技术领域Technical field
本发明属于植物基因工程技术领域。更具体地，涉及一种植物耐低磷重要基因GmCXIP1-1及其应用。The invention belongs to the technical field of plant genetic engineering. More specifically, it relates to GmCXIP1-1, an important plant low-phosphorus tolerance gene and its application.
背景技术Background technique
大豆(Glycine max)是一种重要的豆科作物，是动物饲料中蛋白质和油的主要来源，属于高蛋白粮饲兼用作物，在我国栽培历史悠久，种植区域广泛。同时大豆作为世界上种植最广泛的作物之一，它是人类最重要的植物蛋白和油脂来源，也是目前生物柴油生产的主要原料。随着全国耕地不断减少，大豆种植面积大范围降低，而人们对于大豆的需求量明显增加，导致国内大豆生产供不应求，大量国外质优价廉的大豆涌入国内市场。因此，大力发展大豆种植是解决我国大豆供不应求的重要举措。Soybean (Glycine max) is an important leguminous crop and the main source of protein and oil in animal feed. It is a high-protein grain and feed crop. It has a long history of cultivation in my country and has a wide planting area. At the same time, soybean, as one of the most widely planted crops in the world, is the most important source of vegetable protein and oil for humans, and is also the main raw material for current biodiesel production. With the continuous reduction of cultivated land across the country, soybean planting area has been reduced across a wide range, and people's demand for soybeans has increased significantly, resulting in domestic soybean production being in short supply, and a large number of foreign high-quality and low-price soybeans flooding into the domestic market. Therefore, vigorously developing soybean cultivation is an important measure to solve the shortage of soybeans in our country.
我国南方水热条件充足，适合大豆生产。但南方土壤大多为酸性土壤，在酸性土壤中，低磷胁迫因子严重限制了大豆的产量(Zhang and Xu,2005；Wang et al.,2010；Liu etal.,2020)，施用的磷肥很容易被土壤中的铁和铝离子等固定，形成难溶性磷。虽然土壤中储存了大量的磷素，但能够为植物直接吸收和利用的有效磷的含量远远不能满足大豆正常生长发育的需要。酸性土壤磷有效性低已成为限制大豆生产的重要原因之一(严小龙等,2000；Zhang et al.,2005；Wang et al.,2010；Ham et al.,2018)。因此，提高大豆对酸性土壤中低磷胁迫的适应能力，促进大豆的生产，对我国的农业和经济的持续发展具有十分重要的意义。Southern my country has sufficient water and heat conditions, which are suitable for soybean production. However, most soils in the south are acidic soils. In acidic soils, low phosphorus stress factors severely limit soybean yields (Zhang and Xu, 2005; Wang et al., 2010; Liu et al., 2020). The applied phosphorus fertilizers are easily Iron and aluminum ions in the soil are fixed to form insoluble phosphorus. Although a large amount of phosphorus is stored in the soil, the content of available phosphorus that can be directly absorbed and utilized by plants is far from meeting the needs of normal growth and development of soybeans. Low phosphorus availability in acidic soil has become one of the important reasons limiting soybean production (Yan Xiaolong et al., 2000; Zhang et al., 2005; Wang et al., 2010; Ham et al., 2018). Therefore, improving the adaptability of soybeans to low phosphorus stress in acidic soil and promoting soybean production are of great significance to the sustainable development of my country's agriculture and economy.
钙(Ca2+)是植物生长发育所必需的关键阳离子和主要营养元素，也是细胞信号转导中重要的第二信使，Ca2+通过各种离子输入和输出机制维持细胞质和液泡中的平衡。CAX基因家族属于Ca2+阳离子反转运蛋白(CaCA)超家族成员，可以运输金属阳离子，如Ca、Mn、Zn和Cd，通过液泡膜增强植物的抗旱性(等人，2001；Cheng等，2002；Koren’kov等人，2007；Socha和Guerinot,2014；Yamada et al.，2014)。CXIP基因家族是一组与CAX n端相互作用调节CAX向Ca2+转运的独特蛋白，目前为止对CXIP基因的研究很少，只对模式植物拟南芥中的CXIP1和CXIP4进行了研究(Cheng and Hirschi,2003；Cheng等人，2004)。目前，CXIP基因尚未在任何其他植物物种中被克隆报道，其具体相关的功能和作用未知。而且，CXIP基因是否也参与了植物对土壤中磷胁迫的相关机制尚不清楚，在大豆中对CXIP基因的研究分析也鲜有报道。Calcium (Ca2+ ) is a key cation and major nutritional element necessary for plant growth and development. It is also an important second messenger in cell signal transduction. Ca2+ maintains balance in the cytoplasm and vacuoles through various ion input and output mechanisms. . The CAX gene family belongs to the Ca2+ cation antiporter (CaCA) superfamily, which can transport metal cations, such as Ca, Mn, Zn and Cd, through the tonoplast to enhance plant drought resistance ( et al., 2001; Cheng et al., 2002; Koren'kov et al., 2007; Socha and Guerinot, 2014; Yamada et al., 2014). The CXIP gene family is a group of unique proteins that interact with the N-terminus of CAX to regulate CAX transport to Ca2+ . So far, there are few studies on CXIP genes, and only CXIP1 and CXIP4 in the model plant Arabidopsis thaliana have been studied (Cheng and Hirschi, 2003; Cheng et al., 2004). At present, the CXIP gene has not been cloned and reported in any other plant species, and its specific related functions and roles are unknown. Moreover, it is not clear whether the CXIP gene is also involved in the mechanism of plants responding to phosphorus stress in the soil, and there are few reports on the research and analysis of the CXIP gene in soybeans.
