CN108841855B - 一种编辑水稻Ehd1基因培育长生育期粳稻品种的方法 - Google Patents
一种编辑水稻Ehd1基因培育长生育期粳稻品种的方法 Download PDFInfo
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- CN108841855B CN108841855B CN201810599355.5A CN201810599355A CN108841855B CN 108841855 B CN108841855 B CN 108841855B CN 201810599355 A CN201810599355 A CN 201810599355A CN 108841855 B CN108841855 B CN 108841855B
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Abstract
本发明提供一种培育“北粳南移”水稻品种的方法,包含以下步骤:在水稻Ehd1基因接收结构域起始区设计CRISPR/Cas9基因编辑位点并构建基因编辑载体,转化水稻,实现对Ehd1基因的定点突变,获得Ehd1基因移码突变及接收结构域起始区缺失了3个氨基酸的框内缺失突变株系。实验证明,与野生型相比,所述框内缺失突变株系生育期中度延长两周左右,移码突变体的生育期则延迟近一个月。通过本发明提供的方法,可快速获得不同生育期延长的粳稻材料,扩大现有优良品种的生态适应性。可为选育适应低纬度地区不同生态条件的粳稻品种提供一种高效的基因敲除方法和育种方法。
Description
技术领域
本发明涉及植物转基因技术及作物遗传育种领域,涉及一种利用CRISPR-Cas9技术编辑水稻Ehd1基因接收结构域延长水稻生育期,培育 “北粳南移”水稻品种的育种方法。
背景技术
水稻是主要的粮食作物,是世界上近一半人口的主食,水稻分为籼稻与粳稻两个亚种,其中粳稻品质优良,市场价格要普遍高于籼稻,粳米在许多国家都颇受欢迎。为了满足市场消费需求,中国政府提出了以提高稻米品质为重点的“籼稻改粳稻”项目。
影响北方粳稻品种引入低纬度地区种植主要限制因素是短日高温条件下其抽穗开花时间严重缩短,导致产量降低。目前,粳稻主要种植于中纬度地区,该地区具有亚热带或温带气候,水稻生长期日照较长。大多数适应中纬度地区种植的粳稻品种对光周期或温度敏感,如果将它们直接引入低纬度地区短光照和高温环境条件下种植,其抽穗期会严重缩短,从而导致产量降低。为了解决这个问题,育种者需要延迟粳稻品种抽穗期和提高产量,以适应低纬度地区环境。
在水稻中,存在独特的Ehd1依赖性开花途径,作为开花转换的信号整合因子,Ehd1促进Hd3a和RFT1表达从而诱导开花转换。Ehd1是由341个氨基酸组成的高度保守的B型双组分响应调节因子(RR),该蛋白在其N端具有保守的接收结构域(在第11和第123个残基之间),蛋白中段具有GARP DNA结合域,位于第197个到第255个残基。研究表明,在Ehd1基因的GARP基序中从G到A的单核苷酸替换的自然突变体产生了ehd1 / ef1无功能等位基因,携带ehd1基因的水稻品种可以表现迟开花的特点,ehd1等位基因使台湾粳稻品种适应低纬度短日气候条件,它们都表现出很长的基本营养生长期,并具有很好的产量性状。
ehd1等位基因资源可促进低纬度地区优质粳稻品种的开发应用,但是ehd1等位基因仅分布于台湾品种中。目前,常规杂交育种是主要的水稻育种方法,利用传统技术可通过杂交和回交手段将ehd1突变基因转入优良水稻品种以选育适宜低纬度地区生长生育期的粳稻品种,然而,这些育种方法的主要缺点是劳动强度大,成本高且育种周期长。近来,CRISPR / Cas9基因组编辑技术已被用于创造定向的水稻基因突变并开展快速有效的水稻遗传改良。通过CRISPR/Cas9系统直接对现有优良粳稻Ehd1基因进行定点编辑,可快速获得长生育期的粳稻品种,缩短育种周期,扩大现有优良粳型水稻品种的生态适应性。
