CN113913439B - 一种水稻基因OsAL11应用及其方法 - Google Patents
一种水稻基因OsAL11应用及其方法 Download PDFInfo
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Abstract
本发明公开了一种水稻OsAL11基因的应用,水稻OsAL11基因用于调节水稻种子的大小和水稻的抗旱性;本发明OsAL11基因突变体具有增加或减少水稻种子大小,从而改变水稻产量。
Description
技术领域
本发明涉及植物基因工程技术领域,尤其涉及一种水稻基因OsAL11应用及其方法。
背景技术
水稻是世界上重要的粮食作物之一,也是我国的主粮,供养着超过中国一半的人口。稻谷的生产也是我国的主要农业活动,通过培育高产抗逆水稻新品种对提高我国水稻产量,保障我国粮食安全具有重要意义。
粒重、穗粒数和有效穗数是水稻产量三要素。谷粒重量作为产量的三要素之一,是谷粒长、粒宽与粒厚的综合指标,一般以千粒重表示。谷粒大小决定粒重,不仅是重要的产量性状,也是一个极为重要的品质性状,在禾谷类作物进化上也具有重要的意义。在水稻的驯化过程中人们趋向于选择大粒的品种进行栽培,逐步形成了栽培种比它们的野生亲缘种籽粒偏大的状况。同时菲律宾国际水稻研究所也认为,提高粒重可以增产30%以上(马丽莲等,水稻大粒种质资源和遗传分析,植物学通报,2006, 23 (4): 395-401),培育大粒水稻品种也是当前水稻育种的一个重要方向。
水稻单株产量的还受到外界环境因素的影响。现代工业的发展也给全球气候环境的急剧变化带来了风险,极端气候条件频发,其中干旱是重要的自然灾害之一。研究和发现植物中重要的抗逆基因也是当前的迫切需要。其中Alfin-like基因家族被认为是调控植物的抗逆性的。Alfin-like基因是植物特异性基因,最初是从紫花苜蓿(Medicago sativaL.)盐敏感细胞培养物中发现并克隆了有助于增加苜蓿耐盐性的基因Alfin1(Winicov I,Valliyodan B, Xue L, et al. The MsPRP2 promoter enables strong heterologousgene expression in a root-specific manner and is enhanced by overexpressionof Alfin1[J]. Planta. 2004, 219(6): 925-935)。Alfin-like蛋白具有相似的结构,C端为长度约50个氨基酸的高度保守的植物同源结构域(plant homeodomain,PHD),N端含有一个长度约为150aa的保守结构域DUF3594结构域。PHD是一种交错的Cys4-His-Cys3(C4HC3)锌指结构域,能与一些核伴侣结合,并且PHD蛋白可以识别在4号位赖氨酸上发生了二/三甲基化的组蛋白H3(tri- and dimethylation of histone H3 at lysine 4,H3K4me2/3)并与之结合,故认为可能参与了植物的表观遗传调控。AL蛋白的N端保守的DUF3594结构域的功能尚不是很清楚,但其在植物种仍旧具有高度的保守性 (Lee W Y, Lee D, Chung W,et al. Arabidopsis ING and Alfin1-like protein families localize to thenucleus and bind to H3K4me3/2 via plant homeodomain fingers[J]. The PlantJournal. 2009, 58(3): 511-524)。
Alfin-like蛋白与非生物胁迫是紧密相关的。拟南芥中7个AL基因的研究发现用PEG模拟干旱以及高盐条件下能够诱导表达。对AL3、AL5、AL6三个基因构建突变体发现在干旱和盐胁迫下,AL5起到主要的抗逆作用,过表达AL5株系发现其能够明显增加植物的抗逆性(Wei W, Zhang Y, Tao J, et al. The Alfin-like homeodomain finger proteinAL5 suppresses multiple negative factors to confer abiotic stress tolerancein Arabidopsis[J]. The Plant Journal. 2015, 81(6): 871-883)。来自于大豆的GmPHD2过表达转基因拟南芥具有更高的耐盐性(Wei W, Huang J, Hao Y J, et al.Soybean GmPHD-type transcription regulators improve stress tolerance intransgenic Arabidopsis plants[J]. PLoS One. 2009, 4(9): e7209)。对菠菜进行包括高盐、干旱、寒冷、添加外源ABA处理,发现菠菜的AhAL基因的表达在胁迫响应中有各自特定的作用。对过表达AhAL1转基因拟南芥进行胁迫处理,发现其对盐和干旱具有更强的耐受性(Tao J, Wei W, Pan W, et al. An Alfin-like gene from Atriplex hortensisenhances salt and drought tolerance and abscisic acid response in transgenicArabidopsis[J]. Scientific Reports. 2018, 8(1), 2707.)。
