CN108642077A - 基于CRISPR/Cas9基因编辑技术选育绿豆不育突变体的方法及专用gRNA - Google Patents

基于CRISPR/Cas9基因编辑技术选育绿豆不育突变体的方法及专用gRNA Download PDF

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CN108642077A
CN108642077A CN201810479981.0A CN201810479981A CN108642077A CN 108642077 A CN108642077 A CN 108642077A CN 201810479981 A CN201810479981 A CN 201810479981A CN 108642077 A CN108642077 A CN 108642077A
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陈景斌
林云
袁星星
薛晨晨
顾和平
李群三
陈新
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Abstract

本发明公开了一种基于CRISPR/Cas9基因编辑技术选育绿豆不育突变体的方法及专用gRNA。所述方法包括：将含有gRNA序列的植物CRISPR/Cas9质粒转入绿豆中，从而编辑SEQ ID NO.1所示的绿豆VrPUB4基因的ORF区域，其中所述gRNA对应的DNA序列如SEQ ID NO.2～SEQ NO.21任一条所示。本发明主要通过利用CRISPR/Cas9基因编辑技术对SEQ ID NO.1所示基因VrPUB4的序列进行编辑，使该基因中出现插入/缺少(InDel)，从而发生了移码变异而使基因功能丧失，产生不育的基因编辑植株。本发明提供一种选育雄性不育绿豆的新方法，为绿豆的杂交育种提供新的种质资源。

Description

基于CRISPR/Cas9基因编辑技术选育绿豆不育突变体的方法 及专用gRNA
技术领域
本发明涉及分子育种、基因工程有分子生物学领域，具体地说，涉及一种利用CRISPR/Cas9基因编辑技术选育绿豆不育突变体的方法。
背景技术
绿豆(Vigna radiate L.)是我国主要食用豆类作物之一，其含有丰富的维生素和矿物质、膳食纤维、蛋白，深得消费者喜爱。但是，绿豆品种均为产量较低的常规品种，产量一般只是1000至1500公斤/公倾。由于单产低，难于与其它大田作物竞争，面积不断萎缩。因此如何大幅度提高绿豆产量是广大绿豆育种家所必须面临和解决的迫切问题。
杂种优势的利用是提高作物产量的途径之一。杂种优势是生物界的一种普遍现象，一般是指杂种在生长势、生活力、抗逆性、繁殖力、适应性、产量、品质等方面优于其亲本的现象。目前已经有多种作物的杂交种被利用于生产。在天然自花传粉的作物之中，水稻的杂种优势的利用是最好的例子，杂交稻一般比常规稻增产10-20％。中国在1973年就建立起“三系配套”的水稻育种系统，近年来杂交水稻的种植面积一直都超过水稻种植总面积的50％(Cheng et al.,2007)。Chen 等(2003)发现利用绿豆品种KPS1与Korea7等不同组合配制的杂交组合后代在产量上最高可得到40％以上的超亲优势。
但是，目前绿豆还缺乏可用的雄性不育系。
发明内容
发明目的：为解决现有技术中的问题，本发明的目的之一是提供一种基于CRISPR/Cas9基因编辑技术选育绿豆不育突变体的方法；本发明的目的之二是提供一种特异性靶向绿豆VrPUB4基因的gRNA、其DNA分子、相关载体、重组工程菌及其应用。
技术方案：本发明所述的基于CRISPR/Cas9基因编辑技术选育绿豆不育突变体的方法，包括：将含有gRNA序列的植物CRISPR/Cas9质粒转入绿豆中，从而编辑SEQ ID NO.1所示的绿豆VrPUB4基因的ORF区域，其中所述gRNA 的序列如SEQ ID NO.2～SEQ ID NO.21任一条所示。
进一步优选的，gRNA序列如SEQ ID NO.2所示，该gRNA特异性好，基因编辑效果好，可成功获得雄性不育的绿豆突变体。
含有gRNA序列的植物CRISPR/Cas9质粒的骨架载体可以为但不仅限于 pYAO:hSpCas9，也可采用其他植物基因编辑骨架质粒。以pYAO:hSpCas9为例，说明含有gRNA序列的植物CRISPR/Cas9质粒的构建过程：
(1)合成正反寡聚核苷酸链1)5’-ATTG[20N或21N]-3’和2)5’-AAAC[20n 或21n]-3’，其中“20N或21N”为所述的gRNA序列，20n或21n为gRNA的反向互补序列；
(2)正反寡聚核苷酸链1)和2)退火成双链后连入经内切酶BsaI消化过的AtU6-26-sgRNA-SK载体中，构建得AtU6-26-target-sgRNA载体；
(3)AtU6-26-target-sgRNA载体经内切酶SpeI和NheI进行双酶切，切下 AtU6-26-target-sgRNA目的片段后连入经SpeI酶切的pYAO:hSpCas9载体即可。本发明还提供了一种特异性靶向绿豆VrPUB4基因的gRNA，其序列如SEQ ID NO.2～SEQ ID NO.21任一条所示。
本发明还提供了编码所述特异性靶向绿豆VrPUB4基因的gRNA的DNA分子。
本发明还提供了含有所述的DNA分子的重组载体或重组工程菌。