发明内容Contents of the invention
本发明要解决的技术问题是克服现有CXIP基因的不足，提供一种植物耐低磷重要基因GmCXIP1-1及其应用。The technical problem to be solved by the present invention is to overcome the shortcomings of existing CXIP genes and provide an important plant low-phosphorus tolerance gene GmCXIP1-1 and its application.
本发明的第一个目的是提供一种植物耐低磷胁迫的GmCXIP1-1基因。The first object of the present invention is to provide a GmCXIP1-1 gene that can tolerate low phosphorus stress in plants.
本发明的第二个目的是提供GmCXIP1-1基因的编码蛋白。The second object of the present invention is to provide the protein encoded by the GmCXIP1-1 gene.
本发明的第三个目的是提供述GmCXIP1-1基因或其表达促进剂、或权利要求2所述编码蛋白的应用。The third object of the present invention is to provide the application of the GmCXIP1-1 gene or its expression promoter, or the encoded protein of claim 2.
本发明的第四个目的是提供一种重组表达载体。The fourth object of the present invention is to provide a recombinant expression vector.
本发明的第五个目的是提供一种基因工程菌。The fifth object of the present invention is to provide a genetically engineered bacterium.
本发明的第六个目的是提供一种低磷胁迫下促进植物生长的制剂或提高植物根系耐低磷胁迫能力的制剂。The sixth object of the present invention is to provide a preparation that promotes plant growth under low phosphorus stress or a preparation that improves the ability of plant roots to tolerate low phosphorus stress.
本发明的第七个目的是提供一种促进植物根系生长和/或提高植物对低磷胁迫耐受性的方法。The seventh object of the present invention is to provide a method for promoting plant root growth and/or improving plant tolerance to low phosphorus stress.
本发明上述目的通过以下技术方案实现：The above objects of the present invention are achieved through the following technical solutions:
本发明通过实时荧光定量PCR和同源克隆的方法，首次在大豆中克隆得到一个参与植物体内阳离子转运(与CAX基因家族相关)的CXIP家族的GmCXIP1-1基因，其核苷酸序列如SEQ ID NO:1所示，GmCXIP1-1基因的编码蛋白的氨基酸序列如SEQ ID NO:2所示。本发明研究发现GmCXIP1-1在基因转录水平受低磷胁迫显著上调表达，且随着磷处理时间的延长，其表达量明显增加。再通过拟南芥转基因技术获得超量GmCXIP1-1表达的拟南芥转基因植株材料，显示GmCXIP1-1基因具有调控大豆根系适应低磷胁迫的功能；同时，能减少低磷胁迫对植物根系生长的抑制作用。Through real-time fluorescence quantitative PCR and homologous cloning methods, the present invention clones a GmCXIP1-1 gene of the CXIP family involved in cation transport in plants (related to the CAX gene family) in soybean for the first time. Its nucleotide sequence is as SEQ ID NO: 1 is shown, and the amino acid sequence of the protein encoded by the GmCXIP1-1 gene is shown in SEQ ID NO: 2. The study of the present invention found that the expression of GmCXIP1-1 was significantly up-regulated by low phosphorus stress at the gene transcription level, and its expression increased significantly with the extension of phosphorus treatment time. Arabidopsis transgenic plant materials with excessive GmCXIP1-1 expression were then obtained through Arabidopsis transgenic technology, showing that the GmCXIP1-1 gene has the function of regulating soybean roots to adapt to low phosphorus stress; at the same time, it can reduce the impact of low phosphorus stress on plant root growth. inhibitory effect.
因此，本发明提供SEQ ID NO:1所示GmCXIP1-1基因或其表达促进剂、或SEQ IDNO:2所示GmCXIP1-1蛋白的以下应用：Therefore, the present invention provides the following applications of the GmCXIP1-1 gene shown in SEQ ID NO: 1 or its expression promoter, or the GmCXIP1-1 protein shown in SEQ ID NO: 2:
在低磷胁迫下促进植物生长或在制备低磷胁迫下促进植物生长的制剂中的应用。Use in promoting plant growth under low phosphorus stress or in preparing preparations for promoting plant growth under low phosphorus stress.
在减少低磷胁迫对植物根系生长的抑制作用方面的应用。Application in reducing the inhibitory effect of low phosphorus stress on plant root growth.
在提高植物根系耐低磷胁迫能力中的应用。Application in improving the ability of plant roots to tolerate low phosphorus stress.
在培育耐低磷植株中的应用。Application in cultivating low phosphorus tolerant plants.
本发明提供一种重组表达载体，含上述GmCXIP1-1基因。The invention provides a recombinant expression vector containing the above GmCXIP1-1 gene.
