CN115786346A - Application of Knockout TaSnRK2.10 to Increase Wheat Tiller Number, Ear Grain Number and Grain Width - Google Patents

Abstract

Translated fromChinese

本发明公开了一种利用基因编辑敲除TaSnRK2.10增加小麦分蘖数、穗粒数和籽粒宽度的应用，是设计靶向TaSnRK2.10基因的sgRNA，构建含有sgRNA的pBUE411‑TaSnRK2.10双元重组载体，利用农杆菌侵染小麦幼胚诱导的愈伤组织，将sgRNA与Cas9元件共同转入小麦，获得TaSnRK2.10基因敲除的转基因小麦，即得到分蘖数、穗粒数与籽粒宽度增加的小麦tasnrk2.10敲除突变体。本发明的应用为利用基因工程技术实现小麦快速育种、提高小麦产量提供一种切实可行的方法，具有重要的育种应用价值和广阔市场应用前景。

The invention discloses an application of using gene editing to knock out TaSnRK2.10 to increase the number of tillers, the number of spikes and the width of grains in wheat. It is to design an sgRNA targeting the TaSnRK2.10 gene and construct a pBUE411‑TaSnRK2.10 binary containing the sgRNA. The recombinant vector uses Agrobacterium to infect the callus induced by wheat immature embryos, and transfers sgRNA and Cas9 elements into wheat together to obtain a transgenic wheat with TaSnRK2.10 gene knockout, that is, the number of tillers, the number of grains per panicle and the width of grains are increased The wheat tasnrk2.10 knockout mutant. The application of the invention provides a practical method for realizing rapid wheat breeding and increasing wheat yield by using genetic engineering technology, and has important breeding application value and broad market application prospect.

Description

Translated fromChinese

利用敲除TaSnRK2.10增加小麦分蘖数、穗粒数和籽粒宽度的应用Knockout of TaSnRK2.10 to increase tiller number, grain number per panicle and grain width in wheatapplication

技术领域technical field

本发明涉及小麦TaSnRK2.10基因调控的应用，尤其涉及利用基因编辑敲除TaSnRK2.10增加小麦分蘖数、穗粒数和籽粒宽度的应用，属于基因工程和分子育种技术领域。The invention relates to the application of wheat TaSnRK2.10 gene regulation, in particular to the application of gene editing to knock out TaSnRK2.10 to increase the number of tillers, the number of spikes and the width of grains in wheat, and belongs to the technical field of genetic engineering and molecular breeding.

背景技术Background technique

小麦(Triticum aestivum L.,2n＝6x＝42,AABBDD)作为世界三大粮食作物之一，占人类热量摄入的20％以上，并提供比所有动物来源更多的蛋白质(约23％)。小麦产量的增加对全球粮食和营养安全具有重大影响。然而，目前小麦产量趋势将不足以满足未来的需求，而这种情况可能会因全球气候变化等环境因素而加剧。因此需要采取紧急行动，可持续性地增加全球粮食产量。Wheat (Triticum aestivum L., 2n=6x=42, AABBDD), as one of the world's three major food crops, accounts for more than 20% of human caloric intake and provides more protein (about 23%) than all animal sources. Increases in wheat production have major implications for global food and nutrition security. However, current wheat production trends will not be sufficient to meet future demand, a situation that may be exacerbated by environmental factors such as global climate change. Urgent action is therefore needed to sustainably increase global food production.

ABA(脱落酸)调节农作物生长的许多重要方面，包括种子蛋白和脂质储存，干旱耐受性，休眠以及从胚胎到萌发生长和营养生长到生殖生长的相变调节。植物处于逆境胁迫环境时，体内ABA会迅速积累。维持与不同环境中不同组织一致的游离ABA的基础水平对于整个植物的适当生长和发育状态至关重要。脱落酸通过细胞内受体PYLs的识别在植物中发挥作用，ABA结合的PYLs与分支A中PP2Cs形成复合物，释放PP2Cs抑制的SnRK2s蛋白激酶，通过自磷酸化激活SnRK2s，或者它们可以被其他蛋白激酶(如Raf-like MAPKK)激活。SnRK2s通过磷酸化靶底物(如ABF2，ABF3，ABF4和ABI5)调节ABA响应基因的表达和多种生理反应，包括离子通道，转录因子和转运蛋白等；而在没有ABA的情况下，PP2C与SnRK2s相互作用并抑制SnRK2s的激酶活性以阻断ABA信号。这些成分的调节对于在非生物胁迫条件下管理过度和有害的防御反应至关重要，它们构成了在非最佳生长环境中保持增长的核心信号系统。ABA (abscisic acid) regulates many important aspects of crop growth, including seed protein and lipid storage, drought tolerance, dormancy, and regulation of phase transitions from embryonic to germinal growth and vegetative to reproductive growth. When plants are in a stressful environment, ABA will accumulate rapidly in the body. Maintaining basal levels of free ABA consistent with different tissues in different environments is critical for proper growth and developmental status of the whole plant. Abscisic acid functions in plants through the recognition of intracellular receptors PYLs, ABA-bound PYLs form complexes with PP2Cs in branch A, release SnRK2s protein kinases inhibited by PP2Cs, activate SnRK2s through autophosphorylation, or they can be activated by other proteins. Kinase (such as Raf-like MAPKK) activation. SnRK2s regulate the expression of ABA-responsive genes and various physiological responses, including ion channels, transcription factors, and transporters, by phosphorylating target substrates (such as ABF2, ABF3, ABF4, and ABI5); while in the absence of ABA, PP2C and SnRK2s interact and inhibit the kinase activity of SnRK2s to block ABA signaling. Regulation of these components is critical for managing excessive and deleterious defense responses under abiotic stress conditions, and they constitute a central signaling system for maintaining growth in non-optimal growth environments.

已报道的小麦TaSnRK2.10基因的编码序列见公开号CN102399760A的专利，TaSnRK2.10基因在提高千粒重和植物耐逆性方面也有相关专利。在公开号CN103820476A的专利中，报道了一个与小麦千粒重相关的基因TaSnRK2.10，该基因在不同的小麦品种中存在紧密连锁的3个SNP位点和一个InDel位点，表现为两种单倍型，将单倍型与千粒重性状进行关联分析，发现两种单倍型与小麦千粒重显著相关。申请人研究发现TaSnRK2.10是ABA信号通路中的关键正调控因子AtSnRK2.6(OST1)的同源基因，具有调控小麦分蘖数、穗粒数和籽粒宽度的功能。该调控功能为研究ABA在小麦生长发育及高产育种提供了非常重要的理论依据。但检索发现TaSnRK2.10敲除突变体及利用基因编辑敲除TaSnRK2.10增加小麦分蘖数、穗粒数和籽粒宽度的应用未见报道。The coding sequence of the reported wheat TaSnRK2.10 gene can be found in the patent with the publication number CN102399760A. The TaSnRK2.10 gene also has related patents on improving thousand-grain weight and plant stress tolerance. In the patent of Publication No. CN103820476A, a gene TaSnRK2.10 related to wheat thousand-grain weight was reported. This gene has three closely linked SNP sites and one InDel site in different wheat varieties, showing two haplotypes. The association analysis between haplotypes and thousand-grain weight traits revealed that two haplotypes were significantly correlated with wheat thousand-grain weight. The applicant's research found that TaSnRK2.10 is a homologous gene of AtSnRK2.6 (OST1), a key positive regulator in the ABA signaling pathway, and has the function of regulating tiller number, grain number per panicle and grain width of wheat. This regulatory function provides a very important theoretical basis for the study of ABA in wheat growth and development and high-yield breeding. However, the retrieval found that the TaSnRK2.10 knockout mutant and the application of gene editing to knock out TaSnRK2.10 to increase the number of tillers, grains per panicle and grain width of wheat have not been reported.

发明内容Contents of the invention

针对现有技术的不足，本发明要解决的问题是提供一种利用基因编辑敲除TaSnRK2.10增加小麦分蘖数、穗粒数和籽粒宽度的应用。Aiming at the deficiencies of the prior art, the problem to be solved by the present invention is to provide an application of gene editing to knock out TaSnRK2.10 to increase the number of tillers, grains per spike and grain width of wheat.

本发明所述的利用基因编辑敲除TaSnRK2.10增加小麦分蘖数、穗粒数和籽粒宽度的应用。The application of gene editing to knock out TaSnRK2.10 in the present invention to increase the number of tillers, the number of grains per ear and the width of grains in wheat.

