CRISPR/Cas9基因编辑技术。
进一步地,所述的应用包括以下步骤:
(1)设计HvCBP60b基因的靶序列,构建CRISPR/Cas9载体;
(2)构建含步骤(1)所述CRISPR/Cas9载体的农杆菌基因工程菌;
(3)将步骤(2)所述基因工程菌转化大麦幼胚,获得不含外源Cas9蛋白且稳定遗传的纯合单突变体株系。
与现有技术相比,本发明具有以下有益效果:
(1)本发明首次克隆和分析了大麦中响应盐胁迫的钙调蛋白结合蛋白基因HvCBP60b,公布了该基因的核苷酸序列、氨基酸序列和启动子序列,对阐明大麦耐盐调控的分子机理和耐盐材料的培育具有重要意义。
(2)本发明首次通过基因编辑遗传转化手段,证实了HvCBP60b基因参与大麦耐盐性。其中敲除HvCBP60b基因后可显著增强大麦的耐盐性,有效缓减盐胁迫带来的不利影响,从而维持植株的正常生长。
(3)本发明指出钙调蛋白结合蛋白家族在植物中还存在多种分型,如能得到充分发掘和有效利用,将有利于改良大麦品种的耐盐性,同时亦可提高其他作物的抗逆性。
附图说明
图1是HvCBP60b基因结构图;
其中,数字表示碱基位点,白框表示非编码区域,黑框表示外显子。
图2是CRISPR敲除载体构建示意图;
其中,TaU6启动子驱动sgRNA,玉米Ubi启动子(Ubi pro.)驱动Cas9蛋白的表达,35S启动子驱动抗性基因潮霉素(Hygromycin)。
图3是基因敲除材料水培实验结果;
其中(a)在盐处理(200mM NaCl)三周以后的基因敲除植株表型差异图,WT:野生型;(b)地上部和地下部的干重测量结果,单位:g;(c)茎长和根长的测量结果,单位:g;(d)地上部和地下部的K+含量测量结果,单位:mg.plant-1;(e)地上部和地下部的K+浓度测量结果,单位:mg.g-1DW;(f)K+转运率测量结果,单位:%;(j)地上部和地下部的Na+含量测量结果,单位:mg.plant-1;(h)地上部和地下部的Na+浓度测量结果,单位:mg.g-1DW;(i)Na+转运率测量结果,单位:%。
具体实施方式
下面通过具体实施例,对本发明的技术方案作进一步的具体说明。应当理解,本发明的实施并不局限于下面的实施例,对本发明所做的任何形式上的变通和/或改变都将落入本发明保护范围。
在本发明中,若非特指,所有的份、百分比均为重量单位,所采用的设备和原料等均可从市场购得或是本领域常用的。下述实施例中的方法,如无特别说明,均为本领域的常规方法。KOD One高保真酶,购自日本东洋纺(TOYOBO)有限公司;T载体,购自南京诺唯赞(Vazyme)生物科技股份有限公司;农杆菌AGL1,购自上海唯地生物(Shanghai WeidiBiotech)有限公司。
实施例1大麦钙调蛋白HvCBP60b基因和启动子克隆
1、植物材料的处理
大麦Golden Promise种子用2% H2O2消毒20min后,用ddH2O冲洗5次后,种子腹面朝上在发芽盒中发芽。于培养室(14h/10h,22℃/18℃)暗培养3天,至地上部露白2cm后补光。取7日苗龄的大麦幼苗,液氮速冻后保存于-70℃,用于DNA和总RNA提取。
2、大麦基因组DNA的提取
采用CTAB法提取大麦基因组DNA。
3、大麦总RNA的提取
采用Trizol法提取大麦总RNA;并用DNase I酶(Takara,日本)去除基因组DNA。
4、cDNA的合成
使用Takara公司的RT reagent Kit Perfect Real Time(DRR037A)试剂盒将上述3中提取的总RNA反转录成单链cDNA。
5、HvCBP60b基因、启动子等的克隆
以上述步骤2中的DNA和4中的cDNA为模板,根据目的基因的ORF框分别设计引物,选用KOD One高保真酶分别进行PCR扩增:
PCR体系(50μL):
获得特异性的目的条带,切胶回收后将片段连入T载体(Vazyme),转化大肠杆菌DH5α,PCR验证得到阳性克隆,然后送公司进行测序。引物合成和测序均由生物科技公司完成。
所涉及的引物和序列信息见附图,其中:
以DNA为模板,DNA-F/R扩增的全长基因组序列6070bp,为SEQ ID No:1所示,引物信息为:
DNA-F:5’-CCGACCCACACCGCGC-3’,
DNA-R:5’-TGCCGATGCACCACTTCAACA-3’;
以cDNA为模板,cDNA-F/R为引物扩增全长cDNA序列1845bp,为SEQ ID No:2所示,引物信息为:
cDNA-F:5’-ATGAACGAGAAGCGCGGGCTG-3’,
cDNA-R:5’-TTAATCATCCAGCTCAACAATC-3’;
以DNA为模板,Promoter-F/R扩增的启动子序列3000bp,为SEQ ID No:4所示,引物信息为:
Promoter-F:5’-AAGATGAGAGGGAAGGGGTGC-3’,
Promoter-R:5’-CGCCGGCAGCGAACC-3’;
通过BioXM 2.6将HvCBP60b基因的CDS核苷酸序列翻译成氨基酸序列615aa,为SEQID No:3所示。
通过对基因结构进行分析,发现该基因存在八个外显子和七个内含子,基因结构图见附图1。