目前主要在双子叶植物中报道过相关Alfin-like基因的功能,但在单子叶植物中未见公开报道。我们通过水稻种质资源的研究发现,OsAL11基因与水稻的抗旱系数紧密相关,并可影响水稻种子大小,本发明通过基因编辑和改造,可以用于水稻籽粒精准育种,提高水稻在干旱胁迫下的稳产高产打下基础。
发明内容
本发明的提供一种水稻基因OsAL11及其应用和方法。
本发明是基于一部分来源于水稻的OsAL11基因调控水稻抗旱性和籽粒发育的发现。本发明的目的在于提供一种OsAL11基因改变水稻抗旱性和种子大小的应用。
为此,本发明提供了一种通过基因编辑OsAL11基因来改变水稻种子大小的方法。同时本发明还提供了一种通过超表达OsAL11基因的不同选择性剪切本来改变水稻抗旱性的方法。
本发明的方案是:
一种水稻OsAL11基因的应用,水稻OsAL11基因用于调节水稻种子的大小和水稻的抗旱性。
作为优选的技术方案,所述的水稻OsAL11基因具有两种选择性剪切本OsAL11.1与OsAL11.2,所述选择性剪切本OsAL11.1与OsAL11.2的氨基酸序列分别如SEQ ID NO.1与SEQID NO.2所示。
作为优选的技术方案,所述选择性剪切样本OsAL11.2中用于制备具有抗旱性转基因植物的水稻基因OsAL11.2选择性剪切本编码的蛋白,其编码氨基酸序列如SEQ ID NO.2所示。
作为优选的技术方案,所述OsAL11基因第三外显子区域插入或缺失,该突变使基因翻译提前终止,形成OsAL11基因的突变体knAL11基因,所述的knAL11基因编码的氨基酸序列如SEQ ID NO.3所示。
作为优选的技术方案,所述水稻OsAL11基因引导RNA靶点序列引物在编辑水稻基因组中的应用是以OsAL11基因诱导RNA的核苷酸序列为OsAL11-sgRNA:GTGCGGTTCATAGTGATACC。
作为优选的技术方案,还包括添加接头利用PCR扩增的方法构建所述的OsAL11基因应用于生产具有抗旱性转基因植物的引导RNA靶点序列的sgRNA表达盒;
将sgRNA表达盒装载到CRISPR/Cas9载体上,得到含靶点序列的CRISPR/Cas9-sgRNA载体;
将靶点CRISPR/Cas9-sgRNA载体转化水稻愈伤组织,得到水稻OsAL11基因突变体knAL11植株。
本发明还公开了一种水稻OsAL11基因应用于生产具有抗旱性转基因植物的方法,包括以下步骤:
1)将水稻基因OsAL11.2选择性剪切本可操作地连接于植物表达调控序列,形成植物表达载体,所述水稻基因OsAL11.2的编码的氨基酸序列如SEQ ID NO.2所示;
2)将步骤1)所得的植物表达载体转入植物细胞;
3)经筛选获得的转化细胞,再生为植物及其后代。
本发明还公开了一种OsAL11基因突变体的应用,所述OsAL11基因的突变体中OsAL11基因突变体编码的蛋白质可调控水稻种子的大小。
本发明还公开了一种水稻OsAL11基因应用在生产在干旱条件下改善水稻产量的转基因植物的方法,该方法包括以下步骤:
1)将所述的OsAL11基因、突变基因及其基因编辑可操作地连接于植物表达调控序列,形成植物表达载体;
2)将步骤1)所得的植物表达载体转入植物细胞;
3)经筛选获得的转化细胞,再生为植物及其后代,所述的植物包括植物细胞、植物组织或植物种子。
同时本发明提供了一种通过人工编辑水稻基因组使产生提前终止密码来增加水稻种子大小的方法。本发明也包括采用其它的方法改造编码区5’端序列而得到的OsAL11基因的等位基因或衍生物。本发明提过改造OsAL11基因形成突变等位基因或过表达基因不同选择性转录剪切本,显著改变水稻抗旱性,这些结果表明OsAL11基因在水稻产量育种中和抗逆性稳产育种中具有重要的应用价值。通过精确改良OsAL11基因,构建适当的植物表达载体,可以拓展当前植物生物技术中可应用于水稻稳产高产的基因,为改良水稻的精准育种提供精准育种手段。
本发明提供的OsAL11基因两个选择性剪切本编码的氨基酸序列如SEQ ID NO.1和SEQ ID NO.2所示,所述的OsAL11基因位点进行人工编辑形成knAL11基因,所述knAL11基因编码的氨基酸序列如SEQ ID NO.3所示。
