CN108642078A - 基于CRISPR/Cas9基因编辑技术选育绿豆开花传粉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开花传粉突变体的方法及专用gRNA Download PDF

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CN108642078A
CN108642078A CN201810479983.XA CN201810479983A CN108642078A CN 108642078 A CN108642078 A CN 108642078A CN 201810479983 A CN201810479983 A CN 201810479983A CN 108642078 A CN108642078 A CN 108642078A
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陈景斌
拔杰·宋他
崔晓艳
林云
袁星星
顾和平
李群三
陈新
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Jiangsu Academy of Agricultural Sciences
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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开花传粉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Cha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。本发明利用CRISPR/Cas9基因编辑技术对绿豆的一个编码YUCCA蛋白的基因进行编辑，可以选育得到一个缺失龙骨瓣且柱头、花药外露的绿豆新种质，为绿豆的杂交育种提供基础。

Description

基于CRISPR/Cas9基因编辑技术选育绿豆开花传粉突变体的 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，本发明涉及一种基于CRISPR/Cas9基因编辑技术选育绿豆开花传粉突变体的方法及专用 gRNA。
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。
杂交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。
绿豆(Vigna radiata)属于豆科植物，其花器官为蝶形花，花药和柱头被包裹在龙骨瓣内，阻碍植株之间进行花粉交流。因此，绿豆是一种自花传粉作物，其异交率极低。这不利于绿豆的杂种优势的利用。有研究发现一个缺失龙骨瓣的绿豆突变体能进行开花传粉，并且通过基因定位得到控制开花传粉的突变基因 cha，并发现该基因编码一个YUCCA蛋白(Chen J,Somta P,Chen X,et al.Gene Mapping of a Mutant Mungbean(Vigna radiataL.)Using New Molecular Markers Suggests a Gene Encoding a YUC4-like ProteinRegulates the Chasmogamous Flower Trait[J].Frontiers in plant science,2016,7.)。这为绿豆的杂种优势利用打下了基础。但是通过诱变的方法获得突变体具有随机性和偶然性。
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开花传粉突变体的方法；本发明的目的之二是提供一种特异性靶向绿豆Cha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开花传粉突变体的方法，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 IDNO.1所示的绿豆Cha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.6所示，该gRNA特异性好，基因编辑效果好，可成功获得绿豆开花传粉突变体。
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Cha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Cha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Cha基因以及选育绿豆开花传粉突变体中的应用。
本发明进一步提供了一种用于编辑绿豆Cha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Cha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Cha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：
本发明利用CRISPR/Cas9基因编辑技术对绿豆的一个编码YUCCA蛋白的基因(cha基因)进行编辑，可以选育得到一个缺失龙骨瓣且柱头、花药外露的绿豆新种质，为绿豆的杂交育种提供基础。本发明方法简单、定向、成功率高。
附图说明
图1为获得的基因编辑绿豆与野生型苏绿1号的花器官图片；
图2为获得的基因编辑绿豆与野生型苏绿1号的花器官各部分结构图片。
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cha基因的gRNA的设计
根据CRISPR/Cas9基因编辑的原理，SEQ ID NO.1所示基因cha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cha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1 gRNA序列
实施例2用于编辑cha的CRISPR载体的构建
编辑cha的CRISPR载体的框架为pYAO:hSpCas9(Yan L,Wei S,Wu Y,et al. High-efficiency genome editing in Arabidopsis using YAO promoter-driven CRISPR/Cas9system[J].Molecular plant,2015,8(12):1820-1823.)，用于编辑cha 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中的第五条序列GATGTTGAAGTGTGAGGCGTAGG。
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，以每第分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(2017)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药，观察得到如图1、图2所示的花器官，龙骨瓣和翼瓣缺失且柱头、花药外露。
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开花传粉突变体的方法及专用gRNA
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1206
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 1
atgggttctt gcaaacccca acaagaacat gttcatggac ctatcatcat aggtgccggt 60
ccttcaggcc tagccgtggc tgcgtgtctc tcggagcaca aagtcccttt cgtgattctt 120
gagagaagca actgcatagc ctctctttgg caacacaaaa cctacgaccg tctcaaactc 180
cacctcccaa agcagttctg cgagcttccc ttgaaaggtt ttccccacaa cttccccaag 240
taccccacaa agtaccagtt catatcctac atggagtcct acgcctcaca cttcaacatc 300