本发明又提供了所述的gRNA、所述的DNA分子、所述的重组载体或重组工程菌在靶向修饰绿豆VrPUB4基因以及选育绿豆不育突变体中的应用。
本发明进一步提供了一种用于编辑绿豆VrPUB4基因的试剂盒，含有所述的 gRNA。
本发明进一步提供了一种用于编辑绿豆VrPUB4基因的试剂盒，含有所述的 DNA分子。
本发明进一步提供了一种用于编辑绿豆VrPUB4基因的试剂盒，含有所述的重组载体或重组工程菌。
与现有技术相比，本发明的有益效果为：
本发明主要通过利用CRISPR/Cas9基因编辑技术对SEQ NO.1所示基因 VrPUB4的序列进行编辑，使该基因中出现插入/缺少(InDel)，从而发生了移码变异而使基因功能丧失，产生不育的基因编辑植株。本发明提供一种选育雄性不育绿豆的新方法，为绿豆的杂交育种提供新的种质资源。本发明方法简单、定向、成功率高。
附图说明
图1为实施例3获得的不育突变株相关性状图片；其中，A.不育株与可育株比较，左为可育株，右为不育株；B.不育株的花药与柱头；C.可育株花粉I/KI 溶液染色情况；D.不育株花粉I/KI溶液染色情况。
具体实施方式
下面结合具体实施例，进一步阐明本发明，应理解这些实施例仅用于说明本发明而不用于限制本发明的范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定的范围。
若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明建议的条件。
实施例1用于编辑VrPUB4的gRNA的设计
VrPUB4(LOC106752492)编码一个含有U-Box结构的蛋白。
根据CRISPR/Cas9基因编辑的原理，SEQ ID NO.1所示基因VrPUB4的序列的protospacer adjacent motif(PAM，即“NGG”，其中“N”为任何一种核苷酸) 前的20nt即为gRNA序列。如果gRNA的5’端第一个核苷酸不是鸟嘌呤(G)，则在5’端加上一个G，此时gRNA长21nt。在基因VrPUB4中设计得到的所有 gRNA如表1所示。gRNA序列应该在外显子上，不能离ATG起始子太近，应在整个基因前中段部分。将gRNA在绿豆基因绿数据库 (http://plantgenomics.snu.ac.kr/mediawiki-1.21.3/index.php/Main_Page)进行Blast 比对，确定靶序列在绿豆基因组上是唯一的。
表1gRNA序列
实施例2用于编辑VrPUB4的CRISPR载体的构建
编辑VrPUB4的CRISPR载体的框架为pYAO:hSpCas9(Yan L,Wei S,Wu Y, etal.High-efficiency genome editing in Arabidopsis using YAO promoter-drivenCRISPR/Cas9system[J].Molecular plant,2015,8(12):1820-1823.)，用于编辑 VrPUB4的CRISPR载体的构建方法如下：
合成正反寡聚核苷酸链1)5’-ATTG[20N或21N]-3’和2)5’-AAAC[20n或 21n]-3’。其中“20N或21N”为表1所示的gRNA序列，20n或21n为表1所示的gRNA的反向互补序列，例如GGACCGGCACCTATGATGATAGG的反向互补序列为CCTATCATCATAGGTGCCGGTCC。本实施例及实施例3具体采用的 gRNA为表1中的第一条序列TTCGCCTTGTCTCTAACAATCGG。
将正反两个寡聚核苷酸链1)和2)进行退火，方案为：a.将寡聚核苷酸链溶于超纯水，至100μM；b.在PCR管中加入8μl 1×TE缓冲液，1μl寡聚核苷酸链1，1μl寡聚核苷酸链2，混匀；c.放入PCR仪进行反应，95℃孵育5分钟，以每分1.5℃逐渐从95℃降温到22℃。
将上述退火产物重组连接到经内切酶BsaI消化过的AtU6-26-sgRNA-SK载体上(Yan L,Wei S,Wu Y,et al.High-efficiency genome editing in Arabidopsis usingYAO promoter-driven CRISPR/Cas9system[J].Molecular plant,2015,8(12): 1820-1823.)，方案为：a.在PCR管中加入5.5μl超纯水，1.0μl AtU6-26-sgRNA-SK 载体水溶液，0.5μl退火产物，2.0μl 5×T4连接酶缓冲夜(Takara)，1.0μl 5×T4 连接酶(Takara)，混匀；b.16℃过夜或室温孵育30分钟，得到 AtU6-26-target-sgRNA载体。
将AtU6-26-target-sgRNA载体转化大肠杆菌，方案是：a.从-80℃冰箱中取出大肠杆菌DH5α感受态细胞，立即置于冰上。b.往50μl解冻后感受态细胞轻轻加入10μl的上述连接体系冰浴30分钟。c.42℃水浴热激90秒，然后立即置于冰上孵育2分钟。d.加入100μl的LB培养基，于37℃恒温摇床培养40分钟。 e.取所有菌液均匀涂布于含有50μg/ml的氨苄青霉素的LB琼脂板上，37℃培养过夜。
从LB琼脂板挑出单克隆菌落，接入5ml含有50μg/ml氨苄青霉素的液体 LB培养基中，在37℃恒温摇床培养过夜。用质粒提取试剂盒提取质粒，用内切酶SpeI和NheI进行双酶切，切下AtU6-26-target-sgRNA目的片段。利用琼脂糖电泳回收目的片段(使用Tiangen切胶回收试剂盒)。