其中，应用方法是：设计靶向TaSnRK2.10基因的sgRNA，即基因编辑技术获得小麦TaSnRK2.10基因敲除突变体的特异性引导单链RNA，构建含有sgRNA的pBUE411-TaSnRK2.10双元重组载体，利用农杆菌侵染小麦幼胚诱导的愈伤组织，将sgRNA与Cas9元件共同转入小麦，特异性敲除TaSnRK2.10基因，获得TaSnRK2.10基因敲除的转基因小麦，即得到分蘖数、穗粒数与籽粒宽度增加的小麦tasnrk2.10敲除突变体。Among them, the application method is: design sgRNA targeting TaSnRK2.10 gene, that is, gene editing technology to obtain specific guide single-stranded RNA of wheat TaSnRK2.10 gene knockout mutant, and construct pBUE411-TaSnRK2.10 binary recombination containing sgRNA Vector, use Agrobacterium to infect the callus induced by wheat immature embryos, transfer sgRNA and Cas9 elements into wheat, specifically knock out the TaSnRK2.10 gene, and obtain the transgenic wheat with TaSnRK2.10 knockout, that is, the number of tillers Wheat tasnrk2.10 knockout mutant with increased grain number per panicle and increased grain width.

上述应用中：所述靶向TaSnRK2.10基因的sgRNA属于TaSnRK2.10编码序列或TaSnRK2.10编码序列的互补序列，长度为22～23个脱氧核糖核苷酸，其3’端最后三个脱氧核苷酸序列为NGG，其中N为腺嘌呤或鸟嘌呤或胸腺嘧啶或胞嘧啶，其核苷酸序列如SEQIDNO.5、或SEQ ID NO.6、或SEQ ID NO.7、或SEQ ID NO.8所示。In the above application: the sgRNA targeting the TaSnRK2.10 gene belongs to the TaSnRK2.10 coding sequence or the complementary sequence of the TaSnRK2.10 coding sequence, with a length of 22 to 23 deoxyribonucleotides, and the last three deoxyribonucleotides at its 3' end The nucleotide sequence is NGG, wherein N is adenine or guanine or thymine or cytosine, and its nucleotide sequence is as SEQ ID NO.5, or SEQ ID NO.6, or SEQ ID NO.7, or SEQ ID NO .8 shown.

其中：所述靶向TaSnRK2.10基因的sgRNA优选的核苷酸序列如SEQ ID NO.5所示。Wherein: the preferred nucleotide sequence of the sgRNA targeting the TaSnRK2.10 gene is shown in SEQ ID NO.5.

上述应用中：所述pBUE411-TaSnRK2.10双元重组载体由表达盒E1和表达盒E2构成，其核苷酸序列如SEQ ID NO.3所示；其中所述表达盒E1的核苷酸序列如SEQ ID NO.1所示，其从上游至下游依次包括：来自小麦的TaU3启动子，TaSnRK2.10基因的sgRNA，终止子T1；所述表达盒E2的核苷酸序列如SEQ ID NO.2所示，其从上游至下游依次包括：来自玉米的泛素启动子Ubi，玉米Cas9编码序列，终止子T2；TaSnRK2.10基因的核苷酸序列如SEQIDNO.4所示。In the above application: the pBUE411-TaSnRK2.10 binary recombination vector is composed of expression cassette E1 and expression cassette E2, and its nucleotide sequence is shown in SEQ ID NO.3; wherein the nucleotide sequence of the expression cassette E1 As shown in SEQ ID NO.1, it includes from upstream to downstream: the TaU3 promoter from wheat, the sgRNA of the TaSnRK2.10 gene, and the terminator T1; the nucleotide sequence of the expression cassette E2 is as shown in SEQ ID NO. 2, it includes from upstream to downstream: the ubiquitin promoter Ubi from maize, the maize Cas9 coding sequence, and the terminator T2; the nucleotide sequence of the TaSnRK2.10 gene is shown in SEQ ID NO.4.

其中：所述小麦TaU3启动子的核苷酸序列如SEQ ID NO.9所示；所述TaSnRK2.10基因的sgRNA的核苷酸序列如SEQ ID NO.5所示；所述终止子T1的核苷酸序列如SEQ ID NO.10所示；所述玉米泛素启动子Ubi的核苷酸序列如SEQ ID NO.11所示；所述玉米Cas9编码序列的核苷酸序列如SEQ ID NO.12所示；所述终止子T2的核苷酸序列如SEQ ID NO.13所示。Wherein: the nucleotide sequence of the wheat TaU3 promoter is shown in SEQ ID NO.9; the nucleotide sequence of the sgRNA of the TaSnRK2.10 gene is shown in SEQ ID NO.5; the terminator T1 The nucleotide sequence is shown in SEQ ID NO.10; the nucleotide sequence of the corn ubiquitin promoter Ubi is shown in SEQ ID NO.11; the nucleotide sequence of the corn Cas9 coding sequence is shown in SEQ ID NO .12; the nucleotide sequence of the terminator T2 is shown in SEQ ID NO.13.

本发明所述靶向TaSnRK2.10基因的sgRNA在增加小麦分蘖数、穗粒数和籽粒宽度中的应用，其中所述靶向TaSnRK2.10基因的sgRNA的核苷酸序列如SEQ ID NO.5所示。The application of the sgRNA targeting the TaSnRK2.10 gene of the present invention in increasing the tiller number, grain number and grain width of wheat, wherein the nucleotide sequence of the sgRNA targeting the TaSnRK2.10 gene is as SEQ ID NO.5 shown.

其中：所述应用是通过构建含有该sgRNA的具有敲除TaSnRK2.10基因功能的核苷酸序列如SEQ ID NO.3所示的表达载体pBUE411-TaSnRK2.10，并转化到普通小麦中特异性敲除TaSnRK2.10基因，降低TaSnRK2.10基因的表达，获得TaSnRK2.10基因敲除的转基因小麦实现。Wherein: the application is by constructing the expression vector pBUE411-TaSnRK2.10 containing the sgRNA with the nucleotide sequence of knocking out the TaSnRK2.10 gene function as shown in SEQ ID NO.3, and transforming it into common wheat. The TaSnRK2.10 gene is knocked out, the expression of the TaSnRK2.10 gene is reduced, and the transgenic wheat with the TaSnRK2.10 gene knockout is obtained.

本发明的突出效果是：通过设计TaSnRK2.10基因的引导单链RNA(sgRNA)，构建可以敲除小麦体内TaSnRK2.10基因的表达载体pBUE411-TaSnRK2.10，并通过对小麦遗传转化，得到TaSnRK2.10基因敲除的后代。实验证实：TaSnRK2.10基因敲除的转基因小麦实现了小麦种子分蘖数、穗粒数和籽粒宽度以及最终小麦产量的提高。并且实验结果确定TaSnRK2.10基因对于小麦的分蘖数、穗粒数和籽粒发育具有负向调控作用。本发明提供的利用基因编辑敲除TaSnRK2.10增加小麦分蘖数、穗粒数和籽粒宽度的应用为利用基因工程技术实现小麦快速育种、提高小麦产量提供一种切实可行的方法，具有重要的育种应用价值和广阔市场应用前景。The outstanding effect of the present invention is: by designing the guide single-stranded RNA (sgRNA) of the TaSnRK2.10 gene, constructing the expression vector pBUE411-TaSnRK2.10 that can knock out the TaSnRK2.10 gene in wheat, and obtaining TaSnRK2 through genetic transformation of wheat .10 Knockout offspring. The experiment confirmed that the transgenic wheat with TaSnRK2.10 gene knockout realized the improvement of wheat seed tiller number, ear grain number, grain width and final wheat yield. And the experimental results confirmed that the TaSnRK2.10 gene has a negative regulatory effect on the number of tillers, grains per panicle and grain development of wheat. The application of using gene editing to knock out TaSnRK2.10 to increase the number of tillers, grains per ear and grain width of wheat provided by the present invention provides a practical method for realizing rapid breeding of wheat and increasing wheat yield by using genetic engineering technology, and has important breeding Application value and broad market application prospect.

附图说明Description of drawings

图1为TaSnRK2.10进化分析。Figure 1 shows the evolution analysis of TaSnRK2.10.

图2为TaSnRK2.10-4A/4B/4D基因扩增琼脂糖凝胶电泳图。Fig. 2 is an agarose gel electrophoresis image of TaSnRK2.10-4A/4B/4D gene amplification.