所述OsAL11基因第一个选择性剪切本的OsAL11.1基因的应用是以引物OsAL11.1F和OsAL11R组合,利用水稻反转录cDNA为模板进行PCR扩增,并回收该PCR产物,其中:
OsAL11.1F:atggacggaggcggggcgcac;
OsAL11R: ccatcaagctctggctctcttgc。
OsAL11.1基因全长编码cDNA为OsAL11基因第一个选择性剪切本,其编码氨基酸序列如SEQ ID NO.1所示。
所述截取OsAL11基因第二个选择性剪切本的OsAL11.2基因的应用是以引物OsAL11.2F和OsAL11R组合,利用水稻反转录cDNA为模板进行PCR扩增,并回收该PCR产物,其中:
OsAL11.2F:atggatgagaaagattggctgtcac;
OsAL11R: ccatcaagctctggctctcttgc。
所述OsAL11.2基因全长编码cDNA为OsAL11基因第二个选择性剪切本,其编码氨基酸序列如SEQ ID NO.2所示。
另一方面,所述水稻OsAL11基因引导RNA靶点序列引物在编辑水稻基因组中的应用是以OsAL11基因诱导RNA的核苷酸序列为OsAL11-sgRNA: GTGCGGTTCATAGTGATACC。
本发明公开了一种含有编码核酸的多核苷酸的分离的DNA分子在制备转基因水稻品种中的用途,分离水稻OsAL11基因编码区cDNA两种选择性剪切本。
另一方面,本发明公开了一种通过基因编辑的方式获得的水稻基因组中OsAL11基因等位基因knAL11基因的方法,其特征在于构建含有OsAL11-sgRNA表达盒的CRISP/CAS9载体,利用水稻遗传转化在所述OsAL11基因第三外显子区域产生插入或缺失的水稻突变体。
另一方面,本发明公开了所分离的DNA序列连接至组成性启动子驱动的植物表达载体,利用水稻遗传转化方法获得能超表达OsAL11基因两种选择性剪切本的水稻转基因植株。
本发明还公开了水稻OsAL11基因两种选择性剪切本,其中,所述OsAL11.2选择性剪切本用于调控水稻抗旱性。
由于采用了上述技术方案,一种水稻OsAL11基因的应用,水稻OsAL11基因用于调节水稻种子的大小和水稻的抗旱性。
与现有技术相比,本发明的优点如下:
1.本发明OsAL11基因突变体具有增加或减少水稻种子大小,从而改变水稻产量。
2.本发明将超表达OsAL11选择性剪切本的蛋白编码区导入水稻,实现调控水稻抗旱性。
3.本发明建立了二种利用OsAL11基因来对水稻在干旱条件下水稻产量大小进行遗传改良的方法。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1 OsAL11选择性剪切本及其编码蛋白比对结构图;图1A为OsAL11两个选择性剪切本基因组结构图,其中方框表示外显子,深色方框表示基因编码区,直线表示内含子;图1B为OsAL11选择性剪切本蛋白比对结构图,其中,对SEQ ID NO.1、SEQ ID NO.2和提前终止后knAL11编码蛋白序列比对分析,采用NCBI中BLAST软件(http://blast.ncbi.nlm.nih.gov/),以及相关发表文献绘制OsAL11蛋白保守域图;=表示DUF3594结构域,~~表示PHD结构域。
图2 CRISP/Cas9敲除后转化植物的测序验证结果图,其中,ZH11表示受体亲本中花11部分片段序列,knAL11表示敲除株系部分片段序列。
图3 表达载体pCBH04-OsAL11.1的构建示意图。
图4 表达载体pCBH04-OsAL11.2的构建示意图。
图5 CRISP/Cas9植物转化载体构建示意图。
图6 超表达转基因植株相对表达量示意图,其中,OsAL11.1和OsAL11.2分别表示超表达OsAL11.1和OsAL11.2两个剪切本株系;采用定量PCR方法2-△△ct计算相对表达量,参照为非转基因受体亲本日本晴,其相对表达量参考为1。
图7 超表达OsAL11.1和OsAL11.2转基因植株抗旱性鉴定;其中,处理前表示在正常液体培养基上生长,20%PEG处理后复水表示在液体培养基中添加20%PEG 处理10天后,转移到正常液体培养基中生长5天;WT表示亲本日本晴,***表示t-test分析p<0.001。
图8 knAL11转基因突变植株的种子大小统计;其中,knAL11表示OsAL11基因编辑后功能缺失突变体,ZH11表示受体亲本中花11,**表示t-test分析p<0.01,*表示p<0.05。
具体实施方式
在本文,术语“分离的”、“纯化的” DNA是指,该DNA或片段已从天然状态下位于其两侧的序列中分离出来,还指该DNA或片段已经与天然状态下伴随核酸的组分分开,而且已经与在细胞中伴随其蛋白质分开。
通过本领域已知的方法可以分离和纯化多核苷酸(DNA或RNA)、载体、转化体和生物体。