caccccaggt tcaaccaaac agtcgaaact gctcactttg ataaagcctc tcagctttgg 360
ctcgttagga ctcagcactg tcagcttctc tctccttggc tcgtcgtggc caccggggag 420
aatgctgagc ctgtgcttcc tagaattcat ggcatggacc atttctctgg ctccattgct 480
cacaccagtg tctacaagtc tggctctgag tacacaaacc agaaggttct cgtcattggc 540
tgtggcaatt caggaatgga agttagctta gacctttgca gacacaatgc ctccccttac 600
atggttgcaa ggaacacagt gcatgtcctt cctagggaga tgtttggctt ctcaactttt 660
ggcatagcca tggctcttta caagtggttt cccatcaaag ttgtagacaa aattctctta 720
cttgtgacca acttcatgtt gggaaacaca aatcactatg gcatcaaaag gcctaaaaca 780
ggcccaatag agctgaaact agccacaggg aaaaccccag tccttgatgt gggtcaagtt 840
gcacagatca aatgtggcaa cataaaggtg atggaaggtg tgaaggagat aactagaaaa 900
ggtgcgaaat ttatggatgg acaagaaaag gaatttgatg ctataatatt ggcaacaggg 960
tacaagagca acgtgcctgc ttggcttaag ggttgtgatt ttttcactga ggatggaatg 1020
ccgaaaacac cctttcccca tgggtggaaa ggggagcagg gattgtatac ggtggggttc 1080
accagaagag gcattcaagg aacatcttgt gatgcaatca agatcgctga agacatagcc 1140
tcgcagtgga gaaccgtaga gaacaagaat caatgcaatt cacatatcat ccttctcact 1200
tcataa 1206
<210> 2
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 2
ggaccggcac ctatgatgat agg 23
<210> 3
<211> 24
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 3
gtagagctga aactagccac aggg 24
<210> 4
<211> 24
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 4
gcggtccttc aggcctagcc gtgg 24
<210> 5
<211> 24
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 5
gcacggctag gcctgaagga ccgg 24
<210> 6
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 6
gatgttgaag tgtgaggcgt agg 23
<210> 7
<211> 24
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 7
gctctgagta cacaaaccag aagg 24
<210> 8
<211> 24
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 8
gtgaggcgta ggactccatg tagg 24
<210> 9
<211> 24
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 9
gtggtacttt gtggggtact tggg 24
<210> 10
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 10
ggacctatca tcataggtgc cgg 23
<210> 11
<211> 24
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 11
gctgaagaca tagcctcgca gtgg 24
<210> 12
<211> 24
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 12
gccgaaaaca ccctttcccc atgg 24
<210> 13
<211> 24
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 13
gccatgaatt ctaggaagca cagg 24
<210> 14
<211> 24
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 14
gcgactgttt ggttgaacct gggg 24
<210> 15
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 15
gggttgtgat tttttcactg agg 23
<210> 16
<211> 24
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 16
gtggggtact tggggaagtt gtgg 24
<210> 17
<211> 23
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 17
ggcaacataa aggtgatgga agg 23
<210> 18
<211> 24
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 18
gagctctatt gggcctgttt tagg 24
<210> 19
<211> 24
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 19
gactccatgt aggatatgaa ctgg 24
<210> 20
<211> 24
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 20
gatagagctg aaactagcca cagg 24
<210> 21
<211> 24
<212> DNA
<213> 绿豆(Vigna radiata Linn. Wilczek.)
<400> 21
gtggtccatg ccatgaattc tagg 24

Claims (9)

1.一种基于CRISPR/Cas9基因编辑技术选育绿豆开花传粉突变体的方法，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Cha基因，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Cha基因的gRNA，其特征在于，其序列如SEQ ID NO.2～SEQ IDNO.21任一条所示。
3.编码权利要求2所述的特异性靶向绿豆Cha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Cha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开花传粉突变体中的应用。
7.一种用于编辑绿豆Cha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Cha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Cha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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