CN110343704B - Ap1基因突变体及调控植物花萼和花瓣开放时间的方法 - Google Patents
Ap1基因突变体及调控植物花萼和花瓣开放时间的方法 Download PDFInfo
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Abstract
本发明属于农业生物技术领域,具体涉及调控植物花萼和花瓣开放时间的方法。本发明的调控植物花萼和花瓣开放时间的方法,敲除植物AP1基因的TCCATCAATGAG碱基的步骤,其中,基因AP1的核苷酸序列如SEQ ID No.1所示。本发明的转基因植物表现为萼片及花瓣在植物授粉前提前张开、开花授粉后萼片脱落滞后,同时,其营养生长和生殖生长均表现正常,花器官数目和育性均不受任何影响。因此,本发明应用CRISPR‑Cas9系统编辑基因AP1对于杂交授粉困难的植物具有较大的应用价值。
Description
技术领域
本发明属于农业生物技术领域,具体涉及AP1基因突变体及调控植物花萼和花瓣开放时间的方法。
背景技术
规律成簇间隔短回文重复系统(clustered regularly interspaced shortpalindromic repeat;CRISPR-associated,CRISPR-Cas9)是一种由RNA指导的,利用Cas9核酸酶对靶向基因进行编辑的技术。CRISPR-Cas9系统广泛存在于原核生物基因中,CRISPR-Cas9系统由两部分组成,一部分是用来识别靶基因组的,长度为20bp左右的sgRNA序列,另外一部分是存在于CRISPR位点附近的双链DNA核酸酶-Cas9,能在sgRNA的引导下对靶位点进行切割,最终通过细胞内的非同源性末端连接机制(NHEJ)和同源重组修复机制(HDR)对形成断裂的DNA进行修复,从而形成基因的敲除和插入,最终实现基因的定向编辑,该技术具有非常精准、廉价、易于使用的特点。但是目前,实施CRISPR-Cas9的成功率并不高,其成功与否的关键问题在于gRNA的设计,gRNA和非目标区域过多的非特异性结果,脱靶效率较高,特别是靠近PAM的8-10bp不能和非目的区有高同源性。
基因AP1(APETALA1)属于植物花分生组织特征基因和花器官形态特征基因,既是花分生组织特征基因,又是花器官形态特征基因,在控制植物花分生组织特性与花器官的形成过程中起着重要的作用,其不但能调控花序分生组织向花分生组织的转变,而且是萼片和花瓣正常发育所必需的。
利用基因编辑技术虽然可以靶向改造目的基因,但是由于该技术仍然存在缺陷,导致目标性状被改造的同时出现非目标的性状,例如,对如大豆等杂交授粉困难的植物进行花发育相关基因编辑改造,往往造成转基因植物在营养生长或生殖生长方面的非预期改变。
发明内容
本发明的目的在于提供一种调控植物花萼和花瓣开放时间的方法。
本发明的再一目的在于提供基因AP1的突变体。
本发明的再一目的在于基因AP1突变体的蛋白。
本发明的再一目的在于提供基因AP1突变体的应用。
根据本发明具体实施方式的调控植物花萼和花瓣开放时间的方法,所述方法包括敲除植物基因AP1的TCCATCAATGAG碱基的步骤,其中,基因AP1的cDNA核苷酸序列如SEQ IDNo.1所示:
ATGGGAAGGGGTAGGGTTCAATTGAAGAGGATAGAGAACAAGATCAATAGACAAGTGACATTCTCGAAAAGAAGAGCTGGTCTTTTGAAGAAAGCTCATGAGATCTCTGTTCTCTGTGATGCTGAAGTTGCTCTTGTTGTCTTCTCCCATAAGGGAAAACTCTTCGAATACTCCACTGATTCTTGTATGGAGAAGATACTTGAACGCTATGAGAGGTACTCTTACGCCGAAAGACAGCTTATTGCACCTGAGTCCGACGTCAATACAAACTGGTCGATGGAGTATAACAGGCTTAAGGCTAAGATTGAGCTTTTGGAGAGAAACCAGAGGCATTATCTTGGGGAAGACTTGCAAGCAATGAGCCCTAAAGAGCTTCAGAATCTGGAGCAGCAGCTTGACACTGCTCTTAAGCACATCCGCACTAGAAAAAACCAACTTATGTACGAGTCCATCAATGAGCTCCAAAAAAAGGAGAAGGCCATACAGGAGCAAAACAGCATGCTTTCTAAACAGATCAAGGAGAGGGAAAAAATTCTTAGGGCTCAACAGGAGCAGTGGGATCAGCAGAACCAAGGCCACAATATGCCTCCCCCTCTGCCACCGCAGCAGCACCAAATCCAGCATCCTTACATGCTCTCTCATCAGCCATCTCCTTTTCTCAACATGGGTGGTCTGTATCAAGAAGATGATCCTATGGCAATGAGGAGGAATGATCTCGAACTGACTCTTGAACCCGTTTACAACTGCAACCTTGGCTGCTTCGCCGCATGA