用于本发明的载体可以是如噬菌体、质粒、粘粒、微型染色体、病毒或逆转录病毒载体。可用于克隆和/或表达本发明的多核苷酸的载体是能在需复制和/或表达多核苷酸的宿主细胞中复制和/或表达多核苷酸的载体。一般说来,携带本发明的核酸序列的重组表达载体可以使用Ti质粒、植物病毒载体,直接DNA转化、微注射、电穿孔等常规生物技术方法导入植物细胞(Weissbach, 1998, Method for Plant Molecular Biology VIII, AcademyPress, New York, pp. 411-463; Geiserson and Corey, 1998, Plant MolecularBiology (2nd Edition)。
已开发出多种方法用于经由互补的粘性末端使多核苷酸与载体可操作相连。例如,可在欲插入载体 DNA 内的 DNA 区段添加互补的同聚体序列片段。然后通过互补同聚体尾之间的氢键连接载体和 DNA 区段以形成重组 DNA 分子。
含有一或多种限制性位点的合成接头提供了另一种连接 DNA 区段与载体的方法。用噬菌体 T4 DNA 聚合酶或大肠杆菌 DNA 聚合酶 I 处理通过内切核酸酶限制性消化产生的 DNA 区段,所述的两种聚合酶用其 3',5’-核酸外切活性除去突出的γ-单链末端,并用其聚合活性补平 3’-凹端。因此,这些活性的联合产生了平端 DNA 区段,然后在能催化平端 DNA 分子连接的酶,如噬菌体 T4 DNA 连接酶的存在下将平端区段与摩尔过量的接头分子一起保温。因此,反应产物是末端携有聚合接头序列的 DNA 区段,然后用适当的限制性酶裂解这些 DNA 区段,并连接至已用酶裂解的表达载体中,所述酶能产生与所述DNA 区段相容的末端。从多个商家可以买到含有多个限制性内切核酸酶位点的合成接头。
其它新发展的技术利用同源重组方法,将携带有特定序列接头或同源序列接头的多核苷酸与载体进行同源重组,将欲插入载体 DNA 内的 DNA 区段与同样携带有特定序列或同源序列的载体通过重组酶的作用形成重组DNA分子。
多核苷酸插入物应该可操作地连接于同表达多核苷酸的宿主细胞相容的适当启动子上,启动子可以是强启动子和/或诱导型启动子。列举的一些启动子的例子包括噬菌体PL 启动子、大肠杆菌 lac、trP 、phoA、tac 启动子、SV40 早期和晚期启动子以及逆转录病毒 LTR 启动子;其它适当启动子是本领域技术人员已知的。表达重组载体进一步含有转录起始、终止位点,并在转录区含有用于翻译的核糖体结合位点。重组载体表达的转录物的编码部分可包括位于起点处的翻译起始密码子和适当地位于被翻译多肽的末端的终止密码子(UAA, UGA或UAG)。
如上所述,表达载体可包括至少一个选择标记。所述标记包括编码抗生素的抗性基因,例如:新霉素磷酸转移酶(Neomycin phosphotransferase)基因nptⅡ、潮霉素磷酸转移酶(Hygromycin phosphotransferase)基因hpt 和二氢叶酸还原酶(Dihydrofolatereductase)基因dhfr;另一类是编码除草剂抗性基因,例如,草丁膦乙酰转移酶(Phosphinothricin acetyltransferase)基因bar、5-烯醇丙酮酰草酸-3-磷酸合成酶(5-Enoylpyruvate shikimatr-3-phosphate)基因epsps。适当宿主的代表性例子包括但不限于:原生质体细胞和植物细胞。上述宿主细胞的适当培养基和培养条件是本领域已知的。
目的基因或目的多核苷酸的转化方法:一类是载体介导的转化方法,即将目的基因插入到农杆菌的质粒或病毒的DNA 等载体分子上,随着载体DNA的转移而将目的基因导入到植物基因组中;农杆菌介导和病毒介导法就属于这种方法。第二类为基因直接导入法,是指通过物理或化学的方法直接将外源目的基因导入植物的基因组中。物理方法包括基因枪转化法、电激转化法、超声波法、显微注射法和激光微束法等;化学方法有PEG介导转化方法和脂质体法等。第三类为种质系统法,这包括花粉管通道法、生殖细胞浸染法、胚囊和子房注射法等。
本发明中,使用的术语“转化体”(transformant),即带有异源DNA分子的宿主细胞或生物体。
本发明还包括含有本发明的核苷酸序列的宿主细胞,所述核苷酸序列经本领域已知的技术与一或多种异源控制区(如启动子和/或增强子)可操作相连。可以选择能调节插入的基因序列的表达,或能按照所需的特殊方式修饰和加工基因产物的宿主菌株。在某些诱导物的存在下,某些启动子启动的表达会升高。
通过众所周知的技术可以鉴定出被成功转化的细胞,即含有本发明所述核苷酸序列的重组载体的细胞或生物体。
为了弥补以上不足,本发明提供了一种水稻基因OsAL11应用及其方法以解决上述背景技术中的问题。
一种水稻OsAL11基因的应用,水稻OsAL11基因用于调节水稻种子的大小和水稻的抗旱性。
所述的水稻OsAL11基因具有两种选择性剪切本OsAL11.1与OsAL11.2,所述选择性剪切本OsAL11.1与OsAL11.2编码的氨基酸序列分别如SEQ ID NO.1与SEQ ID NO.2所示。