发明内容
本发明的目的在于提供一种培育 “北粳南移”水稻品种的方法,利用CRISPR/Cas9基因编辑技术定点突变水稻Ehd1基因接收结构域,延长生育期,扩大现有优良粳稻品种的生态适应性,培育适合低纬度地区种植的优质粳稻品种的育种方法。
本发明的另一目的在于提供一种ehd1短等位基因及其应用。
本发明是通过以下技术方案实现的:
所述一种利用CRISPR-Cas9技术编辑水稻Ehd1基因接收结构域,延长水稻生育期并培育“北粳南移”水稻品种的育种方法,特别适合于创制适合低纬度地区的长生育期粳稻品种及选育应用;
包含如下步骤:
1)优选的Ehd1基因第一外显子中的23bp序列 5,-GCCTTATGGACTAAGAGTTCTGG-3,作为sgRNA靶序列。所述sgRNA特异性靶向Ehd1基因接收结构域5,端起始位置。其sgRNA的序列如下:5,-GCCTTATGGACTAAGAGTTC-3,。
2) 参照CRISPR/Cas9编辑技术,构建CRISPR-ED1-RD基因编辑载体。
3)采用农杆菌介导的遗传转化方法,将CRISPR-ED1-RD编辑载体转入北方常规粳稻品种中,获得再生植株。
4)参照所述sgRNA靶向的Ehd1基因序列,依据PCR引物设计原理,在所述sgRNA靶位点处上下游100-200 bp处设计Ehd1基因特异性引物,引物碱基序列如下:
Ehd1F1:5,- CGGAGAAGCAGATGTAGTCG-3, 如SEQ ID NO.1所示
Ehd1R1: 5,- TGTGTAGTGGTGCGTGTTTG-3, 如SEQ ID NO. 2所示
5)利用所述引物PCR扩增Ehd1基因的基因组片段并进行测序鉴定,筛选Ehd1基因移码敲除或碱基缺失数为3的整倍数的框内缺失突变株。
6)对各突变株进行生育期调查鉴定,获得生育期中度或高度延长的突变株,繁殖突变株系。
7)在低纬度地区种植上述生育期延长的株系,考查生育期及各农艺性状,选育适合低纬度地区种植的长生育期粳稻品种。
一种ehd1短等位基因及其应用,通过所述CRISPR-ED1-RD基因编辑载体在Ehd1 接收结构域起始区缺失了第10-12位的Gly Leu Arg三个氨基酸。所形成的新型Ehd1等位基因,具有中等延迟水稻开花两周时间的功能。
所述ehd1短等位基因其编码区的核苷酸序列如SEQ ID NO.3所示,其氨基酸序列如SEQ ID NO.4所示。应用具体是,可利用所述ehd1短等位基因所具有的中等延迟水稻开花两周时间的功能,培育不同于移码突变体生育期特征的水稻品种,可培育具有更加广泛的生态适应性的优质稻品种。
本发明具有如下的有益效果:
1、本发明利用Ehd1基因及其蛋白参与水稻开花期发育调控的特点及CRISPR/Cas9系统的基因组靶向修饰,通过定点突变该基因,可极显著延长水稻的生育期,在农业生产上具有十分重要的应用。
2、本发明利用CRISPR/Cas9基因编辑技术定点突变水稻Ehd1基因接收结构域起始区所产生的3个氨基酸缺失突变,获得了ehd1短等位基因,具有中等延迟水稻开花的功能,是一种全新的复等位基因,具有重要的育种利用价值。
3、本发明为扩大现有优良粳稻品种的生态适应性,培育适合低纬度地区种植的优质粳稻品种提供了一种新的“北粳南移”育种方法。
4、本发明为创造基于水稻Ehd1基因的具有长生育期的水稻品种、种质资源、杂交稻亲本提供了一种高效的育种方式。
附图说明
图1:水稻Ehd1基因结构图以及第一外显子sgRNA靶序列。
图2:转化再生水稻株系中突变株系与野生型Ehd1基因靶序列核苷酸示意图,其中WT表示为野生型基因,“-”表示发生了删除突变的序列,“+”表示发生了插入突变的序列,“-/+”后边的数字表示删除或插入的核苷酸数量。
图3:龙稻16中两个突变株系与野生型的序列测序结果,第一个序列为野生型,中间的序列为插入A的移码突变L-ehd1-#2,第三个序列为缺失9bp的框内缺失突变L-ehd1-#3。