本发明提供一种基因工程菌，含上述重组表达载体。The invention provides a genetically engineered bacterium containing the above recombinant expression vector.
本发明还提供一种低磷胁迫下促进植物生长的制剂或提高植物根系耐低磷胁迫能力的制剂，含GmCXIP1-1基因或其表达促进剂。The invention also provides a preparation for promoting plant growth under low phosphorus stress or a preparation for improving the ability of plant roots to tolerate low phosphorus stress, containing the GmCXIP1-1 gene or its expression promoter.
本发明还提供一种促进植物根系生长和/或提高植物对低磷胁迫耐受性的方法，在植物中超量表达GmCXIP1-1基因。The invention also provides a method for promoting plant root growth and/or improving plant tolerance to low phosphorus stress by overexpressing the GmCXIP1-1 gene in plants.
优选地，通过基因编辑技术，正调控植物中GmCXIP1-1基因表达水平或蛋白活性来促进植物根系生长和/或提高植物对低磷胁迫耐受性。Preferably, gene editing technology is used to positively regulate GmCXIP1-1 gene expression levels or protein activity in plants to promote plant root growth and/or improve plant tolerance to low phosphorus stress.
优选地，构建超量表达GmCXIP1-1基因的表达载体，转化植株，得到促进植物根系生长和/或提高植物对低磷胁迫耐受性的转基因植株。Preferably, an expression vector overexpressing the GmCXIP1-1 gene is constructed and the plants are transformed to obtain transgenic plants that promote plant root growth and/or improve plant tolerance to low phosphorus stress.
本发明具有以下有益效果：The invention has the following beneficial effects:
本发明公开了一种植物耐低磷重要基因GmCXIP1-1及其应用。本发明首次在大豆中克隆得到一个参与植物体内阳离子转运的CXIP家族GmCXIP1-1基因。研究显示GmCXIP1-1基因受低磷胁迫诱导表达上调，且随着磷处理时间的延长，其表达量明显增加；在不同磷浓度处理条件下，超量表达GmCXIP1-1明显能增加转基因植物的生物量，在低磷条件下促进植物生长；同时，超量表达GmCXIP1-1能提高植物对低磷胁迫能力的耐受能力，减少低磷对植物根系生长的抑制作用。The invention discloses an important plant low-phosphorus tolerance gene GmCXIP1-1 and its application. The present invention clones a GmCXIP1-1 gene of the CXIP family involved in cation transport in plants for the first time in soybean. Research shows that the expression of GmCXIP1-1 gene is up-regulated by low phosphorus stress, and its expression increases significantly with the extension of phosphorus treatment time; under different phosphorus concentration treatment conditions, overexpression of GmCXIP1-1 can significantly increase the biological activity of transgenic plants. The amount promotes plant growth under low phosphorus conditions; at the same time, overexpression of GmCXIP1-1 can improve plants' tolerance to low phosphorus stress and reduce the inhibitory effect of low phosphorus on plant root growth.
本发明公开了大豆GmCXIP1-1基因在调控植物适应低磷胁迫促生长中的应用，显示大豆的CXIP基因参与了植物调节土壤磷胁迫的相关机制，GmCXIP1-1正调控植物根系适应低磷的能力，表明GmCXIP1-1对植物适应低磷胁迫具有重要作用，能通过转基因技术提高植物对酸性土壤低磷胁迫的适应能力。The invention discloses the application of the soybean GmCXIP1-1 gene in regulating plant adaptation to low phosphorus stress and promoting growth. It shows that the soybean CXIP gene is involved in the relevant mechanism of plants regulating soil phosphorus stress, and GmCXIP1-1 positively regulates the ability of plant roots to adapt to low phosphorus. , indicating that GmCXIP1-1 plays an important role in plants adapting to low phosphorus stress, and can improve plants' adaptability to low phosphorus stress in acidic soil through transgenic technology.
