CN116622746A - 烟草cbl相互作用的丝氨酸/苏氨酸蛋白激酶23基因及应用 - Google Patents
烟草cbl相互作用的丝氨酸/苏氨酸蛋白激酶23基因及应用 Download PDFInfo
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Abstract
本发明涉及一种烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因,其核苷酸序列如SEQ ID NO.1所示。本发明提供的烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因NtCIPK23,利用CRISPR/Cas9介导的基因编辑技术敲除NtCIPK23基因获得在低钾条件下与对照植株相比钾离子含量降低的基因编辑植株;在低钾条件下,基因编辑植株的长势弱于对照植株,为烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因研究及该基因在烟草钾吸收利用方面的应用提供了遗传材料和理论依据。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及一种烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因及应用。
背景技术
CBL相互作用的丝氨酸/苏氨酸蛋白激酶(CIPK)在植物界广泛存在,其最早是在模式植物拟南芥中被发现的。该家族蛋白结构特点是氨基端含有1个激酶催化结构域,羧基端含可变调节结构域,通过一个中间连接结构域连接在一起,在连接结构域之后含有保守的NAF(又名FISL)基序是CIPK与CBL相互作用所必需的结构。CIPKs和CBLs相互作用构成一个复杂的信号网络,在植物对干旱、低温和盐碱等逆境的应答调控中起核心作用。
现有技术中几乎没有关于烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因NtCIPK23对钾吸收的分子机制调控的研究报道。
发明内容
本发明的目的是为了解决现有技术的不足,提供一种烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因及应用,为研究烟草钾通道吸收利用相关基因功能提供遗传材料和理论依据。
为实现上述目的,本发明采用的技术方案如下:
本发明第一方面提供了一种烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因,核苷酸序列如SEQ ID NO.1所示,包括1452bp个碱基,该基因来源于烟草(Nicotianatabacum,命名为NtCIPK23)。
本发明第二方面提供了一种第一方面所述烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因的编码蛋白,氨基酸序列如SEQ ID NO.2所示,包括483个氨基酸。
本发明第三方面提供了一种第一方面所述烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因在烟草钾吸收利用方面的应用,通过CRISPR/Cas9介导的基因编辑技术,构建了用于敲除烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因的CRISPR/Cas9编辑载体,经遗传转化后获得了烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因发生编辑的烟草植株。
优选的,所述基因发生编辑的烟草植株外排钾离子能力增强,吸收钾离子能力减弱。
优选的,所述基因发生编辑的烟草植株在MS培养条件下长势相当于基因未发生编辑的烟草植株,在低钾条件下长势差于基因未发生编辑的烟草植株。
优选的,所述低钾条件是指钾离子浓度为10-17μM的条件。
本发明的有益效果是:
1、本发明通过同源克隆方法克隆一种烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因NtCIPK23。
2、本发明通过CRISPR/Cas9介导的基因编辑技术,构建了用于敲除NtCIPK23基因的CRISPR/Cas9编辑载体,经遗传转化后获得了NtCIPK23基因发生编辑的红花大金元突变体植株。
3、本发明分别通过在正常和低钾条件下,对NtCIPK23基因编辑苗期植株与基因未发生编辑的烟草植株(下文称对照植株)苗期植株进行表型鉴定,正常条件下两类植株表型无差异,低钾条件下NtCIPK23基因编辑植株长势差于对照植株。同时对二者钾含量进行测定,发现基因编辑植株中钾离子含量显著低于对照植株;对植株中其他钾离子通道基因表达检测,发现钾吸收通道基因表达量在NtCIPK23基因编辑植株中发生不同程度的降低;根系钾离子流速测定发现NtCIPK23基因编辑植株中外排钾离子的能力增强,吸收钾离子的能力减弱。
4、本发明所述的基因发生编辑的烟草植株在低钾条件下长势差于对照植株,因此本发明所述基因发生编辑的烟草植株对低钾更为敏感,可以通过提前小范围种植该基因发生编辑的烟草植株,通过该基因发生编辑烟草植株的苗期长势是否良好提前预测该范围内土壤中是否含有满足烟草植物生长所必需的钾条件,避免在未知条件下大面积种植烟草植株但土壤中钾含量不足造成的损失。
附图说明
图1为对照植株(HD)和基因编辑植株(C1~C3)在正常条件(MS)、低钾条件(LK)下表型鉴定;
图2为对照植株(HD)和基因编辑植株(C1~C3)在正常条件(MS)、低钾条件(LK)下钾离子含量测定;
图3为对照植株(HD)和基因编辑植株(C1)在低钾条件下(LK)处理24h,进行11个与钾吸收相关基因的表达量测定;
图4为对照植株(HD)和基因编辑植株(C1)在苗期低钾条件下,进行植株根系钾离子流速测定。
具体实施方式
以下通过实施例来详细说明本发明的技术方案,以下的实施例仅是示例性的,仅能用来解释和说明本发明的技术方案,而不能解释为是对本发明技术方案的限制。
在本申请的各实施例中,没有注明具体技术或条件者,按照本领域内现有技术或条件进行,所使用的材料或设备未注明生产厂商者,均为可以通过购买获得的常规产品。