其中，泳道M为Transgen 2K plus marker，泳道a、b、c分别为TaSnRK2.10-4A/4B/4D-Inner基因。Among them, lane M is Transgen 2K plus marker, and lanes a, b, and c are TaSnRK2.10-4A/4B/4D-Inner genes, respectively.

图3为小麦JW1品种中TaSnRK2.10基因编码区核苷酸序列。Fig. 3 is the nucleotide sequence of the TaSnRK2.10 gene coding region in wheat JW1 variety.

其中，下划线标注为TaSnRK2.10的sgRNA靶序列。Among them, the underline is marked as the sgRNA target sequence of TaSnRK2.10.

图4为pBUE411-TaSnRK2.10双元表达载体的测序结果。Fig. 4 is the sequencing result of pBUE411-TaSnRK2.10 binary expression vector.

其中，下划线标注为TaSnRK2.10的sgRNA靶序列，其余序列为pBUE411载体骨架。Among them, the underline marks the sgRNA target sequence of TaSnRK2.10, and the rest of the sequence is the pBUE411 vector backbone.

图5为T₀代小麦转基因株系鉴定结果。Figure 5 shows the identification results of transgenic wheat lines of the T₀ generation.

其中，泳道M为Transgen 2K plus marker，泳道1～15分别为T₀代小麦转基因株系，泳道16为野生型材料JW1，泳道17为双元表达载体pBUE411-TaSnRK2.10质粒。Among them, lane M is the Transgen 2K plus marker,lanes 1 to 15 are the T₀ generation wheat transgenic lines,lane 16 is the wild-type material JW1, andlane 17 is the binary expression vector pBUE411-TaSnRK2.10 plasmid.

图6为tasnrk2.10敲除株系基因编辑结果。Figure 6 shows the gene editing results of the tasnrk2.10 knockout strain.

图7为TaSnRK2.10-4A及编辑后的氨基酸序列比对结果。Fig. 7 is the comparison result of the amino acid sequence of TaSnRK2.10-4A and after editing.

图8为tasnrk2.10敲除株系CAPS鉴定基因扩增结果。Figure 8 shows the amplification results of the CAPS identification gene of the tasnrk2.10 knockout strain.

其中，泳道M为Transgen 2K plus marker，泳道1为野生型材料JW1，泳道2～6分别为T₁代小麦敲除株系TaSnRK2.10-4A扩增结果；泳道7为野生型材料JW1，泳道8～12分别为T₁代小麦敲除株系TaSnRK2.10-4B扩增结果；泳道13为野生型材料JW1，泳道14～18分别为T₁代小麦敲除株系TaSnRK2.10-4D扩增结果。Among them, lane M is the Transgen 2K plus marker,lane 1 is the wild-type material JW1,lanes 2 to 6 are the amplification results of the_T1 generation wheat knockout line TaSnRK2.10-4A;lane 7 is the wild-type material JW1, lane 8-12 are the amplification results of the_T1 generation wheat knockout line TaSnRK2.10-4B;lane 13 is the wild-type material JW1, and lanes 14-18 are the amplification results of the_T1 generation wheat knockout line TaSnRK2.10-4D. increase results.

图9为tasnrk2.10敲除株系CAPS鉴定酶切结果。Figure 9 shows the enzyme digestion results of CAPS identification of the tasnrk2.10 knockout strain.

其中，泳道M为Transgen 2K plus marker，泳道1为野生型材料JW1，泳道2～6分别为T₁代小麦敲除株系TaSnRK2.10-4A酶切结果；泳道7为野生型材料JW1，泳道8～12分别为T₁代小麦敲除株系TaSnRK2.10-4B酶切结果；泳道13为野生型材料JW1，泳道14～18分别为T₁代小麦敲除株系TaSnRK2.10-4D酶切结果。Among them, lane M is the Transgen 2K plus marker,lane 1 is the wild-type material JW1,lanes 2 to 6 are the enzyme digestion results of the_T1 generation wheat knockout line TaSnRK2.10-4A;lane 7 is the wild-type material JW1, lane 8-12 are the digestion results of TaSnRK2.10-4B of the_T1 generation wheat knockout line;lane 13 is the wild-type material JW1, and lanes 14-18 are the TaSnRK2.10-4D enzymes of the_T1 generation wheat knockout line Cut the result.

图10为野生型小麦JW1、纯和基因敲除株系tasnrk2.10植株形态和籽粒大小表型。Figure 10 shows the plant morphology and grain size phenotypes of wild-type wheat JW1, pure and gene knockout line tasnrk2.10.

其中，A，野生型小麦JW1、纯和基因敲除株系tasnrk2.10植株形态；B～G，tasnrk2.10的分蘖数、穗粒数、千粒重和单株产量相较于野生型小麦JW1有显著增加，株高和穗长无明显差异；H&I，与野生型小麦JW1相比，tasnrk2.10籽粒长度无明显变化。J&K，与野生型小麦JW1相比，tasnrk2.10籽粒宽度显著增加。Among them, A, the plant morphology of wild-type wheat JW1, pure and gene knockout line tasnrk2.10; B～G, the number of tillers, grains per ear, thousand-grain weight and yield per plant of tasnrk2.10 compared with wild-type wheat JW1 Significantly increased, with no significant difference in plant height and panicle length; H&I, no significant change in grain length in tasnrk2.10 compared with wild-type wheat JW1. J&K, tasnrk2.10 has significantly increased grain width compared to wild-type wheat JW1.

具体实施方式Detailed ways

下面结合附图和具体实施例对本发明内容进行详细说明。如下所述例子仅是本发明的较佳实施方式而已，应该说明的是，下述说明仅仅是为了解释本发明，并非对本发明作任何形式上的限制，凡是依据本发明的技术实质对实施方式所做的任何简单修改，等同变化与修饰，均属于本发明技术方案的范围内。即本发明保护内容应延伸理解为与本发明技术思想雷同的各种实施方式均属于本发明内容的范畴。The content of the present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The examples described below are only preferred implementation modes of the present invention. It should be noted that the following descriptions are only for explaining the present invention, and are not intended to limit the present invention in any form. Any simple modifications, equivalent changes and modifications all fall within the scope of the technical solutions of the present invention. That is to say, the protection content of the present invention should be extended to be understood as that various implementations similar to the technical idea of the present invention all belong to the scope of the content of the present invention.

下述实施例中的实验方法，如无特殊说明，均为常规方法。The experimental methods in the following examples are conventional methods unless otherwise specified.

下述实施例中所用的试验材料、试剂等，如无特殊说明，均从常规生化试剂公司购买得到。The test materials and reagents used in the following examples were all purchased from conventional biochemical reagent companies unless otherwise specified.

PCR扩增所需的高保真酶为KOD-FX NEO(东洋纺)；限制性核酸内切酶BsgI、吉布森组装所需的限制性内切酶BsaI与T4连接酶均购自NEB公司；酶切片段回收所需的凝胶回收试剂盒以及质粒提取试剂盒均购自赛默飞世尔科技公司。培养基配制所需的无机盐购自国药集团，维生素和抗生素购自Sigma公司。植物CRISPR/Cas9基因编辑载体为pBUE411，含有小麦U3启动子TaU3，用以启动sgRNA，Cas9模拟了禾本科植物基因具有5’端GC含量较高的特征，是设计合成的植物密码子优化的基因。所述质粒pBUE411公众可从中国农业大学获得或购自于上海柯雷生物科技有限公司，为17430bp。本发明所用的大肠杆菌菌株是E.coliTransgen5α，购自北京全式金公司；所使用的引物由青岛擎科梓熙生物技术有限公司合成，相关引物序列如表1所示：The high-fidelity enzyme required for PCR amplification is KOD-FX NEO (Toyobo); restriction endonuclease BsgI, restriction endonuclease BsaI and T4 ligase required for Gibson assembly were all purchased from NEB Company; The gel extraction kit and plasmid extraction kit required for fragment recovery were purchased from Thermo Fisher Scientific. Inorganic salts required for medium preparation were purchased from Sinopharm, and vitamins and antibiotics were purchased from Sigma. The plant CRISPR/Cas9 gene editing vector is pBUE411, which contains the wheat U3 promoter TaU3 to activate sgRNA. Cas9 mimics the characteristics of higher GC content at the 5' end of the grass gene, and is a plant codon-optimized gene designed and synthesized . The plasmid pBUE411 is publicly available from China Agricultural University or purchased from Shanghai Kelei Biotechnology Co., Ltd., and is 17430bp. The Escherichia coli strain used in the present invention is E.coliTransgen5α, which was purchased from Beijing Quanshijin Company; the primers used were synthesized by Qingdao Qingke Zixi Biotechnology Co., Ltd., and the relevant primer sequences are shown in Table 1:

表1：本发明使用的引物Table 1: Primers used in the present invention

本发明中使用的小麦品种JW1是山东省农科院作物所自行选育的具有良好组织培养能力的新种质，公众可从山东省农业科学院作物研究所获得。The wheat variety JW1 used in the present invention is a new germplasm with good tissue culture ability selected and bred by the Institute of Crops, Shandong Academy of Agricultural Sciences. The public can obtain it from the Institute of Crops, Shandong Academy of Agricultural Sciences.