附图说明Description of the drawings
图1为大豆GmCXIP1-1表达模式分析(低磷处理时间对GmCXIP1-1在大豆根系表达模式的影响；数据为3次重复的平均值与标准误，星号表示对照(+P)与处理(-P)之间差异显著(Student’s t-test)，*：P<0.05，**：P<0.01，***：P<0.001)；Figure 1 is an analysis of the expression pattern of soybean GmCXIP1-1 (the effect of low phosphorus treatment time on the expression pattern of GmCXIP1-1 in soybean roots; the data are the average and standard error of three repetitions, asterisks indicate control (+P) and treatment ( -P) There is a significant difference (Student's t-test), *: P<0.05, **: P<0.01, ***: P<0.001);
图2为GmCXIP1-1亚细胞定位分析(A：GmCXIP1-1融合GFP蛋白在烟草叶片的亚细胞定位分析结果；B：GmCXIP1-1融合GFP蛋白在洋葱表皮细胞中的亚细胞定位分析结果；图A和B第一排为转化空载体的烟草或者洋葱表皮细胞中的的亚细胞定位图(35S::GFP)；第二排为GmCXIP1-1融合GFP蛋白在烟草叶片或者洋葱表皮细胞中的的亚细胞定位图(35S::GmCXIP1-1-GFP)；图片A和B分别为在激光共聚焦显微镜下绿色荧光通道(GFP)、光镜通道(明场)和重叠后的图片(融合)观察，标尺为20μm)；Figure 2 shows the subcellular localization analysis of GmCXIP1-1 (A: The results of the subcellular localization analysis of GmCXIP1-1 fusion GFP protein in tobacco leaves; B: The results of the subcellular localization analysis of GmCXIP1-1 fusion GFP protein in onion epidermal cells; Figure The first row of A and B is the subcellular localization map of tobacco or onion epidermal cells transformed with the empty vector (35S::GFP); the second row is the location of GmCXIP1-1 fusion GFP protein in tobacco leaves or onion epidermal cells. Subcellular localization map (35S::GmCXIP1-1-GFP); Pictures A and B are the green fluorescence channel (GFP), light microscope channel (bright field) and overlapping pictures (fusion) observed under a confocal laser microscope, respectively. , scale bar is 20μm);
图3为超量表达GmCXIP1-1转基因拟南芥植株鉴定(A：GmCXIP1-1PCR检测；B：GmCXIP1-1在不同株系叶部的表达量检测；WT：野生型株系；OX1和OX2：两个不同的超量表达GmCXIP1-1的转基因大豆株系；图中数据为4次重复的平均值和标准误；“*”表示与WT相比差异显著(Student’s t-test，P<0.05))；Figure 3 shows the identification of overexpressed GmCXIP1-1 transgenic Arabidopsis plants (A: GmCXIP1-1 PCR detection; B: GmCXIP1-1 expression level detection in leaves of different lines; WT: wild-type line; OX1 and OX2: Two different transgenic soybean lines overexpressing GmCXIP1-1; the data in the figure are the average and standard error of 4 repetitions; "*" indicates significant differences compared with WT (Student's t-test, P<0.05) );
图4为高低磷处理对超量表达GmCXIP1-1的拟南芥幼苗生长的影响(A：野生型(WT)与转基因拟南芥株系(OX1，OX2)在高低磷处理下的表型，图中标尺为1cm；B：野生型(WT)与转基因拟南芥株系(OX1，OX2)在高低磷处理下的侧根数C：野生型(WT)与转基因拟南芥株系(OX1，OX2)在高低磷处理条件下的根系生长量；D：野生型(WT)与转基因拟南芥株系(OX1，OX2)在高低磷处理条件下地上部鲜重；E：野生型(WT)与转基因拟南芥株系(OX1，OX2)在高低磷处理下的根系鲜重；星号表示野生型(WT)与转基因拟南芥株系(OX1，OX2)之间差异显著(Student’s t-test)，*：P<0.05，**：P<0.01，***：P<0.001)。Figure 4 shows the effects of high and low phosphorus treatments on the growth of Arabidopsis seedlings overexpressing GmCXIP1-1 (A: Phenotypes of wild type (WT) and transgenic Arabidopsis lines (OX1, OX2) under high and low phosphorus treatments, The scale bar in the figure is 1cm; B: The number of lateral roots of wild type (WT) and transgenic Arabidopsis lines (OX1, OX2) under high and low phosphorus treatments. C: The number of lateral roots of wild type (WT) and transgenic Arabidopsis lines (OX1, OX2). OX2) Root growth under high and low phosphorus treatment conditions; D: Aboveground fresh weight of wild type (WT) and transgenic Arabidopsis lines (OX1, OX2) under high and low phosphorus treatment conditions; E: Wild type (WT) and transgenic Arabidopsis lines (OX1, OX2) Root fresh weight of transgenic Arabidopsis lines (OX1, OX2) under high and low phosphorus treatments; asterisks indicate significant differences between wild type (WT) and transgenic Arabidopsis lines (OX1, OX2) (Student's t-test ), *: P<0.05, **: P<0.01, ***: P<0.001).
具体实施方式Detailed ways
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。The invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.
除非特别说明，以下实施例所用试剂和材料均为市购。Unless otherwise stated, the reagents and materials used in the following examples were all commercially available.
下述实验中采用的大豆(Glycine max(L.)Merr.)材料为粤春03-3(YC03-3)，来源于华南农业大学根系生物学研究中心。The soybean (Glycine max (L.) Merr.) material used in the following experiments is Yuechun 03-3 (YC03-3), which was obtained from the Root Biology Research Center of South China Agricultural University.
实施例1GmCXIP1-1基因表达模式分析Example 1 GmCXIP1-1 gene expression pattern analysis
本发明首次在大豆中克隆得到一个参与植物体内阳离子转运(与CAX基因家族相关)的CXIP家族的GmCXIP1-1基因，其核苷酸序列如SEQ ID NO:1所示，GmCXIP1-1基因的编码蛋白的氨基酸序列如SEQ ID NO:2所示。本发明研究发现GmCXIP1-1在基因转录水平受低磷胁迫调控表达，进一步对GmCXIP1-1基因在大豆协同响应酸性土壤低磷胁迫的过程中的具体功能进行分析研究。For the first time, the present invention clones a GmCXIP1-1 gene of the CXIP family involved in cation transport in plants (related to the CAX gene family) in soybeans. Its nucleotide sequence is shown in SEQ ID NO: 1. The coding of the GmCXIP1-1 gene The amino acid sequence of the protein is shown in SEQ ID NO:2. The present invention has found that the expression of GmCXIP1-1 is regulated by low phosphorus stress at the gene transcription level, and further analyzes and studies the specific function of the GmCXIP1-1 gene in the process of soybean's coordinated response to acidic soil low phosphorus stress.