实施例2CRISPR基因编辑方法验证HvCBP60b基因功能
1、pUB-Cas9-TaU6-sgRNA:HvCBP60b CRISPR敲除载体的构建
为了生成CRISPR/Cas9质粒,设计了两个HvCBP60b的单引导RNA序列,并通过限制酶位点BsaI分别连接到pTaU6:Cas9载体,靶点信息为:
TaU6-CBP60-sg1F:CTTGAGATGGGTCGTTCCATAAG
TaU6-CBP60-sg1R:AAACCTTATGGAACGACCCATCT
TaU6-CBP60-sg2F:CTTGTCACACTAAAAGACGGTGT
TaU6-CBP60-sg2R:AAACACACCGTCTTTTAGTGTGA
经T4连接酶连接之后,最终获得pTaU6-sgRNA-Cas9敲除载体(见附图2)。
2、基因敲除大麦植株的获得
采用热激法将上述步骤1中构建好的表达载体,分别导入农杆菌AGL1中。将含CRISPR敲除载体的农杆菌依次转化大麦Golden Promise幼胚,经潮霉素筛选后,获得数十株基因敲除候选植株。再依次提取基因敲除候选植株和大麦Golden Promise野生型的DNA,通过PCR的验证方法获得基因敲除阳性植株,验证引物为CBP60-SG1/2-F/CBP60-SG1/2-R,扩增片段长度593bp,引物信息为:
CBP60-SG1/2-F:5’-TGATACCAGCAATGGCTGTG-3’,
CBP60-SG1/2-R:5’-GTACGCAACTTCTGTGGATC-3’。
繁种后,收获足够用于实验的基因敲除种子。
3、大麦HvCBP60b CRISPR敲除植株耐盐性的比较
(1)水培实验:依次选取大麦HvCBP60b CRISPR敲除材料各3份、大麦GoldenPromise野生型1份为材料。以上述材料发芽14天以后的幼苗为实验材料,每个基因型移入相同数量的幼苗至15L培养盒,分别设置含NaCl浓度为0和200mM的营养液培养盒各2盆,作为正常条件和盐处理条件,每隔3天换一次NaCl浓度相同的营养液。培养室条件为:光照强度为250μm m-2s-1,日夜温度为22/18℃,日夜时长14/10h。经3周盐处理以后,用去离子水清洗三遍,将实验材料表面的盐分去除,控干多余水分后,再对实验材料通过整体拍照、测地上部和地下部鲜重和离子含量等操作。
水培实验结果表明,被试材料的耐盐性强弱为:HvCBP60b CRISPR敲除材料耐盐性显著强于野生型。经PCR(验证引物为2中所述CBP60-SG1/2-F/R,)验证,试验所用大麦基因敲除材料均为阳性植株(见附图3)。
在盐处理(200mM NaCl)三周以后,CRISPR敲除植株和野生型WT出现明显的表型差异(见附图3a)。敲除植株的耐盐性优于野生型植株,生长表现为:敲除植株地上部仅有1~2片老叶黄化,新叶生长放缓,侧生3~4个分蘖;而野生型植株地上部3~4片老叶发黄,新叶萎蔫,侧生1~2个分蘖。地上部和地下部的干重、根长茎长以及Na+含量的测定也表明,HvCBP60b负调控大麦耐盐性,且这种负调控作用可能是由于HvCBP60b参与到了根系Na+分配中(见附图3)。
以上所述的实施例只是本发明的一种较佳的方案,并非对本发明作任何形式上的限制,在不超出权利要求所记载的技术方案的前提下还有其它的变体及改型。所有涉及HvCBP60b基因的转录本、基因组和启动子的核苷酸序列,编码蛋白的氨基酸序列,以及含任一上诉基因的生物材料,均属于本发明的保护范畴。
Claims (9)
1.钙调蛋白结合蛋白基因HvCBP60b,其特征在于,其核苷酸序列为SEQ ID NO.1所示。
2.钙调蛋白结合蛋白基因HvCBP60b的编码蛋白,其特征在于,编码蛋白的氨基酸序列如SEQ ID NO.3所示。
3.包含权利要求1所述的钙调蛋白结合蛋白基因HvCBP60b的CRISPR敲除重组载体,其特征在于,所述CRISPR敲除重组载体由编码基因HvCBP60b编码区域的19bp基因片段连入pTaU6:Cas9载体构建得到。
4.一种基因工程菌,其特征在于,所述基因工程菌中包含有编辑HvCBP60b钙调蛋白结合蛋白基因的CRISPR/Cas9载体;所述HvCBP60b钙调蛋白结合蛋白基因的核苷酸序列为SEQID NO.1所示。
5.如权利要求4所述的基因工程菌在调控大麦耐盐性中的应用。
6.如权利要求1所述的钙调蛋白结合蛋白基因HvCBP60b作为负调控因子在提高大麦耐盐性中的应用。
7.如权利要求5所述的应用,其特征在于,所述应用的途径为通过沉默HvCBP60b基因、敲除HvCBP60b基因或抑制HvCBP60b基因的表达获得突变体,从而提高大麦的耐盐性。
8.如权利要求5所述的应用,其特征在于,敲除的手段为CRISPR/Cas9基因编辑技术。
9.如权利要求5所述的应用,其特征在于,包括以下步骤:
(1)设计HvCBP60b基因的靶序列,构建CRISPR/Cas9载体;
(2)构建含步骤(1)所述CRISPR/Cas9载体的农杆菌基因工程菌;
(3)将步骤(2)所述基因工程菌转化大麦幼胚,获得不含外源Cas9蛋白且稳定遗传的纯合单突变体株系。
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