所述选择性剪切样本OsAL11.2中用于制备具有抗旱性转基因植物的水稻基因OsAL11.2选择性剪切本编码的蛋白,其编码氨基酸序列如SEQ ID NO.2所示,或编码氨基酸序列与SEQ ID NO.2中从1-519序列长度有至少70%、80%、90%或95%的同一性。
所述OsAL11基因第三外显子区域插入或缺失,该突变使基因翻译提前终止,形成OsAL11基因的突变体knAL11基因,所述的knAL11基因编码的氨基酸序列如SEQ ID NO.3所示。
所述水稻OsAL11基因引导RNA靶点序列引物在编辑水稻基因组中的应用是以OsAL11基因诱导RNA的核苷酸序列为OsAL11-sgRNA: GTGCGGTTCATAGTGATACC。
还包括添加接头利用PCR扩增的方法构建所述的OsAL11基因应用于生产具有抗旱性转基因植物的引导RNA靶点序列的sgRNA表达盒;
将sgRNA表达盒装载到CRISPR/Cas9载体上,得到含靶点序列的CRISPR/Cas9-sgRNA载体;
将靶点CRISPR/Cas9-sgRNA载体转化水稻愈伤组织,得到水稻OsAL11基因突变体knAL11植株。
本发明还公开了一种水稻OsAL11基因应用于生产具有抗旱性转基因植物的方法,包括以下步骤:
1)将水稻基因OsAL11.2选择性剪切本可操作地连接于植物表达调控序列,形成植物表达载体,所述水稻基因OsAL11.2编码的氨基酸序列如SEQ ID NO.2所示;
2)将步骤1)所得的植物表达载体转入植物细胞;
3)经筛选获得的转化细胞,再生为植物及其后代。
本发明还公开了一种OsAL11基因突变体的应用,所述OsAL11基因的突变体中OsAL11基因突变体编码的蛋白质可调控水稻种子的大小。
本发明还公开了一种水稻OsAL11基因应用在生产在干旱条件下改善水稻产量的转基因植物的方法,该方法包括以下步骤:
1)将所述的OsAL11基因、突变基因及其基因编辑可操作地连接于植物表达调控序列,形成植物表达载体;
2)将步骤1)所得的植物表达载体转入植物细胞;
3)经筛选获得的转化细胞,再生为植物及其后代,所述的植物包括植物细胞、植物组织或植物种子。
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体实施例,进一步阐述本发明。
实施例1:分离克隆OsAL11基因
1.1OsAL11基因克隆
依据Rice Genome Annotation Project website (http://rice.uga.edu/)数据库提供的OsAL11基因数据,发现存在两个选择性剪切本,分别设计引物合成两个剪切本编码cDNA序列。OsAL11.1对应的引物为OsAL11.1F(5’-atggacggaggcggggcgcac-3’)和OsAL11R(5’- ccatcaagctctggctctcttgc-3’),OsAL11.2对应的引物为OsAL11.2F(5’atggatgagaaagattggctgtcac-3’)剪和OsAL11R。取水稻叶片,采用TRIzol试剂 (GIBCOBRL, USA) 抽提总RNA。利用反转录酶MLV(Tiangen,China)将其反转录成cDNA。引物OsAL11.1F和引物OsAL11R,扩增出基因的全长OsAL11.1编码cDNA,引物OsAL11.2F和引物OsAL11R,扩增出基因的全长OsAL11.2编码cDNA。PCR反应条件为:94℃预变性3min;94℃30sec,60℃30sec,72℃60sec共35个循环;72℃延伸1min。将扩增获得的PCR产物连入pGEM-T载体(Promega, USA),筛选阳性克隆并测序,获得OsAL11基因两个剪切本编码的氨基酸序列(SEQ ID NO. 1和SEQ ID NO.2)。OsAL11推测的蛋白序列进行同源基因的比对分析,发现选择性剪切本1OsAL11.1基因为全长转录本,编码蛋白在N端保守的DUF3594结构域和C端保守的PHD结构域的转录因子(见图1)。但选择性剪切本2OsAL11.2为一截断转录本,在N端产生了部分缺失,使其DUF3594结构域不完整(见图1)。故推测这种序列的差异对两个剪切本的基因功能产生了影响。
实施例2:OsAL11基因相关表达载体的构建
2.1含目的基因超表达载体的构建:
根据OsAL11基因2个选择性剪切本的序列(SEQ ID NO. 1和SEQ ID NO.2),设计扩增出完整编码阅读框的引物,并在上游引物和下游引物上分别添加接头引物,以便构建表达载体。以实施例1中获得的扩增产物为模板,经高保真Taq酶pfu酶(Tiangen,China)进行PCR扩增后,将实施例1中获得的全长cDNA克隆至中间载体(如pDONR207),进一步转化大肠杆菌DH5α,在保证阅读框架正确的前提下鉴定中间载体,然后抽提质粒,然后使用LRClonase重组酶与包含启动子和终止子蛋白植物表达载体载体pCBH04进行重组反应,构成了一个完整的表达单元(见图3和图4),转化农杆菌EHA105,最后进行水稻愈伤组织转化实验。