1、植物样品1. Plant samples
采用卷纸育苗法，挑选种皮无破损、大小均一的大豆种子，用100mL次氯酸钠加4.2mL的盐酸反应产生的氯气消毒12h，再于超净工作台吹1h备用。剪裁20×20cm的方形滤纸，配制pH为5.8的1/4大豆全营养液(具体配方参见：刘国选,陈康,陆星,田江&梁翠月.(2021).大豆GmPIN2b调控根系响应低磷胁迫的功能研究.华南农业大学学报(04),33-41.)以及无菌水，灭菌待用。Use the roll paper seedling method to select soybean seeds with undamaged seed coats and uniform sizes, disinfect them with chlorine gas produced by the reaction of 100 mL sodium hypochlorite and 4.2 mL hydrochloric acid for 12 hours, and then blow them on a clean workbench for 1 hour before use. Cut a 20×20cm square filter paper and prepare 1/4 soybean complete nutrient solution with a pH of 5.8 (for specific recipes, see: Liu Guoxuan, Chen Kang, Lu Xing, Tian Jiang & Liang Cuiyue. (2021). Soybean GmPIN2b regulates root response to low phosphorus stress Functional research. Journal of South China Agricultural University (04), 33-41.) and sterile water, sterilize and set aside.
进行卷纸培时，将保鲜膜铺在试验台面，用配制好的1/4大豆全营养液浸透滤纸，在距离滤纸一边约1cm处摆放7颗消毒后的豆子，豆子种脐朝下，从第一颗豆子卷至末端。卷好后的滤纸将没有豆子的一端朝下，放入装有1/4大豆全营养液的500mL烧杯中，用保鲜膜将卷有豆子的上端滤纸包起。将烧杯放入培养箱中24～26℃，先暗培养1d，再光照/黑暗(12h/12h)培养3～4d，至胚根5～6cm。When performing paper roll cultivation, lay the plastic wrap on the test table, saturate the filter paper with 1/4 of the prepared soybean complete nutrient solution, and place 7 sterilized beans about 1cm away from one side of the filter paper, with the bean navel facing down. Roll from the first bean to the end. Put the rolled filter paper with the end without beans facing down into a 500mL beaker filled with 1/4 of the soybean complete nutrient solution, and wrap the upper end of the filter paper with the beans in it with plastic wrap. Place the beaker in an incubator at 24-26°C, first cultivate it in the dark for 1 day, and then cultivate it in light/dark (12h/12h) for 3-4 days until the radicle is 5-6cm.
挑选生长一致的幼苗，转移到高低磷处理条件：+P(250μM KH2PO4)与-P(5μMKH2PO4)的营养液中，每个处理4个重复，每个重复8株苗。每两天将营养液pH调至5.8左右，每周换一次营养液，分别在6d、12d和18d分别收获根系样品，液氮冷冻后，置于-80℃冰箱中保存，备用。Seedlings with consistent growth were selected and transferred to high and low phosphorus treatment conditions: +P (250 μM KH2 PO4 ) and -P (5 μMKH 2 PO4 ) nutrient solutions. There were 4 replicates for each treatment, and 8 seedlings per replicate. The pH of the nutrient solution was adjusted to about 5.8 every two days, and the nutrient solution was changed once a week. Root samples were harvested on 6d, 12d, and 18d respectively. After freezing in liquid nitrogen, they were stored in a -80°C refrigerator for later use.
2、实时荧光定量PCR(qRT-PCR)分析2. Real-time fluorescence quantitative PCR (qRT-PCR) analysis
使用TRIzol试剂盒(Invitrogen，美国)分别提取上述处理后的植物样品的总RNA。采用MMLV-逆转录试剂盒(Promega,美国)将经DNase I处理后的RNA逆转录合成为cDNA。再采用SYBR(Promega，美国)试剂盒进行qRT-PCR分析。完成逆转录后将样品稀释15倍后通过Applied Biosystems StepOnePlus Real-Time PCR system进行实时荧光定量PCR分析。Total RNA from the above-treated plant samples was extracted using TRIzol kit (Invitrogen, USA). The DNase I-treated RNA was reverse transcribed into cDNA using MMLV-Reverse Transcription Kit (Promega, USA). Then SYBR (Promega, USA) kit was used for qRT-PCR analysis. After completing reverse transcription, the sample was diluted 15 times and analyzed by real-time fluorescence quantitative PCR using the Applied Biosystems StepOnePlus Real-Time PCR system.