实施例1重组双元表达载体pBUE411-TaSnRK2.10的构建Example 1 Construction of recombinant binary expression vector pBUE411-TaSnRK2.10

1.TaSnRK2.10进化分析1. Evolutionary analysis of TaSnRK2.10

以TaSnRK2.10-4A(TraesCS4A02G235600)的蛋白质序列在Ensemble Plant网站(http://plants.ensembl.org/index.html)进行Blast-P序列比对，搜索的数据库包括：拟南芥(TAIR10)，小麦(IWGSC)，水稻(IRGSP-1.0)和玉米(Zm-B73-REFERENCE-NAM-5.0)，获得比对结果后，选择同源性较高的基因，并下载其FASTA格式的蛋白质序列。Use the protein sequence of TaSnRK2.10-4A (TraesCS4A02G235600) to perform Blast-P sequence alignment on the Ensemble Plant website (http://plants.ensembl.org/index.html). The searched databases include: Arabidopsis thaliana (TAIR10) , wheat (IWGSC), rice (IRGSP-1.0) and maize (Zm-B73-REFERENCE-NAM-5.0), after obtaining the alignment results, select genes with higher homology and download their protein sequences in FASTA format.

用MEGA7软件进行ClustalW比对分析，并构建neighbor-joining树，结果如图1所示。TraesCS4A02G235600，TraesCS4B02G079300和TraesCS4D02G078100三个基因与OsSAPK10同源性最高，Guo等人(2019)报道Os03g0610900(SAPK10)为拟南芥中OST1的同源基因，故构建同时编辑三个基因拷贝的CRISPR/Cas9株系。The ClustalW comparison analysis was performed with MEGA7 software, and the neighbor-joining tree was constructed. The results are shown in Figure 1. The three genes TraesCS4A02G235600, TraesCS4B02G079300 and TraesCS4D02G078100 have the highest homology with OsSAPK10. Guo et al. (2019) reported that Os03g0610900 (SAPK10) is the homologous gene of OST1 in Arabidopsis, so a CRISPR/Cas9 strain that simultaneously edits three gene copies was constructed Tie.

2.靶向TaSnRK2.10的sgRNA的设计2. Design of sgRNA targeting TaSnRK2.10

为设计能够在TaSnRK2.10基因编码区发生编辑的sgRNA，本实验首先在小麦品种JW1里分别扩增TaSnRK2.10-4A/4B/4D基因的序列。由于TaSnRK2.10-4A/4B/4D的序列高度保守，难以直接扩增，因此本实施例采用巢式PCR技术方法扩增，具体实施方法如下：In order to design sgRNA capable of editing in the coding region of TaSnRK2.10 gene, this experiment first amplified the sequence of TaSnRK2.10-4A/4B/4D gene in wheat variety JW1. Since the sequence of TaSnRK2.10-4A/4B/4D is highly conserved, it is difficult to amplify directly, so this embodiment adopts nested PCR technique for amplification, and the specific implementation method is as follows:

首先扩增Outer片段，30μL PCR反应体系为：15μL 2×KOD-FX Buffer；6μL 2mMdNTP；0.9μL 10μM TaSnRK2.10-4A/4B/4D-OF；0.9μL 10μM TaSnRK2.10-4A/4B/4D-OR；1μLJW1cDNA；1μL KOD-FX DNA Polymerase；5.2μL ddH₂O。待各成分充分混匀后，离心数秒，使反应混合物沉到管底，然后将反应管置于PCR仪中进行扩增。PCR反应程序为：95℃预变性3min(Step 1)，95℃变性20s(Step 2)，58℃退火20s(Step 3)，72℃延伸1min(Step 4)，Step 2～4反应35个循环，72℃复性5min，4℃保存。向PCR产物中加入6*上样缓冲液进行琼脂糖凝胶电泳检测，TaSnRK2.10-4A/B/D-Outer条带大小分别为：1482bp、1757bp、1411bp。切取对应大小的单一条带，使用thermo GeneJET Gel Extraction Kit回收DNA片段。Firstly amplify the Outer fragment, the 30μL PCR reaction system is:15μL 2×KOD-FX Buffer; 6μL 2mMdNTP; 0.9μL 10μM TaSnRK2.10-4A/4B/4D-OF; 0.9μL 10μM TaSnRK2.10-4A/4B/4D -OR; 1 μL JW1 cDNA; 1 μL KOD-FX DNA Polymerase; 5.2 μL ddH₂ O. After the components are fully mixed, centrifuge for a few seconds to allow the reaction mixture to sink to the bottom of the tube, and then place the reaction tube in a PCR instrument for amplification. The PCR reaction program is: pre-denaturation at 95°C for 3 min (Step 1), denaturation at 95°C for 20 s (Step 2), annealing at 58°C for 20 s (Step 3), extension at 72°C for 1 min (Step 4), and 35 cycles of reaction at Step 2-4 , renatured at 72°C for 5 minutes, and stored at 4°C. Add 6* loading buffer to the PCR product for agarose gel electrophoresis detection, and the band sizes of TaSnRK2.10-4A/B/D-Outer are: 1482bp, 1757bp, 1411bp respectively. Cut out a single band of the corresponding size, and use the thermo GeneJET Gel Extraction Kit to recover the DNA fragments.

分别以TaSnRK2.10-4A/4B/4D的Outer片段为模板进行Inner片段扩增，30μL PCR反应体系为：15μL 2×KOD-FX Buffer；6μL 2mM dNTP；0.9μL 10μM TaSnRK2.10-IF；0.9μL10μM TaSnRK2.10-IR；1μL Outer fragment；1μL KOD-FX DNA Polymerase；5.2μL ddH₂O。待各成分充分混匀后，离心数秒，使反应混合物沉到管底，然后将200μL PCR管置于PCR仪中进行扩增。PCR反应程序为：95℃预变性3min(Step 1)，95℃变性20s(Step 2)，58℃退火20s(Step 3)，72℃延伸1min(Step 4)，Step 2～4反应35个循环，72℃复性5min，4℃保存。向PCR产物中加入6*上样缓冲液进行琼脂糖凝胶电泳检测，结果如图2所示，泳道M为Transgen2K plus marker，泳道a、b和c分别为TaSnRK2.10-4A/4B/4D的目的条带。切取目的条带送至青岛睿博兴科生物技术有限公司测序，测序结果表明TaSnRK2.10-4A/4B/4D在JW1中的序列与CS参考基因组序列相似性高达100％。Inner fragments were amplified using the Outer fragments of TaSnRK2.10-4A/4B/4D as templates. The 30 μL PCR reaction system was: 15μL 2×KOD-FX Buffer; 6 μL 2mM dNTPs; 0.9μL 10 μM TaSnRK2.10-IF; 0.9μL 10 μM TaSnRK2.10-IR; 1 μL Outer fragment; 1 μL KOD-FX DNA Polymerase; 5.2 μL ddH₂ O. After the components are fully mixed, centrifuge for a few seconds to allow the reaction mixture to sink to the bottom of the tube, and then place the 200 μL PCR tube in a PCR instrument for amplification. The PCR reaction program is: pre-denaturation at 95°C for 3 min (Step 1), denaturation at 95°C for 20 s (Step 2), annealing at 58°C for 20 s (Step 3), extension at 72°C for 1 min (Step 4), and 35 cycles of reaction at Step 2-4 , renatured at 72°C for 5 minutes, and stored at 4°C. Add 6* loading buffer to the PCR product for agarose gel electrophoresis detection, the results are shown in Figure 2, lane M is the Transgen2K plus marker, and lanes a, b and c are TaSnRK2.10-4A/4B/4D respectively the purpose strip. The target bands were cut and sent to Qingdao Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the sequence of TaSnRK2.10-4A/4B/4D in JW1 was as high as 100% similar to the CS reference genome sequence.