2.2 CRISP/Cas9植物转化载体构建
2.2.1、引导RNA靶点序列选择与引物设计
根据控制水稻基因OsAL11的基因组序列(LOC_Os11g14010),设计基因OsAL11的sgRNA。20nt的核苷酸sgRNA靶点序列按照5’-N20-NGG-3’序列进行设计,同时设计靶点序列引物gRT+ 和OsU6aT-,其3’端有15-17 nt分别与sgRNA和U6a启动子配对。具体靶点核苷酸序列如下,见图2。
OsAL11-sgRNA: GTGCGGTTCATAGTGATACC
gRT+: 5’-ATCACTATGAACCGCACgttttagagctagaaat-3’
OsU6aT-:5’-GTGCGGTTCATAGTGATggcagccaagccagca-3’
2.2.2、靶点序列sgRNA表达盒的构建
参考Ma等人的方法(Ma et al., 2015, Molecular Plant,8 (8) : 1274-1284),取2-5 ng pYLgRNA-OsU6a/LacZ质粒(1ul)为模板,在两个反应体系中分别进行PCR扩增:U-F和OsU6aT-用于扩增OsU6a-靶点片段,gR-R 和gRT+用于扩增sgRNA-靶点片段。一般使用KOD plus聚合酶(TOYOBO),反应体系为1ul质粒模板,2.5 uL 10×Buffer,0.5 uL KODplus聚合酶,1ul 25mM的MgSO4,2.5 uL 2mM的dNTPs,10 uM的前后引物各0.5 uL,补充ddH2O至25 uL ;PCR扩增程序为:95℃ 2min,98 ℃ 10s,58℃ 15s ,68 ℃ 20 s 25个循环。电泳检测PCR产物,OsU6a-靶点片段为700bp左右,gRNA-靶点片段为131bp左右。取第一轮两个PCR产物各1μl为模板,用引物U-GAL和Pgs-GAR按以上步骤进行第二轮PCR, 30循环。产物大小为830bp左右。凝胶电泳检测,切胶回收,此产物即为基因OsAL11靶点序列-sgRNA表达盒。
所述的Gibson assembly的引物为:
U-F: 5’- ctccgttttacctgtggaatcg -3’
gR-R: 5’- cggaggaaaattccatccac -3’
U-GAL: 5’-accggtaaggcgcgccgtagtgctcgactagtatggaatcggcagcaaagg-3’
Pgs-GAR: 5’-tagctcgagaggcgcgccaatgataccgacgcgtatccatccactccaagctcttg-3’
2.2.3、靶点CRISPR/Cas9-sgRNA载体构建
组装靶点序列sgRNA表达盒到pYLCRISPR/Cas9Pubi-H载体,以南京诺唯赞生物科技有限公司的ClonExpress快速克隆重组酶为例:4 μl 5 × CE II Buffer,200 ng线性化pYLCRISPR/Cas9Pubi-H质粒,上面回收的含OsAL11靶点序列-sgRNA的PCR产物200 ng,2 μlExnaseTM II,最后加水到20 μl,37℃ 温浴30min,将5μl反应混合物转化大肠杆菌,涂布含卡那霉素的LB平板筛选阳性克隆,次日挑取阳性单克隆进行测序验证并抽提质粒保存。质粒所示载体示意图见图6,转化农杆菌EHA105,最后进行水稻愈伤组织转化实验。其所获得的转基因植株命名为knAL11。
实施例3:水稻遗传转化
3.1种子消毒
成熟的日本晴水稻种子去壳后放入无菌三角瓶中,用75%酒精浸泡1-2 min,无菌水冲洗2次;再用30% NaClO消毒30 min,其间需经常摇动,再用无菌水洗3-4次,用无菌滤纸吸干多余的水分,将种子接种到愈伤组织诱导培养基(MS + 2,4-D 2.0 mg/L)上,每皿约30粒,于28℃暗培养。
3.2继代培养
经过近1月的诱导,水稻长出黄色膨大的愈伤组织,去其盾片,将愈伤转至新鲜的愈伤组织诱导培养基(MS + 2,4-D 2.0 mg/L)上进行继代。每2周继代一次,继代2-4次即可获得适合转基因的嫩黄色、颗粒状的胚性愈伤组织。在继代培养2周后,挑选胚性颗粒用于遗传转化。
3.3农杆菌的培养
在转化平板上挑取单菌落在1ml农杆菌培养基中培养。在50ml农杆菌培养基(含相应抗生素)中加入1ml上述培养物,200rpm,28℃培养5-6hr至OD600为0.6-1.0,培养结束前2hr加入乙酰丁香酮(AS,终浓度100uM)。取上述菌液在室温下,4000rpm,10min,弃上清,加入MS液体培养基(含AS 100uM)重悬菌体,在与上相同的条件下培养2hr,使菌液的OD600=0.5-1,此时可用来转化愈伤组织。AS=acetosringone。
3.4共培养
将水稻胚性愈伤组织浸入农杆菌菌液20-30min,再用无菌吸水纸吸干水分,将侵染的愈伤组织置于共培养培养基(MS + 2,4-D 2.0 mg/L + AS 100 uM)上,28℃暗培养三天。