图4:框内缺失突变株系L-ehd1-#3与野生型Ehd1基因蛋白结构示意图。
图5:龙稻16(左) L-ehd1-#3-1(中)和移码突变株系L-ehd1-#1-1生育期对比图。
图6:两类突变体及野生型龙稻16抽穗期差异分析。
图7:L-ehd1-#3 T1代分离群体中Ehd1基因框内缺失9bp的纯合突变株,杂合突变株及野生型的PCR基因型鉴定及各单株的抽穗期。
图8:龙稻16(左) L-ehd1-#3-1(中)和移码突变株系L-ehd1-#1-1主穗长度及单株产量对比。
具体实施方式
下面结合一个实施例对本发明作进一步的说明。下述实施例中所涉及的仪器、试剂、材料等,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料等,可通过正规商业途径获得。下述实施例中所涉及的实验方法,检测方法等,若无特别说明,均为现有技术中已有的常规实验方法,检测方法等。
本发明通过构建水稻Ehd1基因CRISPR/Cas9基因编辑载体CRISPR-ED1-RD,再通过农杆菌介导的转化方法将CRISPR-ED1-RD导入东北优质粳稻品种龙稻16中,从后代中筛选Ehd1基因移码敲除或碱基缺失数为3的整倍数的框内缺失突变株,对各基因突变株进行生育期调查鉴定,获得生育期延长的突变株,繁殖突变株系。在低纬度地区种植上述生育期延长的株系,考查生育期及各农艺性状,选育适合低纬度地区种植的优质粳稻品种。具体包括如下步骤:
一、 CRISPR-ED1-RD载体的构建
1、sgRNA靶点引物的设计和合成
运用http://www.e-crispr.org/筛选并优选Ehd1基因第一外显子中的23bp序列5,-GCCTTATGGACTAAGAGTTCTGG-3,作为sgRNA靶序列。所述sgRNA特异性靶向所述Ehd1基因receiver domain起始位置。合成人工序列,其中正向引物与靶序列一致,但不包括PAM序列NGG三个碱基,5’末端需加上接头CAG。反向引物则与不包括NGG的靶序列互补,其5’末端需加上接头AAC。
2、引物二聚体的形成
将正、反向引物稀释成10uM的浓度,按如下体系加入各样品。放入PCR仪95℃变性3min,以每分钟减3℃的降温速率缓慢降至室温,16℃再5 min即可完成引物二聚体的形成。
3、引物二聚体与VK005-1载体的连接
VK005-1载体由北京唯尚立德生物技术公司提供。按如下体系分别加入各样品后,混匀放入16℃水浴锅连接2 h。
4、将连接产物用热激法转化大肠杆菌感受态细胞DH5α,步骤如下:
(1)大肠杆菌感受态100μl,冰上融化5 min。
(2)加入6 μl连接产物,用移液枪轻轻吸打均匀,冰上放置30分钟。
(3)42℃水浴90秒(热激),快速转移到冰上放置2分钟。
(4)加800 μl无抗生素的液体 LB,37℃摇床温和摇动培养一小时,使细胞复苏。
(5)制备筛选培养基:制备含50 μg/mL Amp或kan的LB平板。
(6)将上述培养菌液摇匀后分别取100 μl 涂布于筛选培养基上,正面向上放置半小时,待菌液完全被培养基吸收后倒置培养皿,37℃培养16-24小时。挑取单克隆培养并测序,测序引物序列:5,-AGCCATGAATAGGTCTATGAC- 3’验证其表达盒sgRNA 序列5,-GCCTTATGGACTAAGAGTTC-3’正确性。该克隆的编辑载体命名为CRISPR-ED1-RD。
5、碱法小量提取质粒DNA
(1)取1.5mL CRISPR-ED1-RD培养液于1.5 mL eppendorf管中,4℃下9000g离心2分钟。
(2)弃上清,将管倒置于滤纸上数分钟,使液体流尽。
(3)菌体沉淀重悬浮于100 μl溶液Ⅰ中(需剧烈振荡),室温下放10分钟。
(4)加入新配制的溶液Ⅱ200 μl,盖紧管口,快速温和颠倒eppendorf管数次混匀内容物(千万不要振荡),冰浴2分钟。