下载TraesCS4A02G235600，TraesCS4B02G079300和TraesCS4D02G078100三个基因的基因组序列，在CRISPRdirect网站(http://crispr.dbcls.jp/)进行靶序列预测，选择IWGSC小麦基因组数据库。从生成的结果数据中，筛选出符合条件的靶序列信息。筛选条件如下：Download the genome sequences of the three genes TraesCS4A02G235600, TraesCS4B02G079300 and TraesCS4D02G078100, perform target sequence prediction on the CRISPRdirect website (http://crispr.dbcls.jp/), and select the IWGSC wheat genome database. From the generated result data, the qualified target sequence information is screened out. The filter criteria are as follows:

a.GC含量45～70％为宜；a. The appropriate GC content is 45-70%;

b.5’端开头为A的靶序列，以适应U3启动子的转录起始；b. The target sequence at the beginning of the 5' end is A, to adapt to the transcription initiation of the U3 promoter;

c.选择包含PAM区上游第3、4两个碱基处的酶切位点，便于后续的酶切鉴定；c. Select a restriction site that includes the 3rd and 4th bases upstream of the PAM region to facilitate subsequent restriction identification;

d.选择靠近基因5’端的靶序列，以增加编辑后对基因的破坏程度，同时兼顾靶序列位置是否位于该基因蛋白的关键结构域；d. Select the target sequence close to the 5' end of the gene to increase the degree of damage to the gene after editing, while taking into account whether the target sequence is located in the key structural domain of the gene protein;

e.选择20mer脱靶数为3的靶序列，以便于敲除ABD三个基因组中的基因。e. Select the target sequence with 20mer off-target number of 3, so as to knock out the genes in the three genomes of ABD.

f.将靶序列与小麦基因组数据库进行BLAST分析，进一步验证靶序列的特异性。f. Perform BLAST analysis on the target sequence and the wheat genome database to further verify the specificity of the target sequence.

最终本实施例共找到4个合适的靶序列，选择靠近激酶结构域N端的一条靶序列进行后续分析，其核苷酸序列信息如SEQ ID NO.5所示，具体核苷酸序列是：AGATTGACGAGAACGTGCAGCGG，下划线标记为靶向TaSnRK2.10基因的sgRNA序列，序列长度为20bp，其3’端的CGG为PAM序列(图3)。Finally, four suitable target sequences were found in this example, and a target sequence close to the N-terminus of the kinase domain was selected for subsequent analysis. The nucleotide sequence information is shown in SEQ ID NO.5, and the specific nucleotide sequence is: AGATTGACGAGAACGTGCAGCGG , the underline marks the sgRNA sequence targeting the TaSnRK2.10 gene, the sequence length is 20bp, and the CGG at the 3' end is the PAM sequence (Figure 3).

3.含有sgRNA片段的扩增3. Amplification of sgRNA-containing fragments

用于构建pBUE411-TaSnRK2.10双元表达载体的2条引物TaSnRK2.10-gR1-F和TaSnRK2.10-gR1-R(表1)，由青岛睿博兴科生物技术有限公司合成。分别将两条引物进行磷酸化，直接退火形成双链，反应体系为：4μL TaSnRK2.10-gR1-F(10μM)，4μLTaSnRK2.10-gR1-R(10μM)，1.5μL 10×T4 PNK buffer，1μL PNK，1μL ATP，ddH₂O补足至15μL。混匀各成分后瞬离，置于PCR仪中反应，反应程序为：37℃，30min；95℃，5min；ramp to 25℃at 5℃/min。The two primers TaSnRK2.10-gR1-F and TaSnRK2.10-gR1-R (Table 1) used to construct the pBUE411-TaSnRK2.10 binary expression vector were synthesized by Qingdao Ruibo Xingke Biotechnology Co., Ltd. Phosphorylate the two primers and directly anneal to form double strands. The reaction system is: 4 μL TaSnRK2.10-gR1-F (10 μM), 4 μL TaSnRK2.10-gR1-R (10 μM), 1.5 μL 10×T4 PNK buffer, 1 μL PNK, 1 μL ATP, ddH₂ O make up to 15 μL. After mixing the components, they were separated and placed in a PCR instrument for reaction. The reaction program was: 37°C, 30min; 95°C, 5min; ramp to 25°C at 5°C/min.

4.sgRNA与pBUE411载体连接4. sgRNA is connected to pBUE411 vector

采用吉布森组装完成pBUE411与sgRNA的连接，具体反应体系：2μL pBUE411质粒(100ng/μL)，2μL步骤3的产物片段，1.5μL 10×NEB T4 Buffer，1.5μL 10×BSA，1μL BsaI，1μL T4 Ligase，ddH₂O补齐15μL。混匀后置于37℃恒温水浴锅中反应5小时，得到pBUE411载体与sgRNA连接产物。Use Gibson assembly to complete the connection between pBUE411 and sgRNA. The specific reaction system: 2 μL pBUE411 plasmid (100ng/μL), 2 μL the product fragment ofstep 3, 1.5 μL 10×NEB T4 Buffer, 1.5 μL 10×BSA, 1 μL BsaI, 1 μL T4 Ligase , ddH₂ O to make up 15 μL. After mixing, put it in a constant temperature water bath at 37°C for 5 hours to react to obtain the pBUE411 carrier and sgRNA ligation product.

5.转化与鉴定5. Transformation and identification

将连接产物转化大肠杆菌，具体方案如下：将15μL连接产物加入大肠杆菌感受态Transgen 5α中冰浴30min，42℃热激45s后迅速冰浴2min，加入无抗生素的液体LB培养基，37℃摇床复苏40min，之后用涂布器涂于含有卡那霉素的固体LB平板上，37℃倒置培养12h，挑3个单克隆进行测序，测序引物为pBUE411-F和pBUE411-R。The ligation product was transformed into Escherichia coli, and the specific scheme was as follows: add 15 μL of the ligation product to E. coli competent Transgen 5α and ice-bath for 30 min, heat shock at 42°C for 45 s, then quickly ice-bath for 2 min, add liquid LB medium without antibiotics, and shake at 37°C The bed was revived for 40 minutes, and then spread on a solid LB plate containing kanamycin with a spreader, cultured upside down at 37°C for 12 hours, and three single clones were picked for sequencing, and the sequencing primers were pBUE411-F and pBUE411-R.

测序结果(如图4)检测到了sgRNA的靶序列，同时在靶序列上游检测到了TaU3启动子序列，测序结果说明含有sgRNA的表达盒E1成功组装入pBUE411双元表达载体，证明了TaSnRK2.10的CRISPR/Cas9基因编辑载体即重组双元表达载体pBUE411-TaSnRK2.10构建成功。The sequencing results (as shown in Figure 4) detected the target sequence of the sgRNA, and at the same time detected the TaU3 promoter sequence upstream of the target sequence. The sequencing results showed that the expression cassette E1 containing the sgRNA was successfully assembled into the pBUE411 binary expression vector, proving the function of TaSnRK2.10. The CRISPR/Cas9 gene editing vector, the recombinant binary expression vector pBUE411-TaSnRK2.10, was successfully constructed.

实施例2转基因后代获得与鉴定Example 2 Acquisition and identification of transgenic offspring

1.TaSnRK2.10转基因后代获得1. TaSnRK2.10 transgenic offspring obtained

将实施例1构建好的重组双元表达载体pBUE411-TaSnRK2.10转化至农杆菌EHA105感受态细胞，EHA105菌株携带一无自身转运功能的琥珀碱型Ti质粒pEHA105(pTiBo542DT-DNA)，此质粒含有vir基因，从而使该菌株在质粒自身的T-DNA转移功能被破坏的前提下，能够帮助转入的双元载体T-DNA顺利转移至植物体内。The recombinant binary expression vector pBUE411-TaSnRK2.10 constructed in Example 1 was transformed into Agrobacterium EHA105 competent cells, and the EHA105 strain carried a succinic alkali type Ti plasmid pEHA105 (pTiBo542DT-DNA) without self-transport function, which contained vir gene, so that under the premise that the T-DNA transfer function of the plasmid itself is destroyed, the bacterial strain can help the transferred binary vector T-DNA to transfer smoothly to the plant body.