本发明除非另有说明,否则百分号为体积百分数,比例为体积比。
本申请中所使用的烟草品种为红花大金元,一种商品化烟草品种。
本申请中所述的正常培养条件是指MS培养条件,该条件下钾离子浓度能保证植物正常生长状态。
实施例1
本实施例主要就烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因NtCIPK23的获得过程,简要介绍如下。
以栽培种烟草红花大金元叶片为样品,利用RNA提取试剂盒提取烟草叶片总RNA,反转录为cDNA备用:
按照植物RNA提取试剂盒说明书提取烟草总RNA。
1μg从叶片中提取总RNA用于反转录,转录体系如下:
TotalRNA 1μg
Oligo(dT)(10μM) 1.5μL
ddH2O upto15μL
将上述体系混匀后置于PCR中,70℃保温5min,去除后立即置于冰上5min,之后向体系中加入以下试剂:
上述体系放入PCR仪中,42℃65min,65℃10min,4℃保温,之后置于-20℃冰箱中保存使用。
通过同源比对的方法,参考拟南芥基因的序列及已知烟草部分基因序列,设计扩增引物序列如下:
F:5’-ATGGGTTCAAGATCAAAT-3’,(SEQ ID No.3)
R:5’-TCAATTATGAGGGTCATTGATG-3’;(SEQ ID No.4)
以上述所制备cDNA为模板,利用上述引物进行PCR扩增:
扩增体系(50μL):
混匀离心后进行PCR扩增,PCR反应条件为:95℃10sec,52℃30sec,72℃2.5min,共30个循环;72℃10min;12℃Hold。
对扩增产物进行提纯后测序,获得烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因NtCIPK23序列,其碱基序列如SEQ ID No.1所示,共包括1452bp个碱基。对该基因序列进行翻译后,其所编码蛋白序列如SEQ ID No.2所示,共包括483个氨基酸,进一步对比分析表明,该蛋白含有同源性很高的序列,高度保守。
实施例2
利用实施例1中所获得烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23相关基因NtCIPK23,本发明进一步构建了CRISPR/Cas9载体,以及利用叶盘法转化获得基因编辑植株。
选择NtCIPK23基因中较特异的23nt核苷酸序列(SEQ ID No.5)为CRISPR/Cas9的引导序列,并将该序列片段与CRISPR/Cas9载体(由西南大学提供)进行连接,获得转化的克隆子,进行PCR扩增检测,后将PCR阳性克隆送测序公司进行测序确认,最后得到CRISPR/Cas9-NtCIPK23编辑载体。
利用上一步所构建的CRISPR/Cas9-NtCIPK23编辑载体质粒,以红花大金元为例,进行遗传转化试验,以敲除植物体内的烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23相关基因NtCIPK23。
将获得的基因编辑植株进行转化植株叶片取样,送华大基因进行分子检测,确定获得NtCIPK23基因编辑植株。
实施例3
种植编辑植株与对照植株,把在土中生长30天的烟草材料,进行水培处理15天,结果表明在正常培养条件下,对照植株和编辑植株的生长没有明显差异,但是在低钾处理(10μMK+)后,编辑植株长势明显差于对照植株,说明NtCIPK23编辑植株对低钾敏感(结果如图1)。
(1)将上述种植植株整株进行烘干,测定钾离子含量,结果表明在正常培养条件下,对照植株和编辑植株中钾离子含量没有明显差异,但是在低钾处理后,编辑植株中钾离子含量明显低于对照植株(结果如图2),说明NtCIPK23编辑植株吸收钾离子的能力较弱。
(2)将上述种植植株进行总RNA提取,然后进行荧光定量PCR实验,结果表明在编辑植株中,8个与钾吸收相关基因的表达量明显降低,包括TPKla,TPK3a,TPK3b,SKOR1,SKOR3,SKOR4,NKT4,NKT6。3个与钾吸收相关基因的表达量明显升高包括TPKlb,SKOR2,KC1(结果如图3),表明大多数钾吸收相关基因的表达量降低可能导致编辑植株钾吸收能力减弱。
(3)将上述种植植株根部清洗,进行钾离子流速测定,检测时间是5分钟,检测部位是根毛区,展示结果是钾离子外排的情况,结果表明NtCIPK23编辑植株的钾离子流速高于对照植株(结果如图4)。说明NtCIPK23编辑植株钾离子外排能力增强,钾离子吸收能力减弱。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。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。
Claims (6)
1.一种烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因,其特征在于,核苷酸序列如SEQ ID NO.1所示。
2.权利要求1所述烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因的编码蛋白,其特征在于,氨基酸序列如SEQ ID NO.2所示。
3.权利要求1所述烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因在烟草钾吸收利用方面的应用,其特征在于,通过CRISPR/Cas9介导的基因编辑技术,构建了用于敲除烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因的CRISPR/Cas9编辑载体,经遗传转化后获得了烟草CBL相互作用的丝氨酸/苏氨酸蛋白激酶23基因发生编辑的烟草植株。
4.根据权利要求3所述的应用,其特征在于,所述基因发生编辑的烟草植株外排钾离子能力增强,吸收钾离子能力减弱。
5.根据权利要求3所述的应用,其特征在于,所述基因发生编辑的烟草植株在MS培养条件下长势相当于基因未发生编辑的烟草植株,在低钾条件下长势差于基因未发生编辑的烟草植株。
6.根据权利要求5所述的应用,其特征在于,所述低钾条件是指钾离子浓度为10-17μM的条件。
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