后将上述AtU6-26-target-sgRNA目的片段连接入经SpeI酶切的 pYAO:hSpCas9载体(Yan et al.,2015)，方案为：a.在PCR管中加入5.5μl超纯水，1.0μl pYAO:hSpCas9载体水溶液，0.5μl AtU6-26-target-sgRNA目的片段， 2.0μl 5×T4连接酶缓冲夜(Takara)，1.0μl 5×T4连接酶(Takara)，混匀；b.16℃过夜或室温孵育30分钟，得到pYAO:hSpCas9-target-sgRNA载体。
将pYAO:hSpCas9-target-sgRNA载体连接产物转化大肠杆菌，方案是：a.从 -80℃冰箱中取出大肠杆菌DH5α感受态细胞，立即置于冰上。b.往50μl解冻后感受态细胞轻轻加入10μl的上述连接体系冰浴30分钟。c.42℃水浴热激90秒，然后立即置于冰上孵育2分钟。d.加入100μl的LB培养基，于37℃恒温摇床培养40分钟。e.取所有菌液均匀涂布于含有50μg/ml的卡那霉素的LB琼脂板上，37℃培养过夜。
从LB琼脂板挑出单克隆菌落，经测序检验方向正确，然后接入5ml含有 50μg/ml氨苄青霉素的液体LB培养基中，在37℃恒温摇床培养过夜。用质粒提取试剂盒提取质粒，备用。
实施例3将CRISPR质粒转化绿豆植株
参考Zhao等(Zhao X,Meng Z,Wang Y,et al.Pollen magnetofection forgenetic modification with magnetic nanoparticles as gene carriers[J].Natureplants, 2017:1.)的方法，利用磁性纳米载体介导将pYAO:hSpCas9-target-sgRNA质粒转化绿豆植株。用超纯水将MNP(PolyMag1000，购自Chemicell公司)的质粒稀释至1μg/μl，按1:4混合并在室温中孵育30分钟使MNP-质粒复合体形成。将 MNP-质粒复合加入到1ml花粉培养基(每100ml含15g蔗糖，0.03g Ca(NO3)2·4H2O，0.01g H3BO3)之中。
清晨从绿豆花器官中收集得到100mg的花粉于培养皿中，加入MNP-质粒复合体悬浮液使花粉充分浸润。盖上培养皿，置于MagnetoFACTOR-24磁板(购自Chemicell公司)上0.5小时进行花粉磁转化。然后，用移液器小心去除上层的水，将磁转化的花粉置于滤纸上，30℃干燥15至30分钟。收集干燥后的磁转化花粉。
将绿豆花器官去雄，授予磁转化的花粉。10天后，收获成熟的种子。将种子种于含有50μg/ml潮霉素的Murashige&Skoog(MS)培养基上，长成带有1 对真叶的幼苗后移载至花盆。长出3对真叶后收取叶子提取DNA进行PCR鉴定。令阳性转化植株继续生长至开花，观察得到如图1所示的雄性不育突变体，I/KI 溶液对花粉染色确定为不育。
序列表
<110> 江苏省农业科学院
<120> 基于CRISPR/Cas9基因编辑技术选育绿豆不育突变体的方法及专用gRNA
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2511
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 1
atggagatat cattgttaaa aatgatatta aatggaatat cctcgttttt gcatttatca 60
atttctggaa acaagagctc tgaacctgtc tcaaagtatt accacaaggc agaggagata 120
cttaagctgt tgaagccaat cattgatgag attgttaatt ctgagttagc ttctgatgaa 180
gtgcttaata agatattgga agaaatcggt cttgctgtta atgaattaaa ggagcatgtc 240
cagaactggc acctattgtc tagcaaagta tactttgttt tgcaagttga accccttata 300
tcaagaattc gcacttcagg gctcaatatt ttccagcagc tgaaggtttc tcagcattct 360
ctccctgatg aattgagttc tgaagattta cagcaatgtt cacataaact taagcttttg 420
gggcatgaag aaacctcatc agttattaag gaagctattg cagaacaact ggaatatgca 480
ggacccagtc cagaggtcct gacaaaaatt gctgataggc tggacctcgt gtctaatcag 540
gatgttctta ttgaggctgt ggcccttgaa aggttgaagg aaaatgctga acaatctgaa 600
aagactgatg aggtggaata catcgatcaa atgattgctg tcataacacg tatgcttgag 660
cgtctcgtta tgcttaagca agctcagagt agcagcccag ttcctatacc ggctgatttt 720