具体实施方案如下：The specific implementation plan is as follows:

取-80℃保存的EHA105农杆菌感受态细胞于冰水浴中融化，在无菌条件下向感受态细胞中加入1μg质粒DNA，轻轻混匀，冰水浴中静置5min。将离心管置于液氮中速冻5min，快速将离心管置于37℃水浴中保持5min，期间不要晃动水面，再将离心管放置于冰水浴中静置5min。无菌条件下加入800μL无抗生素的YEB培养基，于28℃振荡培养2～3h使菌体复苏。5000rpm离心3min弃上清收集菌体，加入100μL无菌水，轻轻吹打重悬菌体，涂布于相应抗生素、利福平和链霉素的YEB平板上，28℃培养箱中倒置培养2～3天。Take the EHA105 Agrobacterium competent cells stored at -80°C and melt them in an ice-water bath, add 1 μg of plasmid DNA to the competent cells under sterile conditions, mix gently, and let stand in the ice-water bath for 5 minutes. Quickly freeze the centrifuge tube in liquid nitrogen for 5 minutes, quickly place the centrifuge tube in a 37°C water bath for 5 minutes without shaking the water surface, and then place the centrifuge tube in an ice-water bath for 5 minutes. Add 800 μL of antibiotic-free YEB medium under aseptic conditions, shake and culture at 28°C for 2-3 hours to recover the bacteria. Centrifuge at 5000rpm for 3 minutes, discard the supernatant to collect the bacteria, add 100 μL of sterile water, gently blow and blow the resuspended bacteria, spread on the YEB plate of the corresponding antibiotics, rifampicin and streptomycin, and culture it upside down in a 28°C incubator for 2~ 3 days.

小麦遗传转化培养基配制：见文献Kan Wang(ed.),Agrobacterium Protocals:Volume 1,Methods in Molecular Biology,vol.1223DOI10.007/978-1-4939-1695-5_15,Spring Science+Businedd Media New York 2015。Wheat genetic transformation medium preparation: see literature Kan Wang (ed.), Agrobacterium Protocols:Volume 1, Methods in Molecular Biology, vol.1223DOI10.007/978-1-4939-1695-5_15, Spring Science+Busined Media New York 2015.

取开花后10～15天的JW1小麦种子，在无菌环境下剥取幼胚。吸取1ml农杆菌悬浮液于1.5ml EP管中，加入1.4ul乙酰丁香酮(0.1M)混匀。加入准备好的菌液侵染5分钟后放到共培培养基上，23℃暗培养3天。共培后放到休息培养基上25℃暗培养5天。将愈伤组织转移至筛选培养基1上，用封口膜封好培养皿，在25.5℃培养箱中暗培养2周。将愈伤切割后转移到筛选培养基2上，用封口膜再次封好培养皿，在25.5℃培养箱中继续暗培养2周。愈伤切割筛选2周后，表现有绿色芽点的抗性愈伤转移到再生培养基上。封好培养皿，放到25℃培养箱中光照/黑暗(16h/8h)培养2周。再生2周后，将健康成长的小苗转移到新的抗性再生小盒里。待苗长到一定大小可以取样检测。The JW1 wheat seeds 10-15 days after flowering were taken, and the immature embryos were peeled off under a sterile environment. Pipette 1ml of Agrobacterium suspension into a 1.5ml EP tube, add 1.4ul acetosyringone (0.1M) and mix well. Add the prepared bacterial solution to infect for 5 minutes, put it on the co-cultivation medium, and incubate in the dark at 23°C for 3 days. After co-cultivation, they were cultured in the dark at 25°C for 5 days on rest medium. Transfer the callus to thescreening medium 1, seal the culture dish with a parafilm, and culture it in the dark at 25.5°C for 2 weeks. After the callus was cut, it was transferred to thescreening medium 2, and the petri dish was sealed again with a parafilm, and the culture was continued in the dark at 25.5°C for 2 weeks in an incubator. Two weeks after callus excision selection, resistant calli exhibiting green bud spots were transferred to regeneration medium. Seal the culture dish and put it in a light/dark (16h/8h) incubator at 25°C for 2 weeks. After 2 weeks of regrowth, transfer healthy seedlings to new resistant regrowth boxes. When the seedlings grow to a certain size, samples can be taken for testing.

取再生小麦幼嫩叶片，CTAB法提取基因组DNA，利用BUE-DF1与BUE-DR1两个载体上的引物进行PCR鉴定，PCR反应程序为：10μL 2×PCR master mix，0.5μL 10μM pBUE411-F，0.5μL 10μM pBUE411-R，1μL gDNA(50ng/μL)，8μL ddH₂O。PCR反应程序为：95℃预变性5min，95℃变性30sec；58℃退火30sec，72℃延伸30sec，反应32个循环；72℃复性5min。结果如图5所示，条带代表转化成功的T₀代小麦转基因株系，共得到27株阳性转基因株系。The young leaves of regenerated wheat were taken, and genomic DNA was extracted by CTAB method. The primers on the two vectors BUE-DF1 and BUE-DR1 were used for PCR identification. The PCR reaction program was: 10μL 2×PCR master mix, 0.5μL 10 μM pBUE411-F, 0.5μL 10 μM pBUE411-R, 1 μL gDNA (50 ng/μL), 8 μL ddH₂ O. The PCR reaction program was: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 sec; annealing at 58°C for 30 sec, extension at 72°C for 30 sec, and 32 cycles of reaction; renaturation at 72°C for 5 min. The results are shown in Figure 5, the bands represent the successfully transformed T₀ generation wheat transgenic lines, and a total of 27 positive transgenic lines were obtained.

2.TaSnRK2.10转基因敲除后代鉴定2. Identification of TaSnRK2.10 transgene knockout offspring

本实施例设计的靶序列能够直接靶向编辑A/B/D三个基因组上的同源基因，因此需同时检测TaSnRK2.10-4A/B/D三个同源拷贝的基因编辑情况。本实验采用购自西安青雪生物科技有限公司的Hi-TOM基因编辑位点检测试剂盒，该试剂盒通过PCR完成高通量建库过程，并用Hi-TOM在线软件直接解析出多样品多位点的变异信息。The target sequence designed in this example can directly target and edit the homologous genes on the three genomes of A/B/D, so it is necessary to simultaneously detect the gene editing of the three homologous copies of TaSnRK2.10-4A/B/D. In this experiment, the Hi-TOM gene editing site detection kit purchased from Xi'an Qingxue Biotechnology Co., Ltd. was used. The kit completed the high-throughput library construction process by PCR, and directly analyzed the multiple samples and multiple sites with Hi-TOM online software. Point variation information.

本实施例分别针对sgRNA1靶序列设计了特异性引物(即表1中的tasnrk2.10-gRNA1-F、tasnrk2.10-gRNA1-R)，能够同时扩增基因TaSnRK2.10-4A、TaSnRK2.10-4B和TaSnRK2.10-4D，另外，扩增片段含有SNP序列能够区分ABD基因组，扩增产物建库后送到西安青雪生物科技有限公司测序。In this example, specific primers (namely tasnrk2.10-gRNA1-F and tasnrk2.10-gRNA1-R in Table 1) were designed for the sgRNA1 target sequence, which can simultaneously amplify the genes TaSnRK2.10-4A and TaSnRK2.10 -4B and TaSnRK2.10-4D. In addition, the amplified fragments contain SNP sequences that can distinguish the ABD genome.

以tasnrk2.10小麦敲除突变体植株叶片DNA为模板进行第一轮PCR反应体系：1μLgDNA(约20ng/μL)，10μL 2×Taq Master Mix，0.5μL 10μM Seq-F和Seq-R(表1)，sterileWater补足至20μL。PCR反应条件为：94℃预变性2min；94℃变性30s，64℃退火30s，72℃延伸20s，共32个循环；最后72℃延伸5min。PCR结束后取5μL琼脂糖凝胶电泳检测PCR产物，确保目标产物存在且特异性良好。之后进行第二轮PCR反应：12μL Hi-TOM Mix，1μL第一轮PCR产物，sterile Water补足体积至20μL。PCR反应程序：94℃变性2min；94℃变性30s，58℃退火30s，72℃延伸25s，共33个循环；最后72℃延伸5min。扩增产物混合进行胶回收，胶回收产物即为建库测序样本，随后送到西安青雪生物科技有限公司测序。Using tasnrk2.10 wheat knockout mutant plant leaf DNA as a template for the first round of PCR reaction system: 1 μL gDNA (about 20 ng/μL), 10μL 2×Taq Master Mix, 0.5μL 10 μM Seq-F and Seq-R (Table 1 ), the sterileWater was made up to 20μL. The PCR reaction conditions were: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 64°C for 30 s, and extension at 72°C for 20 s, a total of 32 cycles; finally, extension at 72°C for 5 min. After PCR, 5 μL of agarose gel electrophoresis was used to detect the PCR product to ensure the presence of the target product and good specificity. Then carry out the second round of PCR reaction: 12 μL Hi-TOM Mix, 1 μL of the first round of PCR product, and sterile Water to make up the volume to 20 μL. PCR reaction program: denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 25 s, a total of 33 cycles; finally extension at 72°C for 5 min. The amplified products were mixed for gel recovery, and the gel recovery products were the samples for library construction and sequencing, which were then sent to Xi'an Qingxue Biotechnology Co., Ltd. for sequencing.