标准曲线制备：先从每个样品的cDNA原液中吸取1～2μL样品至一个新的PCR离心管中，然后将混合液再稀释10倍为第一个标样S1，再将第一个标样稀释10倍作为第二个标样S2，以此类推稀释为5个浓度梯度标样，即S1、S2、S3、S4、S5。Standard curve preparation: First draw 1 to 2 μL sample from the cDNA stock solution of each sample into a new PCR centrifuge tube, then dilute the mixture 10 times into the first standard sample S1, and then add the first standard sample Dilute 10 times as the second standard S2, and so on and dilute it to 5 concentration gradient standards, namely S1, S2, S3, S4, and S5.
定量PCR引物采用GmCXIP1-1基因定量引物和内参基因EF1-α为大豆的看家基因引物如下所示：The quantitative PCR primers use the GmCXIP1-1 gene quantitative primer and the internal reference gene EF1-α as the soybean housekeeping gene primers as follows:
GmCXIP1-1-RT1-F：5’-CAAACTCAAACGCGCTTCCA-3’；GmCXIP1-1-RT1-F: 5’-CAAACTCAAAACCGCTTCCA-3’;
GmCXIP1-1-RT1-R：5’-TACTCTTTCAGCCCTTGGCG-3’；GmCXIP1-1-RT1-R: 5’-TACTCTTTCAGCCCTTGGCG-3’;
EF1-α-F：5’-TGCAAAGGAGGCTGCTAACT-3’；EF1-α-F: 5’-TGCAAAGGAGGCTGCTAACT-3’;
EF1-α-R：5’-CAGCATCACCGTTCTTCAAA-3’。EF1-α-R: 5’-CAGCATCACCGTTCTTCAAA-3’.
配制反应混合液：每20微升反应体系中，加入10微升SYBR Premix Ex Taq(2×)，0.5微升正向/反向引物，7微升ddH2O和2微升模板。先计算所需反应的用量，将除了cDNA以外的试剂混匀，每管分装18微升，再加入2微升的cDNA模板使终体积为20微升。Prepare reaction mixture: For every 20 μl of reaction system, add 10 μl of SYBR Premix Ex Taq (2×), 0.5 μl of forward/reverse primer, 7 μl of ddH2 O and 2 μl of template. Calculate the required amount of reaction first, mix the reagents except cDNA, and dispense 18 μl into each tube, then add 2 μl of cDNA template to make the final volume 20 μl.
定量PCR反应程序为：95℃预变性30s，PCR反应(95℃变性30秒，60℃复性15秒，72℃延伸30秒)40个循环。检测结果用Rotor-Gene的Real-Time Analysis Software计算每个样品的表达量。The quantitative PCR reaction program was: pre-denaturation at 95°C for 30 seconds, followed by 40 cycles of PCR reaction (denaturation at 95°C for 30 seconds, renaturation at 60°C for 15 seconds, and extension at 72°C for 30 seconds). The detection results were calculated using Rotor-Gene's Real-Time Analysis Software to calculate the expression level of each sample.
检测结果如图1所示，在高低磷处理条件下，与高磷处理相比，低磷处理6dGmCXIP1-1基因的表达水平受低磷胁迫明显上调，且随着磷处理时间的延长，其表达量明显增加，到第18d其表达量下降。The test results are shown in Figure 1. Under high and low phosphorus treatment conditions, compared with the high phosphorus treatment, the expression level of the 6dGmCXIP1-1 gene in the low phosphorus treatment was significantly increased by low phosphorus stress, and as the phosphorus treatment time increased, its expression The amount increased significantly, and its expression decreased on the 18th day.
实施例2GmCXIP1-1亚细胞定位分析Example 2 GmCXIP1-1 subcellular localization analysis
根据GmCXIP1-1基因序列，设计特异引物GmCXIP1-1-GFP-F：5’-CAGACCTTAATTAAGAGCT CATGTCGTGGTGTTACTGCGCTA-3’，GmCXIP1-1-GF P-R：5’-CATGGTGGCGACCGGTCGTCAGGACAACATTGCCTTCTCC-3’。以大豆基因型YC03-3的根系cDNA为模板对大豆GmCXIP1-1基因ORF全长进行扩增。反应条件为98℃预变性5min，98℃变性30s，58℃退火30s，72℃复性1～3min，这一过程循环30次，72℃延伸10min。According to the GmCXIP1-1 gene sequence, design specific primers GmCXIP1-1-GFP-F: 5’-CAGACCTTAATTAAGAGCT CATGTCGTGGTGTTACTGCGCTA-3’, GmCXIP1-1-GF P-R: 5’-CATGGTGGCGACCGGTCGTCAGGACAACATTGCCTTCTCC-3’. The full length ORF of soybean GmCXIP1-1 gene was amplified using the root cDNA of soybean genotype YC03-3 as a template. The reaction conditions were pre-denaturation at 98°C for 5 min, denaturation at 98°C for 30 s, annealing at 58°C for 30 s, renaturation at 72°C for 1 to 3 min, this process was cycled 30 times, and extension at 72°C for 10 min.