根据本发明具体实施方式的调控植物花萼和花瓣开放时间的方法,所述方法利用CRISPR-Cas9系统敲除植物基因AP1上位点。
根据本发明具体实施方式的调控植物花萼和花瓣开放时间的方法,CRISPR-Cas9系统中的靶向基因AP1的sgRNA,其核苷酸序列如SEQ ID No.2所示:
Agaatagtaccaagttgtccgg
根据本发明具体实施方式的基因AP1突变体,其由植物基因AP1敲除TCCATCAATGAG碱基后得到。
基因AP1突变体的cDNA序列如SEQ ID No.3所示:
ATGGGAAGGGGTAGGGTTCAATTGAAGAGGATAGAGAACAAGATCAATAGACAAGTGACATTCTCGAAAAGAAGAGCTGGTCTTTTGAAGAAAGCTCATGAGATCTCTGTTCTCTGTGATGCTGAAGTTGCTCTTGTTGTCTTCTCCCATAAGGGAAAACTCTTCGAATACTCCACTGATTCTTGTATGGAGAAGATACTTGAACGCTATGAGAGGTACTCTTACGCCGAAAGACAGCTTATTGCACCTGAGTCCGACGTCAATACAAACTGGTCGATGGAGTATAACAGGCTTAAGGCTAAGATTGAGCTTTTGGAGAGAAACCAGAGGCATTATCTTGGGGAAGACTTGCAAGCAATGAGCCCTAAAGAGCTTCAGAATCTGGAGCAGCAGCTTGACACTGCTCTTAAGCACATCCGCACTAGAAAAAACCAACTTATGTACGAGCTCCAAAAAAAGGAGAAGGCCATACAGGAGCAAAACAGCATGCTTTCTAAACAGATCAAGGAGAGGGAAAAAATTCTTAGGGCTCAACAGGAGCAGTGGGATCAGCAGAACCAAGGCCACAATATGCCTCCCCCTCTGCCACCGCAGCAGCACCAAATCCAGCATCCTTACATGCTCTCTCATCAGCCATCTCCTTTTCTCAACATGGGTGGTCTGTATCAAGAAGATGATCCTATGGCAATGAGGAGGAATGATCTCGAACTGACTCTTGAACCCGTTTACAACTGCAACCTTGGCTGCTTCGCCGCATGA
APETALA1(AP1)基因编码区发生了12bp连续的碱基的缺失后,编码蛋白较野生型缺少了“SINE”4个氨基酸。
根据本发明具体实施方式的AP1突变体的蛋白,其氨基酸序列如SEQ ID No.4所示:
MGRGRVQLKRIENKINRQVTFSKRRAGLLKKAHEISVLCDAEVALVVFSHKGKLFEYSTDSCMEKILERYERYSYAERQLIAPESDVNTNWSMEYNRLKAKIELLERNQRHYLGEDLQAMSPKELQNLEQQLDTALKHIRTRKNQLMYELQKKEKAIQEQNSMLSKQIKEREKILRAQQEQWDQQNQGHNMPPPLPPQQHQIQHPYMLSHQPSPFLNMGGLYQEDDPMAMRRNDLELTLEPVYNCNLGCFAA*
根据本发明具体实施方式的基因AP1突变体的应用,尤其是在调控植物花萼和花瓣开放时间,以及植物杂交授粉方面的应用。
本发明的有益效果:
本发明设计的基因AP1的CRISPR-Cas9靶向RNA,靶向明确,不易脱靶,将其克隆到CRISPR-Cas9的双元载体上,定位分析发现,APETALA1(AP1)基因编码区发生了12bp碱基的缺失,导致编码蛋白缺少了4个氨基酸,对阳性转基因植物进行鉴定,发现转基因植物的表型为萼片及花瓣在植物授粉前提前张开、开花授粉后萼片脱落滞后,同时,其营养生长和生殖生长均表现正常,花器官数目和育性也不受任何影响。因此,可以利用基因编辑技术敲除基因AP1上的特定位置,从而对植物花萼和花瓣开放时间进行调控,尤其对于杂交授粉困难的植物具有较大的应用价值。
附图说明
图1显示Col-0野生型拟南芥与ap1-16的花序及荚果对比图,其中,左图显示Col-0野生型的花序及荚果,右图显示ap1-16的花序和荚果。
具体实施方式
实施例1
设计带有接头的靶向RNA引物(上游引物:gtcAagaatagtaccaagttgtc;下游引物:aaacgacaacttggtactattct),其序列如SEQ ID No.2所示。将该靶向序列克隆到pYL-U3-gRNA载体上。随后经过两轮PCR。
第一轮PCR引物:
U-F:5-CTCCGTTTTACCTGTGGAATCG-3;
gRNA-R:5-CGGAGGAAAATTCCATCCAC-3。
第二轮PCR引物:
Uctcg-B1’:TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG-3;
gRctga-B2:AGCGTGggtctcGtcagGGTCCATCCACTCCAAGCTC-3。