测序结果发现共有3个三编辑转基因株系，均发生插入或缺失导致的移码突变，TaSnRK2.10-4A、TaSnRK2.10-4B和TaSnRK2.10-4D的基因编辑结果如图6所示，经过比对发现突变体蛋白tasnrk2.10-4A/B/D均自第64个氨基酸开始出现移码。以1^#株系为例，其TaSnRK2.10-4A编码的氨基酸序列如图7所示。结果表明，sgRNA与Cas9元件转化成功，并发挥了功能，对TaSnRK2.10基因进行了编辑，导致TaSnRK2.10-4A、TaSnRK2.10-4B和TaSnRK2.10-4D三个蛋白移码突变，保守的激酶结构功能域不能正确表达，基因相应的功能丧失。The sequencing results revealed that there were three triple-edited transgenic lines, all of which had frameshift mutations caused by insertions or deletions. The gene editing results of TaSnRK2.10-4A, TaSnRK2.10-4B and TaSnRK2.10-4D are shown in Figure 6. After comparison, it was found that the mutant protein tasnrk2.10-4A/B/D had a frame shift from the 64th amino acid. Takingstrain 1^# as an example, the amino acid sequence encoded by its TaSnRK2.10-4A is shown in FIG. 7 . The results showed that the transformation of sgRNA and Cas9 elements was successful and functioned, and the TaSnRK2.10 gene was edited, resulting in three protein frameshift mutations, TaSnRK2.10-4A, TaSnRK2.10-4B and TaSnRK2.10-4D, which were conserved The kinase domain cannot be expressed correctly, and the corresponding function of the gene is lost.

实施例3小麦敲除TaSnRK2.10基因后表型鉴定Example 3 Phenotypic Identification of Wheat After Knockout of TaSnRK2.10 Gene

为得到TaSnRK2.10-4A、TaSnRK2.10-4B和TaSnRK2.10-4D三个同源拷贝的纯合敲除株系，针对编辑位点开发酶切扩增多态性序列(CAPS)标记，对基因编辑突变体的自交后代进行鉴定，直至得到TaSnRK2.10-4A、TaSnRK2.10-4B和TaSnRK2.10-4D同时敲除的纯合突变体。In order to obtain homozygous knockout lines with three homologous copies of TaSnRK2.10-4A, TaSnRK2.10-4B and TaSnRK2.10-4D, an enzyme cleavage amplified polymorphic sequence (CAPS) marker was developed for the editing site, The selfed offspring of the gene editing mutants were identified until homozygous mutants with simultaneous knockout of TaSnRK2.10-4A, TaSnRK2.10-4B and TaSnRK2.10-4D were obtained.

分别利用TaSnRK2.10_4A-OF和cr210A-R，TaSnRK2.10_4B-OF和cr210B-R，TaSnRK2.10_4D-OF和cr210D-R(表1)对基因编辑后代的TaSnRK2.10-4A、TaSnRK2.10-4B和TaSnRK2.10-4D基因进行PCR扩增。PCR反应体系为：15μL KOD-FX Neo buffer，6μL 2mMdNTP，0.9μL 10μM TaSnRK2.10_4A-OF(或TaSnRK2.10_4B-OF或TaSnRK2.10_4D-OF)，0.9μL10μM cr210A-R(或cr210B-R或cr210D-R)，1μL gDNA(约20ng/μL)，0.3μL KOD-FX NEO，ddH₂O补齐至30μL。PCR反应程序为：98℃预变性2min，98℃变性12s；58℃退火20s，68℃延伸45s，反应35个循环；68℃复性5min，4℃保存。The TaSnRK2.10-4A, TaSnRK2.10- 4B and TaSnRK2.10-4D genes were amplified by PCR. The PCR reaction system is: 15 μL KOD-FX Neo buffer, 6 μL 2mMdNTP, 0.9μL 10 μM TaSnRK2.10_4A-OF (or TaSnRK2.10_4B-OF or TaSnRK2.10_4D-OF), 0.9μL 10 μM cr210A-R (or cr210B-R or cr210D -R), 1 μL gDNA (about 20 ng/μL), 0.3 μL KOD-FX NEO, and ddH₂ O to make up to 30 μL. The PCR reaction program was as follows: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 12 s; annealing at 58°C for 20 s, extension at 68°C for 45 s, and 35 cycles of reaction; renaturation at 68°C for 5 min, and storage at 4°C.

PCR结束后取5ul PCR产物进行琼脂糖凝胶电泳电泳检测，检测结果如图7所示。After the PCR was completed, 5 ul of the PCR product was taken for agarose gel electrophoresis detection, and the detection results are shown in FIG. 7 .

扩增产物使用美国New England Biolabs,Inc.(NEB)的限制性内切酶BsgI进行酶切，酶切反应体系为：15μL PCR产物，2μL 10×NEB Buffer，0.5μL限制性内切酶BsgI。反应体系于37℃水浴锅中酶切1h 30min，之后立即进行琼脂糖凝胶电泳检测，结果如图8所示。电泳鉴定结果中的野生型基因均可被切开，基因编辑成功的位点均不能被切开。对于sgRNA1，野生型植株的TaSnRK2.10-4A、TaSnRK2.10-4B和TaSnRK2.10-4D可被切割为413bp+246bp、599bp+278bp、413bp+264bp大小的片段，而基因编辑突变体不能被切开。由此可筛选出转基因纯合三拷贝基因敲除株系tasnrk2.10。The amplified product was digested with restriction endonuclease BsgI from New England Biolabs, Inc. (NEB), USA. The enzyme digestion reaction system was: 15 μL PCR product, 2 μL 10×NEB Buffer, 0.5 μL restriction endonuclease BsgI. The reaction system was digested in a water bath at 37°C for 1h 30min, and then immediately performed agarose gel electrophoresis detection, the results are shown in Figure 8. All the wild-type genes in the electrophoresis identification results can be cut, and none of the successfully edited genes can be cut. For sgRNA1, TaSnRK2.10-4A, TaSnRK2.10-4B and TaSnRK2.10-4D of wild-type plants could be cut into 413bp+246bp, 599bp+278bp, 413bp+264bp fragments, while the gene editing mutants could not be cut. cut open. Thus, the transgenic homozygous three-copy gene knockout line tasnrk2.10 can be screened out.

分别将野生型受体品种JW1和筛选得到的纯合敲除株系tasnrk2.10种植于山东大学青岛校区温室(120.41°E，36.07°N)，培养条件为：光照/黑暗＝16/8h；白天温度22℃，夜间温度16℃；湿度介于40％～50％之间；CO₂浓度为500ppm～700ppm。灌浆期间统计小麦株高和分蘖数和主穗长，使用佳能高性能单反照相机对野生型和敲除突变体的植株形态进行拍照。待蜡熟期的中晚期之时，单株收获小麦，将穗部和小穗的形态进行拍照，统计小穗数。将收获的小麦种子置于37℃恒温培养箱中持续烘干14d，拍照并统计JW1和tasnrk2.10小麦籽粒的长度和宽度，最后对单株小麦籽粒称重并使用光电自动数粒仪统计粒数，以便于单株产量和千粒重的计算。结果表明纯和敲除突变体的株高、主穗长和粒长与野生型相比差异不显著，而分蘖数、穗粒数、籽粒宽度、千粒重和单株产量相较于野生型JW1均有显著上升。上述结果表明TaSnRK2.10对于小麦的分蘖数、穗粒数和籽粒发育具有负向调控作用。The wild-type recipient variety JW1 and the screened homozygous knockout line tasnrk2.10 were planted in the greenhouse of Qingdao Campus of Shandong University (120.41°E, 36.07°N), and the culture conditions were: light/dark=16/8h; The daytime temperature is 22°C and the nighttime temperature is 16°C; the humidity is between 40% and 50%; the_CO2 concentration is 500ppm to 700ppm. During the grain filling period, the wheat plant height, tiller number and main panicle length were counted, and the plant morphology of the wild type and knockout mutants were photographed with a Canon high-performance SLR camera. At the middle and late stages of the wax ripening period, the wheat was harvested from a single plant, and the morphology of the ears and spikelets was photographed, and the number of spikelets was counted. The harvested wheat seeds were placed in a 37°C constant temperature incubator and dried continuously for 14 days, and the length and width of JW1 and tasnrk2.10 wheat kernels were counted by taking pictures. Number, in order to calculate the yield per plant and 1000-grain weight. The results showed that the plant height, main panicle length and grain length of the pure and knockout mutants were not significantly different from those of the wild type, while the tiller number, grain number per panicle, grain width, thousand-grain weight and yield per plant were significantly lower than those of the wild type JW1. There was a significant increase. The above results indicated that TaSnRK2.10 had a negative regulatory effect on tiller number, grain number per panicle and grain development of wheat.