(6)加入150μl预冷的溶液Ⅲ,盖紧管口,温和振荡10秒混匀,冰浴10分钟,4℃下12000g离心5分钟。
(7)上清液移入干净eppendorf管中,加入等体积的氯仿\异戊醇(24:1),振荡混匀,室温抽提5 min。4℃下12000 rpm离心5分钟。
(8)将水相移入干净eppendorf管中,加入2倍体积的预冷的无水乙醇,振荡混匀后室温放置5分钟,然后4℃下12000 g离心10分钟。
(9)弃上清液,将管口敞开倒置于滤纸上使所有液体流出,加入1 mL 70%乙醇洗沉淀一次,4℃下12000 g离心5分钟。
(10)弃上清液,将管倒置于滤纸上使液体流尽,室温干燥。将沉淀溶于20 μl TE缓冲液(pH8.0,含20μg /mL RNaseA)中,储于-20℃冰箱中。
溶液Ⅰ:葡萄糖 50 mmol/L,Tris.HCl (pH 8.0) 25 mmol/L,EDTA(pH8.0灭菌)10mmol/L。
溶液Ⅱ:(现配现用)10 mL配比:10N NaOH 200 μl,10%SDS 1 mL,H2O 8.8 mL 。
溶液Ⅲ:5mol/L乙酸钾 60 mL,冰乙酸 11.5 mL,水28.5 mL。
TE: NaCl 0.1 mol/L,Tris.HCl (pH 8.0) 10 mmol/L,EDTA (pH 8.0) 1 mmol/L。
6、电激法转化农杆菌感受态细胞
(1)每管40μl农杆菌LBA4404感受态细胞加入2-10 μl(50-100 ng)提取的重组质粒CRISPR-ED1-RD,冰上混匀。
(2)电激槽(0.2cm)于-20度预冷10 min,将混和物加入电激槽,轻轻敲至槽底。
(3)电激参数:电压2.5 KV ,电容 25 Uf ,电阻 200 Ω 。同时按下两个Pulse按钮,听到“吱”声后放开。
(4)立即加入YEB培养基1 mL 轻轻迅速混匀。转入灭菌的 eppendof管,28度保温培养2小时。
(5)设50 μl、250 μl、600 μl三个梯度涂板,28度培养2天。挑取单克隆培养并测序,测序引物序列:5,-AGCCATGAATAGGTCTATGAC- 3’验证其表达框内sgRNA序列5,-GCCTTATGGACTAAGAGTTC-3’正确。
二、获取Ehd1基因变异及生育期延长的水稻
1、水稻遗传转化
取龙稻16的幼胚,消毒后置于NB培养基上27℃暗培养诱导水稻愈伤组织,愈伤组织每2周继代一次,继代3次后的愈伤组织用于农杆菌的转化实验。包括如下步骤:
共培养(整个操作过程需8天)
第一天(预培养):选取自然分散,硬度适中,颜色鲜黄,直径约为2-3mm的颗粒愈伤,置于NB培养基上于27℃暗培养4天。
第三天(划菌):将含有重组VK005-1-ED1的农杆菌LBA4404接种于YEB培养基(含50μg/ml卡那霉素和20μg/ml利福平)中,28℃暗条件下培养48小时。
第五天(转化):将农杆菌悬浮于添加有100uM乙酰丁香酮(AS)20ML的AMM液体培养基中,剧烈震荡1min后,静置1h,让农杆菌形成悬浮液,调整浓度至OD600=1-1.5,取菌液于培养瓶中加入经预培养的愈伤,略微摇动静置30min,于无菌纸上晾干愈伤后置于添加有100uM的乙酰丁香酮的NB培养基上27℃暗培养3天。
第八天(洗菌):待愈伤下农杆菌生长至可见的菌斑,但未长满愈伤时,挑取愈伤于无菌培养瓶中,用无菌水冲洗,直至无可见菌斑为止,最后用含250mg/l的羧苄无菌水静置1h,置于无菌滤纸上晾干后,转移至潮霉素浓度30mg/L的筛选培养基上,筛选。
每两周将愈伤转移到新的选择培养基上,约需三周即可见瘤状抗性愈伤从褐化干瘪的愈伤中长出,当抗性愈伤在30mg/L潮霉素培养基上生长一个月后,将新长出的抗性愈伤转移至50mg/L潮霉素的培养基上,生长一周后,将继续增生分裂的抗性愈伤转入分化培养基上再生,成团放置,在光照培养箱生长一周后愈伤开始转绿,三周后开始长出幼芽,随后根也长出,当芽长至2-3cm时,就移至1/2MS培养基上,每个培养瓶中只放一个抗性愈伤诱导出来的苗,待幼苗在生根培养基上生长10天后,在清水中炼苗三天后移至大田。