3.5洗菌
共培养的愈伤组织先用无菌水冲洗3遍,再浸泡在含Cef/CN 400 mg/L的MS液体培养基中20-30min后,将愈伤组织转入无菌滤纸上吸干。
3.6选择培养
将吸干水分的愈伤组织接种于选择培养基(MS + 2,4-D 2.0 mg/L + Hyg 30 mg/L + Cef 400 mg/L)上。3周后,挑选新长出的愈伤接种于选择培养基(MS + 2,4-D 2.0 mg/L + Hyg 50 mg/L + Cef 250 mg/L)上,再选择2周。
3.7分化培养
将经过2次选择得到的抗性愈伤组织转入到预分化培养基(N6 + KT 2.0 mg/L +NAA 0.2 mg/L + 6-BA 2.0 mg/L + Hyg 30 mg/L + Cef 200 mg/L +琼脂 9g/L + 蔗糖45g/L)上暗培养10天左右,再转到分化培养基(N6 + KT 2.0 mg/L + NAA 0.2 mg/L + 6-BA 2.0 mg/L + Hyg 30 mg/L +琼脂 4.5g/L +蔗糖30 g/L)上光照培养。
3.8生根培养
约1-2个月,将2cm左右高的幼苗转到生根培养基(1/2MS + Hyg 15 mg/L +琼脂4.5g/L + 蔗糖20g/L)上诱导不定根的发生。
3.9转基因苗的移栽
当幼苗长至10cm高时,将幼苗取出,用无菌水洗净附着的固体培养基,移入泥土中,刚开始用玻璃罩罩几天,待植株健壮后再取下玻璃罩,温室中培养。
实施例4:OsAL11基因在转基因植株中的表达分析
4.1材料准备
转基因T1代水稻种子发芽后,移植于液体培养基(自来水配制成1/5MS大量元素)。幼苗生长15 d后,剪取叶片快速投入液氮保存,用于RNA的抽提。
4.2 无DNA的总RNA制备
按上海全式金生物技术有限公司提供的植物叶RNA小量抽提试剂盒使用说明书抽提。使用Beckman Coulter™DU®640紫外分光光度计测定RNA浓度。为除去残留在RNA中的DNA,每个总RNA样品取5 μg,加入1 μL DNAase I (美国Invitrogen公司) 和1 μL10×反应缓冲液,补足体积至10 μL,常温反应30 min,然后每管加入1μL 2 mmol L-1 EDTA终止反应,最后在70℃加热10 min使DNAase I失活。
4.3 第一链cDNA的合成
将上述RNA样品各取2 μL,按美国Promega公司反转录试剂盒提供的试剂依次添加4 μL 25 mmol L-1 MgCl2,2 μL10×RT 缓冲液,2 μL dNTP混和液和1 μL oligo(dT)15,加水补足体积到18.5 μL,在70℃加热变性10 min,快速在冰上冷却。然后加0.5 μL RNaseinhibitor 和1 μL AMVRTase,在42℃水浴60 min,70℃下加热10 min终止反应。
4.4定量PCR
根据基因OsAL11的序列设计特异性引物,由于两个选择性剪切本3’端是相同的,故设计同一套引物进行检测,正向引物为QF: 5’- ggatctgctgcgatgcttgtg -3’,反向引物为QR: 5’- ctggctctcttgctactactgc -3’,用于荧光定量PCR,根据水稻Actin基因(GenBank accession No. AY212324)的cDNA序列设计特异性引物AF: 5’-cttcctcatgccatcctgc-3’,AR: 5’-gcaagcttctccttgatgtcc-3’,用于参照基因的荧光定量PCR。PCR使用美国BioRad定量PCR仪,每一个PCR设置3次重复。反应体系包含Tiangen SYBRPremix Taq™(2×)10μL,正反向引物各0.5 μL,各种处理的cDNA模板1 μL,加水补足体积至25 μL。反应程序为:95℃30 s,然后在95℃10 s,61℃34 s下循环40次,设定在每个循环中60℃34 s时读取荧光值,同时进行ROX值校正,最后添加荧光PCR产物融解曲线分析,其他操作详见仪器使用说明书。为了检测RNA样品中是否存在DNA的污染,随机选取3个样品,各取1 μL RNA作为模板进行PCR,方法同上。
4.5分析方法
Ct是通过BioRad定量PCR仪软件在PCR的荧光域值自动产生的,将数据输入到EXCEL进行计算分析。数据分析采用方法为2-ΔΔCT,然后利用EXCEL表作表达差异柱状图。
4.6 分析结果
以空白非转基因日本晴品种为参照,分别检测了6个独立的转基因株系T1-T6,发现转基因株系均有较强的增强表达,其中OsAL11.1增强表达倍数最高超过20倍以上,OsAL11.1增强表达倍数最高超过5倍以上(见图6),说明该基因导入到水稻后在叶片中得到明显的增强表达,可应用于进一步的转基因水稻研究。
实施例5:knAL11植株鉴定