将PCR扩增的产物通过凝胶电泳，利用试剂盒进行回收纯化，获得纯化PCR产物后，使用同源重组试剂盒II将PCR产物与经Age I酶切后的线性化载体pEGAD进行重组连接反应。反应体系为20μL，包含PCR产物6μL，pEGAD线性化载体质粒8μL，用重组连接酶Exnase II 2μL，反应缓冲液4μL。将试剂混合物于37℃反应30min，将重组质粒转化大肠杆菌并进行测序，无误后提取质粒。将35S::GmCXIP1-1-GFP质粒转化农杆菌GV3101和发根农杆菌K599，检测无误后保存备用。Pass the PCR amplified product through gel electrophoresis and use a kit to recover and purify it. After obtaining the purified PCR product, use a homologous recombination kit II Perform a recombination ligation reaction between the PCR product and the linearized vector pEGAD digested by Age I enzyme. The reaction system is 20 μL, including 6 μL of PCR product, 8 μL of pEGAD linearized vector plasmid, 2 μL of recombinant ligase Exnase II, and 4 μL of reaction buffer. The reagent mixture was reacted at 37°C for 30 minutes, and the recombinant plasmid was transformed into E. coli and sequenced. After the plasmid was found to be correct, the plasmid was extracted. The 35S::GmCXIP1-1-GFP plasmid was transformed into Agrobacterium tumefaciens GV3101 and Agrobacterium rhizogenes K599, and stored for later use after detection.
通过农杆菌转化法进行烟草表皮细胞瞬时表达亚细胞定位分析，将35S::GmCXIP1-1-GFP或pEGAD空载(35S:GFP)导入农杆菌菌株GV3101。转化成功后含载体的GV3101接种于YEP培养基，28℃振荡培养16h后离心(5000rpm，10min)，用浸润液(含10mMMgCl2、10mM MES和10mM乙酰丁香酮，pH＝5.6)重悬至OD600的分光光度值为0.4～0.5，将菌体悬浮液于43℃避光静置3h后，通过注射器渗入菌液的方法共转化5～6周龄烟草的叶片下表皮。转化的烟草正常培养3d后，用激光共聚扫描显微镜(Zeiss LSM780，德国)观察荧光信号在烟草表皮细胞中的分布情况。The subcellular localization analysis of transient expression in tobacco epidermal cells was performed by Agrobacterium transformation method, and 35S::GmCXIP1-1-GFP or pEGAD empty (35S:GFP) was introduced into Agrobacterium strain GV3101. After successful transformation, the vector-containing GV3101 was inoculated into YEP medium, cultured with shaking at 28°C for 16 hours, then centrifuged (5000rpm, 10min), and resuspended to OD in infiltration solution (containing 10mMgCl2 , 10mM MES and 10mM acetosyringone, pH=5.6) The spectrophotometric value of600 is 0.4-0.5. After the bacterial suspension was allowed to stand in the dark at 43°C for 3 hours, the bacterial solution was infiltrated into the lower epidermis of 5-6 week-old tobacco leaves through a syringe. After the transformed tobacco was cultured normally for 3 days, a laser condensation scanning microscope (Zeiss LSM780, Germany) was used to observe the distribution of fluorescence signals in tobacco epidermal cells.
而对于洋葱鳞片内表皮细胞亚细胞定位，则将菌液OD值调为0.6，洋葱鳞片内表皮细胞需预先在MS固体培养基上培养24h，转化时用菌液浸没洋葱鳞片内表皮细胞4～6h。转化完成的洋葱分别培养48h和24h后在激光共聚焦显微镜(Zeiss LSM780，德国)下观察亚细胞定位，质壁分离时二者分别用30％蔗糖溶液和7％氯化钠溶液。激光共聚焦显微镜的激发光波长为488nm。For the subcellular localization of the inner epidermal cells of onion scales, the OD value of the bacterial solution is adjusted to 0.6. The inner epidermal cells of onion scales need to be cultured on MS solid medium in advance for 24 hours. During transformation, the inner epidermal cells of onion scales are immersed in the bacterial solution for 4~ 6h. The transformed onions were cultured for 48 h and 24 h respectively, and the subcellular localization was observed under a laser confocal microscope (Zeiss LSM780, Germany). During plasmolysis, 30% sucrose solution and 7% sodium chloride solution were used for plasmolysis. The excitation light wavelength of the laser confocal microscope is 488nm.