本发明通过构建含有sgRNA并能够靶向TaSnRK2.10的pBUE411-TaSnRK2.10双元重组载体，利用农杆菌侵染小麦幼胚诱导愈伤组织，特异性靶向编辑TaSnRK2.10基因，使其在激酶结构域处发生移码突变进而丧失功能，显著增加了小麦的分蘖数、穗粒数、籽粒宽度和产量，为培育高产密植小麦提供了新的手段。The present invention constructs the pBUE411-TaSnRK2.10 binary recombination vector containing sgRNA and capable of targeting TaSnRK2.10, uses Agrobacterium to infect young wheat embryos to induce callus, and specifically targets and edits the TaSnRK2.10 gene so that it can be A frameshift mutation occurred in the kinase domain and then lost its function, which significantly increased the number of tillers, grains per panicle, grain width and yield of wheat, providing a new method for cultivating high-yield densely planted wheat.

Claims (8)

Translated fromChinese

1.利用基因编辑敲除TaSnRK2.10增加小麦分蘖数、穗粒数和籽粒宽度的应用。1. The use of gene editing to knock out TaSnRK2.10 to increase the number of tillers, grains per panicle and grain width in wheat.2.根据权利要求1所述的应用，其特征在于，应用方法是：设计靶向TaSnRK2.10基因的sgRNA，即基因编辑技术获得小麦TaSnRK2.10基因敲除突变体的特异性引导单链RNA，构建含有sgRNA的pBUE411-TaSnRK2.10双元重组载体，利用农杆菌侵染小麦幼胚诱导的愈伤组织，将sgRNA与Cas9元件共同转入小麦，特异性敲除TaSnRK2.10基因，获得TaSnRK2.10基因敲除的转基因小麦，即得到分蘖数、穗粒数与籽粒宽度增加的小麦tasnrk2.10敲除突变体。2. The application according to claim 1, characterized in that the application method is: designing the sgRNA targeting the TaSnRK2.10 gene, that is, the gene editing technology to obtain the specific guide single-stranded RNA of the wheat TaSnRK2.10 gene knockout mutant , to construct the pBUE411-TaSnRK2.10 binary recombinant vector containing sgRNA, use Agrobacterium to infect the callus induced by wheat immature embryos, transfer sgRNA and Cas9 elements into wheat, and specifically knock out the TaSnRK2.10 gene to obtain TaSnRK2 .10 gene knockout transgenic wheat, that is, a wheat tasnrk2.10 knockout mutant with increased tiller number, panicle number and grain width.3.根据权利要求2所述的应用，其特征在于：所述靶向TaSnRK2.10基因的sgRNA属于TaSnRK2.10编码序列或TaSnRK2.10编码序列的互补序列，长度为22～23个脱氧核糖核苷酸，其3’端最后三个脱氧核苷酸序列为NGG，其中N为腺嘌呤或鸟嘌呤或胸腺嘧啶或胞嘧啶，其核苷酸序列如SEQ ID NO.5、或SEQ ID NO.6、或SEQ ID NO.7、或SEQ ID NO.8所示。3. The application according to claim 2, characterized in that: the sgRNA targeting the TaSnRK2.10 gene belongs to the TaSnRK2.10 coding sequence or the complementary sequence of the TaSnRK2.10 coding sequence, and has a length of 22 to 23 deoxyribose nuclei Nucleotide, the last three deoxynucleotide sequences of its 3' end are NGG, wherein N is adenine or guanine or thymine or cytosine, and its nucleotide sequence is as SEQ ID NO.5, or SEQ ID NO. 6. Or shown in SEQ ID NO.7, or SEQ ID NO.8.4.根据权利要求3所述的应用，其特征在于：所述靶向TaSnRK2.10基因的sgRNA的核苷酸序列如SEQ ID NO.5所示。4. The application according to claim 3, characterized in that: the nucleotide sequence of the sgRNA targeting the TaSnRK2.10 gene is shown in SEQ ID NO.5.5.根据权利要求2所述的应用，其特征在于：所述pBUE411-TaSnRK2.10双元重组载体由表达盒E1和表达盒E2构成，其核苷酸序列如SEQ ID NO.3所示；其中所述表达盒E1的核苷酸序列如SEQ ID NO.1所示，其从上游至下游依次包括：来自小麦的TaU3启动子，TaSnRK2.10基因的sgRNA，终止子T1；所述表达盒E2的核苷酸序列如SEQ ID NO.2所示，其从上游至下游依次包括：来自玉米的泛素启动子Ubi，玉米Cas9编码序列，终止子T2；TaSnRK2.10基因的核苷酸序列如SEQ ID NO.4所示。5. The application according to claim 2, characterized in that: the pBUE411-TaSnRK2.10 binary recombination vector is composed of expression cassette E1 and expression cassette E2, and its nucleotide sequence is shown in SEQ ID NO.3; Wherein the nucleotide sequence of the expression cassette E1 is shown in SEQ ID NO.1, which includes from upstream to downstream: the TaU3 promoter from wheat, the sgRNA of the TaSnRK2.10 gene, and the terminator T1; the expression cassette The nucleotide sequence of E2 is shown in SEQ ID NO.2, which includes from upstream to downstream: the ubiquitin promoter Ubi from maize, the maize Cas9 coding sequence, and the terminator T2; the nucleotide sequence of the TaSnRK2.10 gene As shown in SEQ ID NO.4.6.根据权利要求5所述的应用，其特征在于：所述小麦TaU3启动子的核苷酸序列如SEQID NO.9所示；所述TaSnRK2.10基因的sgRNA的核苷酸序列如SEQ ID NO.5所示；所述终止子T1的核苷酸序列如SEQ ID NO.10所示；所述玉米泛素启动子Ubi的核苷酸序列如SEQ IDNO.11所示；所述玉米Cas9编码序列的核苷酸序列如SEQ ID NO.12所示；所述终止子T2的核苷酸序列如SEQ ID NO.13所示。6. The application according to claim 5, characterized in that: the nucleotide sequence of the wheat TaU3 promoter is as shown in SEQ ID NO.9; the nucleotide sequence of the sgRNA of the TaSnRK2.10 gene is as SEQ ID Shown in NO.5; the nucleotide sequence of the terminator T1 is shown in SEQ ID NO.10; the nucleotide sequence of the maize ubiquitin promoter Ubi is shown in SEQ ID NO.11; the maize Cas9 The nucleotide sequence of the coding sequence is shown in SEQ ID NO.12; the nucleotide sequence of the terminator T2 is shown in SEQ ID NO.13.7.靶向TaSnRK2.10基因的sgRNA在增加小麦分蘖数、穗粒数和籽粒宽度中的应用，其中所述靶向TaSnRK2.10基因的sgRNA的核苷酸序列如SEQ ID NO.5所示。7. The application of the sgRNA targeting the TaSnRK2.10 gene in increasing the number of wheat tillers, the number of spikes and the grain width, wherein the nucleotide sequence of the sgRNA targeting the TaSnRK2.10 gene is shown in SEQ ID NO.5 .8.根据权利要求7所述的应用，其特征在于：所述应用是通过构建含有该sgRNA的具有敲除TaSnRK2.10基因功能的核苷酸序列如SEQ ID NO.3所示的表达载体pBUE411-TaSnRK2.10，并转化到普通小麦中特异性敲除TaSnRK2.10基因，获得TaSnRK2.10基因敲除的转基因小麦实现。8. The application according to claim 7, characterized in that: the application is by constructing the expression vector pBUE411 containing the sgRNA with the nucleotide sequence of knocking out the TaSnRK2.10 gene function as shown in SEQ ID NO.3 -TaSnRK2.10, and transformed into common wheat to specifically knock out the TaSnRK2.10 gene, so as to obtain the realization of transgenic wheat with TaSnRK2.10 gene knockout.

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