将CRISPR/Cas9-sgRNA载体转化水稻所获得的转基因植株移栽,剪切20mg叶片,按照快捷型植物基因组DNA提取系统(Tiangen,China)提供的说明方法,提取叶片DNA,取1-2ul DNA为模板,CasF:5’- ggcacagttgcagtcgttacc -3’, CasR:5’-tgaacaaccacaagcaactgac -3’,经Taq酶(Tiangen,China)进行PCR扩增后,回收纯化PCR片段,提交公司测序,寻找位点纯合突变的单株。通过序列分析,发现在OsAL11基因翻译密码起始位点下游285的位置缺失4个碱基tgat(见图2),导致发生移码突变,引起转录本的提前终止,形成99个氨基酸多肽,而全长OsAL11蛋白序列254个氨基酸。推测基因编辑后OsAL11基因可能由于移码突变提前终止而使该基因丧失功能(见图1),故该试验中所产生的KO突变体植株可为功能敲除缺失突变植株进行下一步研究工作,形成植株命名为knAL11。
实施例6:OsAL11基因过表达转基因在模拟干旱胁迫条件下的生长状况
选取了3个实施例4中OsAL11.1和OsAL11.2超量表达转基因T3代家系植株进行了苗期渗透胁迫实验。具体步骤如下:在96孔PCR板上每份材料种植半板约48株,当植株长至4叶期时,将幼苗移置到含20%PEG6000营养液中室温(夜25℃/白30℃)生长10天后复水处理,转换为正常营养液中培养5天,观察转基因植株的生长状况。结果表明,本发明克隆的两个选择性转录本OsAL11.1和OsAL11.2基因超表达的转基因植株株系的生长在正常条件下与对照无显著差异,但在渗透迫处理后,叶片都明显卷曲,在处理10天后,转移到正常生长条件下,第二个转录本OsAL11.2的成活率明显高于非转基因对照植株,而全长转录本OsAL11.1转基因植株成活率明显低于非转基因对照植株(见图7),说明了过表达该截断转录本OsAL11.2可以增强水稻耐渗透胁迫能力,过表达该全长转录本OsAL11.1降低了植株的抗旱能力。
实施例7:OsAL11基因功能敲除突变体的种子大小鉴定
选取了3个实施例5中所获得的knAL11转基因T1代植株和受体中花11在大田种植,到植株生长成熟时收获转基因T2代植株种子,对种子大小进行观察测量。
将野生型水稻和敲除knAL11种子放在一起进行拍照和统计,我们发现敲除knOsAL11种子呈明显变大,尤其是粒长发生了显著变化,粒宽没有发生显著变化,最后导致长宽比明显高于对照(见图8)。同时也观察了超表达转基因材料的种子大小,没有发生明显变化。
综上所述,敲除OsAL11基因功能,使水稻种子变大。
以上显示和描述了本发明的基本原理、主要特征及本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
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Claims (5)
1.一种水稻OsAL11基因用于调节水稻抗旱性的应用,其特征在于:所述OsAL11基因登录号为LOC_Os11g14010,其具有两种选择性剪切本OsAL11.1与OsAL11.2,所述选择性剪切本OsAL11.1与OsAL11.2编码的氨基酸序列分别如SEQ ID NO.1与SEQ ID NO.2所示;所述调节水稻抗旱性具体为在水稻中过表达OsAL11.2能够增强水稻抗旱性,过表达OsAL11.1能够降低水稻抗旱性。
2.一种水稻OsAL11基因用于调节水稻种子大小的应用,其特征在于:所述OsAL11基因登录号为LOC_Os11g14010,其具有两种选择性剪切本OsAL11.1与OsAL11.2,所述选择性剪切本OsAL11.1与OsAL11.2编码的氨基酸序列分别如SEQ ID NO.1与SEQ ID NO.2所示;所述调节水稻种子大小具体为敲除水稻中OsAL11基因后种子变大。
3.如权利要求2所述的一种水稻OsAL11基因用于调节水稻种子大小的应用,其特征在于:所述OsAL11基因第三外显子区域插入或缺失突变,该突变使基因翻译提前终止,形成OsAL11基因的突变体knAL11基因,所述的knAL11基因编码的氨基酸序列如SEQ ID NO.3所示。
4.如权利要求3所述的一种水稻OsAL11基因用于调节水稻种子大小的应用,其特征在于:用于使所述OsAL11基因第三外显子区域插入或缺失的诱导RNA的核苷酸序列如SEQ IDNO.4所示。
5.一种如权利要求1所述的水稻OsAL11基因应用于生产在干旱条件下改善产量的转基因水稻的方法,其特征在于,包括以下步骤:
1)将所述的剪切本OsAL11.2可操作地连接于植物表达调控序列,形成植物超表达载体;
2)将步骤1)所得的植物超表达载体转入植物细胞;
3)经筛选获得的转化细胞,再生为植物及其后代,所述的植物包括植物细胞、植物组织或植物种子。
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