tgttgtccac tttctttgga gctgatgact gatcctgtga ttgtggcatc agggcaaaca 780
tatgagcgag ctttcatcaa gaactggatt gatcttgggc taactgtttg tccaaagaca 840
cgtcaaactc tggctcatac caacctaata cctaattata ctgtaaaagc tctaattgca 900
aattggtgtg aatctaacaa tgtgcaacta gttgatccca caaaatcccc aaatttaaat 960
ccaccatccg tccttcatgg gtatatggaa tccggtacga ccagggagtc acctgttttt 1020
gctcacccca ggagcaacca gccgtcctca cctgagtcag cacgttctcg ttcttttagt 1080
tcaccaggta ataacataac ttctgttggc attcagctag aggaaacaac atcacctttg 1140
catccccgtt caacttcaga aggttcctta agtggtataa ttaatggaca atacatggat 1200
cttgcaagaa tatctcccgc tggtttggat gacaggtctg ctagctcaga tgaaagtact 1260
gtggattcag ctagccaacc atcaatgtcg ccatctagaa gggaatcatc cagtgccttt 1320
agctctgaac aatctcaaac ccatattaga gctgtttctg actccagtgc actttctact 1380
gcaaactttc ctcaagaaac ccaagatgat gataacaatg ctcggctatc aataagtcca 1440
ggccacagta gagatgcttc tggtgaatta aatgcagggc cagaaactgc tggtactact 1500
gtcatgccat caactcatag agaagctgag tccccagccc gattgttaga gacaaggcga 1560
aatcaaggca tatggaggcg gccaccagaa aggcttgttc ctaggataac atcgcctgct 1620
attgaaacaa gagctgatct ttcaggtatt gaagcccagg tccggaattt ggttgagggc 1680
ctgaggagct ctgatcttga tactcagaaa gaggcaacag cagaactccg ccttcttgca 1740
aagcacaata tggataatag aattgcgatt gcaaactgtg gagccattaa cttgttagtt 1800
gatttactta gatcagctga tacagcaatc caagaaaatg ctgttaccgc acttctaaac 1860
ttatcaatca atgataacaa caaaactgca attgcaaatg ctggtgcaat tgaacctctg 1920
attcacgtgc ttgagactgg gagcccagaa gccaaggaga attctgccgc cactcttttc 1980
agcttatccg tgattgagga aaacaagatt ttcataggga ggtctggggc aattagacca 2040
ctggtagatt tattggggaa tggaacccct aggggaaaga aagatgctgc cactgctttg 2100
tttaatttgt caatatttca tgaaaacaag aataggattg tgcaagctgg tgctgtgagg 2160
caccttgtgg agttaatgga cccagcagct ggaatggttg acaaggcagt tgctgtctta 2220
gcaaatcttg ccacaattca agaaggaaga aacgcaattg gtgaagaagg tgggattcct 2280
gtgctggttg aggttgttga gttgggttct gcgagaggaa aggagaatgc agccgcagct 2340
cttctacatc tttgtttaca tagtaacaaa tttttaggca aggtgcttca acaaggagct 2400
gtccctcctt tagtagcttt atctcagtca ggcaccccaa gagccaaaga aaaggcccag 2460
gctctcctca atcaatttag aagtcaaaga catgggaatg ctgggagggg c 2511
<210> 2
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 2
ttcgccttgt ctctaacaat cgg 23
<210> 3
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 3
agtgcgaatt cttgatataa ggg 23
<210> 4