2、转基因苗及突变体的鉴定
获得的再生植株移栽成活后,提取再生植株的叶片总DNA,分别以基于重组载体CRISPR-ED1-RD的引物CZTF ,CZTR和基于潮霉素的引物HptF ,HptR进行PCR扩增,筛选阳性转化植株。鉴定出的转基因阳性植株再以Ehd1F1和Ehd1R1引物对或引物Ehd1F4 和Ehd1R6引物对PCR扩增Ehd1基因的基因组片段并进行测序鉴定,鉴定结果如图3所示。鉴定所使用引物序列如下所示:
CZTF: 5’-GGGAGATCCAGCTAGAGGTC-3’ 如SEQ ID NO.5所示。
CZTR: 5’-GGAAGGAGGAAGACAAGG-3’ 如SEQ ID NO.6所示。
HptF: 5’-TACACAGCCATCGGTCCAGA-3’ 如SEQ ID NO.7所示。
HptR: 5’-TAGGAGGGCGTGGATATGTC-3’ 如SEQ ID NO.8所示。
Ehd1F1:5,- CGGAGAAGCAGATGTAGTCG-3,如SEQ ID NO.1所示。
Ehd1R1: 5,- TGTGTAGTGGTGCGTGTTTG-3,如SEQ ID NO.2所示。
Ehd1F4:5,- CGGAGAAGCAGATGTAGTCG -3, 如SEQ ID NO.9所示。
Ehd1R6:5,- GACAAGTATGAACAGTCGTC-3,如SEQ ID NO.10所示。
3、突变体生育期表型调查鉴定
分别收获各转基因植株的种子,在处于低纬度的海南三亚冬季短日照条件及福州夏季长日照条件下田间种植T1代,用Ehd1F1和Ehd1R1引物对或引物Ehd1F4 和Ehd1R6引物对扩增Ehd1基因片段,根据测序结果筛选突变杂合子与纯合子。各单株抽穗时进行生育期鉴定及调查统计,获得生育期延长的突变株,生育期调查结果如图5,图6所示。其中移码突变体生育期比野生型延长约1个月,而缺失9个碱基的突变体生育期延迟约两周时间。
4、一种ehd1短等位基因及生育期表型
本次试验通过所述CRISPR-ED1-RD基因编辑载体在Ehd1 接收结构域起始区缺失了第10-12位的Gly Leu Arg三个氨基酸,形成了一个ehd1短等位基因,其编码区的核苷酸序列如SEQ ID NO.3所示,其氨基酸序列如SEQ ID NO.4所示,其蛋白结构如图4所示。经过生育期表型调查,发现所述缺失3个氨基酸所形成的ehd1短等位基因具有中等延迟水稻开花大约两周时间的功能,结果如图5-7所示。因此可利用所述ehd1短等位基因,选育不同于移码突变体生育期特征的水稻品种,可培育具有更加广泛的生态适应性的优质粳稻品种。
由此可见,通过本发明提供的方法,龙稻16Ehd1基因突变后,无论在海南短日照条件及福州长日照条件下,其生育期可极显著延长,而通过选择Ehd1基因的移码突变或框内缺失突变等不同突变类型,可快速获得不同生育期延长的龙稻16水稻材料,本发明可为选育适应低纬度地区不同生态条件的粳稻品种提供一种高效的基因敲除方法和育种方法。
上述虽然结合实施例对本发明的具体实施方式进行了描述,但并非对本发明保护范围的限制,所属领域技术人员应该明白,在本发明的技术方案的基础上,本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。
SEQUENCE LISTING
<110> 福建省农业科学院生物技术研究所
<120> 一种培育 "北粳南移"水稻品种的方法
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agagaagctg tgcctttcat attggacaat ccacaaatag ttgacctagt aatcagtgat 180