回收第二轮PCR产物,并连接到带有Cas9的双元载体pYLCRISPR/Cas9-MTmono中并进行测序鉴定。最后,将构建好的带有靶向RNA序列的pYLCRISPR/Cas9-Mtmono载体转化进农杆菌GV3101中,随后通过农杆菌介导的遗传转化将上述载体导入Col-0野生型拟南芥基因组中。
通过潮霉素抗性对T1代转基因植株进行筛选,并对阳性转基因植物的表型进行鉴定,如图1所示,转基因植物的萼片及花瓣在植物授粉前提前张开,开花授粉后萼片脱落滞后,同时,其营养生长和生殖生长均表现正常,花器官数目和育性也不受任何影响。
将阳性转基因植物与Col-0野生型植物进行回交实验,对回交后F3代群体进行抗性基因的PCR扩增鉴定,挑选PCR扩增为阴性即不带有转基因构建的植株,同时对该植株中的靶向基因进行PCR扩增和测序检测,发现APETALA1(AP1)基因编码区发生了12bp碱基的缺失,导致编码蛋白缺少了4个氨基酸,说明图1所示表型是由于AP1基因12bp碱基缺失造成的。将AP1基因的一个新突变类型,命名为ap1-16。
序列表
<110> 中国科学院植物研究所
<120> AP1基因突变体及调控植物花萼和花瓣开放时间的方法
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ccgcagcagc accaaatcca gcatccttac atgctctctc atcagccatc tccttttctc 660
aacatgggtg gtctgtatca agaagatgat cctatggcaa tgaggaggaa tgatctcgaa 720
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gctgaagttg ctcttgttgt cttctcccat aagggaaaac tcttcgaata ctccactgat 180
tcttgtatgg agaagatact tgaacgctat gagaggtact cttacgccga aagacagctt 240
attgcacctg agtccgacgt caatacaaac tggtcgatgg agtataacag gcttaaggct 300
aagattgagc ttttggagag aaaccagagg cattatcttg gggaagactt gcaagcaatg 360
agccctaaag agcttcagaa tctggagcag cagcttgaca ctgctcttaa gcacatccgc 420
actagaaaaa accaacttat gtacgagctc caaaaaaagg agaaggccat acaggagcaa 480
aacagcatgc tttctaaaca gatcaaggag agggaaaaaa ttcttagggc tcaacaggag 540
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caaatccagc atccttacat gctctctcat cagccatctc cttttctcaa catgggtggt 660
ctgtatcaag aagatgatcc tatggcaatg aggaggaatg atctcgaact gactcttgaa 720
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Pro Val Tyr Asn Cys Asn Leu Gly Cys Phe Ala Ala
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Claims (7)
1.调控拟南芥花萼和花瓣开放时间的方法,其特征在于,所述方法包括敲除拟南芥AP1基因的TCCATCAATGAG碱基的步骤,其中,AP1基因的核苷酸序列如SEQ ID No.1所示。
2.根据权利要求1所述的调控拟南芥花萼和花瓣开放时间的方法,其特征在于,所述方法利用CRISPR-Cas9系统敲除拟南芥基因AP1的位点。
3.根据权利要求2所述的调控拟南芥花萼和花瓣开放时间的方法,其特征在于,CRISPR-Cas9系统包括基因AP1的特异性gRNA,其核苷酸序列如SEQ ID No.2所示。
4.AP1基因突变体,其特征在于,其由AP1基因敲除TCCATCAATGAG碱基后得到,其中, 所述AP1基因的核苷酸序列如SEQ ID No.1所示。
5.AP1蛋白突变体,其特征在于,其由权利要求4所述的AP1基因突变体编码。
6.权利要求4所述的AP1基因突变体在调控植物花萼和花瓣开放时间方面的应用,其中,所述植物为拟南芥。
7.权利要求4所述的AP1基因突变体在植物杂交授粉中的应用,其中,所述植物为拟南芥。
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