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 4
aaagtattac cacaaggcag agg 23
<210> 5
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 5
tatggaatcc ggtacgacca ggg 23
<210> 6
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 6
tgccctgatg ccacaatcac agg 23
<210> 7
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 7
tgtctcaaag tattaccaca agg 23
<210> 8
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 8
aatcttgcca caattcaaga agg 23
<210> 9
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 9
aatcttgttt tcctcaatca cgg 23
<210> 10
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 10
cttctggtga attaaatgca ggg 23
<210> 11
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 11
ttccatatac ccatgaagga cgg 23
<210> 12
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 12
tcttcagaac tcaattcatc agg 23
<210> 13
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 13
agagctttta cagtataatt agg 23
<210> 14
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 14
cacggataag ctgaaaagag tgg 23
<210> 15
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 15
aataagatat tggaagaaat cgg 23
<210> 16
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 16
agacatggga atgctgggag ggg 23
<210> 17
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 17
aagacatggg aatgctggga ggg 23
<210> 18
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 18
ttaggcaagg tgcttcaaca agg 23
<210> 19
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 19
catccccgtt caacttcaga agg 23
<210> 20
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 20
gctcggctat caataagtcc agg 23
<210> 21
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 21
gcagcatctt tctttcccct agg 23

Claims (9)

1.一种基于CRISPR/Cas9基因编辑技术选育绿豆不育突变体的方法，其特征在于，包括：将含有gRNA序列的植物CRISPR/Cas9质粒转入绿豆中，从而编辑SEQ ID NO.1所示的绿豆VrPUB4基因的ORF区域，其中所述gRNA的序列如SEQ ID NO.2～SEQ NO.21任一条所示。
2.一种特异性靶向绿豆VrPUB4基因的gRNA，其特征在于，其序列如SEQ ID NO.2～SEQID NO.21任一条所示。
3.编码权利要求2所述的特异性靶向绿豆VrPUB4基因的gRNA的DNA分子。
4.含有权利要求3所述的DNA分子的重组载体或重组工程菌。
5.权利要求2所述的gRNA、权利要求3所述的DNA分子、权利要求4所述的重组载体或重组工程菌在靶向修饰绿豆VrPUB4基因中的应用。
6.权利要求2所述的gRNA、权利要求3所述的DNA分子、权利要求4所述的重组载体或重组工程菌在选育绿豆不育突变体中的应用。
7.一种用于编辑绿豆VrPUB4基因的试剂盒，其特征在于，含有权利要求2所述的gRNA。
8.一种用于编辑绿豆VrPUB4基因的试剂盒，其特征在于，含有权利要求3所述的DNA分子。
9.一种用于编辑绿豆VrPUB4基因的试剂盒，其特征在于，含有权利要求4所述的重组载体或重组工程菌。
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