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agatcaacgg ccaccgaagc ttcgctagcg cctctagaaa atgaggtgag agatgacatg 540
gtcaactaca atggcgagat cacggacata cgagacctcg gaaagtccag gctgacctgg 600
accacgcagt tgcaccgtca gttcattgca gcagtgaacc acctcggaga agacaaggca 660
gttccaaaga agatactagg gataatgaag gtcaaacatt tgacaagaga gcaagttgcc 720
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tcaagtggct cagagtgcat gcttgaagaa ctgaacgatt actcatccga aggtttccaa 960
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Claims (8)
1.一种编辑水稻Ehd1基因培育长生育期粳稻品种的方法,其特征在于:包括如下步骤:利用CRISPR/Cas9基因编辑系统编辑水稻Ehd1基因接收结构域,减弱或抑制水稻中Ehd1基因的表达,从而培育生育期中等或高度延长水稻品种的方法;
所述CRISPR/Cas9基因编辑系统由CRISPR-ED1-RD质粒载体组成;
所述CRISPR-ED1-RD基因编辑载体是特异性针对水稻Ehd1 基因接收结构域起始区进行定点编辑,其sgRNA的序列如下:5,-GCCTTATGGACTAAGAGTTC-3,;所述CRISPR-ED1-RD质粒载体是由VK005-1载体与引物二聚体连接得到;
所述引物二聚体由正向引物和反向引物组成,其中正向引物为5, -CAGGCCTTATGGACTAAGAGTTC-3,,反向引物为3‘-CGGAATACCTGATTCTCAAGAAC-5’。
2.根据权利要求1所述的方法,其特征在于:sgRNA所靶标的水稻基因序列如下:5,-GCCTTATGGACTAAGAGTTCTGG-3,。
3.根据权利要求1中所述的方法,其特征在于:所述抑制水稻中Ehd1基因的表达的方法,包括如下步骤:将含有所述CRISPR-ED1-RD质粒的根癌农杆菌转入水稻品种,通过对水稻中Ehd1基因进行定点编辑并经过对再生植株的鉴定,选择纯合移码突变实现的。
4.根据权利要求1中所述的方法,其特征在于:所述减弱水稻中Ehd1基因的表达的方法,包括如下步骤:将含有所述CRISPR-ED1-RD质粒的根癌农杆菌转入水稻品种,通过对水稻中Ehd1基因接收结构域进行基因编辑,并经过对再生植株的鉴定,选择纯合型的接收结构域框内缺失突变实现的。
5.如权利要求3或4中所述的方法,其特征在于:所述再生植株的鉴定为突变植株鉴定,具体包括如下步骤:通过Ehd1基因的特异性引物扩增Ehd1基因的基因组片段并进行测序实现的;所述特异性引物序列如SEQ ID NO.1-2所示。
6.一种ehd1短等位基因,其特征在于:通过基因编辑在Ehd1接收结构域起始区产生GlyLeu Arg 3个氨基酸缺失所形成的短等位基因,具有中等延迟水稻开花两周时间的功能;所述ehd1短等位基因其编码区核苷酸序列如SEQ ID NO.3所示。
7.如权利要求6所述的ehd1短等位基因,其特征在于:所述ehd1短等位基因的蛋白为如序列SEQ ID NO.4所示的氨基酸序列组成的蛋白质。
8.如权利要求6-7任一所述ehd1短等位基因在中等延长水稻生育期育种中的应用。
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