结果如图2所示，将35S::GmCXIP1-1-GFP载体转入烟草叶片表皮细胞进行瞬时表达后，空载对照(35S::GFP)在细胞核、细胞质和质膜中均存在GFP的绿色荧光信号，而35S::GmCXIP1-1-GFP在细胞边缘有很强的GFP荧光，与细胞轮廓重合，说明GmCXIP1-1可能定位于细胞细胞壁上(图2A)。同样在洋葱内表皮细胞也观察到类似结果，在其细胞壁上具有较强的GFP荧光信号(图2B)。这些结果证明了GmCXIP1-1蛋白定位在细胞壁，是细胞壁结合蛋白。实施例3高低磷处理对超量表达GmCXIP1-1的拟南芥幼苗生长的影响The results are shown in Figure 2. After the 35S::GmCXIP1-1-GFP vector was transferred into tobacco leaf epidermal cells for transient expression, the green color of GFP was present in the empty control (35S::GFP) in the nucleus, cytoplasm and plasma membrane. Fluorescence signal, while 35S::GmCXIP1-1-GFP had strong GFP fluorescence at the cell edge, coinciding with the cell outline, indicating that GmCXIP1-1 may be localized on the cell wall (Figure 2A). Similar results were also observed in onion inner epidermal cells, which had strong GFP fluorescence signals on their cell walls (Figure 2B). These results prove that GmCXIP1-1 protein is localized in the cell wall and is a cell wall-binding protein. Example 3 Effects of high and low phosphorus treatment on the growth of Arabidopsis seedlings overexpressing GmCXIP1-1
1、GmCXIP1-1超量表达载体的构建(OX-GmCXIP1-1-pTF101s)1. Construction of GmCXIP1-1 overexpression vector (OX-GmCXIP1-1-pTF101s)
以大豆基因型YC03-3根系cDNA为模板，设计GmCXIP1-1特异引物，并用上游特异引物：5’-CCGGGGATCCTCTAGAATGTCGTGGTGTTACTGCGCT A-3’和下游特异引物：5’-GCAGGTCGACTCTAGATCAGGACAACATTGCCT TCTCC-3’，扩增出GmCXIP1-1编码区片段。PCR反应体系为：总共50μL体系，包含43μL Master mix(2×)、基因正向/反向引物各0.5μL、2μL cDNA模板、1μLdNTPs(10μM)和1μL高保真酶，其余用水补足。PCR程序为：98℃预变性5min，98℃变性30s，58℃退火30s，72℃复性1～3min，这一过程循环30次，72℃延伸10min。PCR片段回收测序无误后，将扩增得到的G mCXIP1-1的CDS片段经同源重组试剂盒II kits连接到经XbaⅠ酶切后的线性化载体pTF101s上，得到的重组载体转化大肠杆菌并测序验证，测序结果正确后，抽取重组质粒35S::GmCXIP1-1转入发根农杆菌K599和农杆菌GV3101备用。Using soybean genotype YC03-3 root cDNA as a template, GmCXIP1-1 specific primers were designed, and the upstream specific primer: 5'-CCGGGGATCCTCTAGAATGTCGTGGTGTTACTGCGCT A-3' and the downstream specific primer: 5'-GCAGGTCGACTCTAGATCAGGACAACATTGCCT TCTCC-3' were used to amplify GmCXIP1 -1 coding region fragment. The PCR reaction system is: a total of 50 μL system, including 43 μL Master mix (2×), 0.5 μL each of gene forward/reverse primers, 2 μL cDNA template, 1 μL dNTPs (10 μM) and 1 μL high-fidelity enzyme, and the rest is made up with water. The PCR program was: pre-denaturation at 98°C for 5 minutes, denaturation at 98°C for 30 seconds, annealing at 58°C for 30 seconds, renaturation at 72°C for 1 to 3 minutes, this process was cycled 30 times, and extension was performed at 72°C for 10 minutes. After the PCR fragments were recovered and sequenced correctly, the amplified CDS fragment of G mCXIP1-1 was subjected to homologous recombination kit II kits were connected to the linearized vector pTF101s digested by Bacillus GV3101 was used as a spare.
2、转基因拟南芥的获得2. Obtaining transgenic Arabidopsis thaliana
采用花序侵染法，将上述构建好的OX-GmCXIP1-1-pTF101s载体质粒转入农杆菌GV3101，挑取阳性克隆于5mL YEP培养液(含壮观和利福平)，28℃培养过夜；之后再转入100mL YEP培养液扩大培养至OD600为1.6～2.0；然后6000rpm，离心10min，弃上清收集菌体，用等体积的5％的蔗糖水或者1/2MS培养液重悬菌体，加0.005～0.02％ SilwetL-77配成转化液；转化时将拟南芥(转化前一天浇水保持植株湿润)花絮全部浸入转化液1min，取出后用稍用滤纸擦去多余的转化液，然后盖上保鲜膜保持植株湿润，先用黑色袋子罩住暗培养18h后转到正常培养条件下培养至收种(培养期间每隔1周转化一次，一共转化3次即可)。Using the inflorescence infection method, the OX-GmCXIP1-1-pTF101s vector plasmid constructed above was transferred into Agrobacterium GV3101, and positive clones were picked out and placed in 5 mL YEP culture medium (containing spectacular and rifampicin), and cultured at 28°C overnight; then Then transfer to 100mL YEP culture medium to expand the culture until the OD600 is 1.6~2.0; then centrifuge at 6000rpm for 10 minutes, discard the supernatant to collect the cells, and resuspend the cells with an equal volume of 5% sucrose water or 1/2MS culture medium. Add 0.005~0.02% SilwetL-77 to prepare the transformation solution; during transformation, immerse all the Arabidopsis thaliana catkins (water the day before transformation to keep the plant moist) in the transformation solution for 1 minute, take it out and wipe off the excess transformation solution with filter paper, and then Cover with plastic wrap to keep the plants moist. Cover with a black bag and cultivate in the dark for 18 hours, then transfer to normal culture conditions and culture until harvest (transform every 1 week during the culture period, for a total of 3 times).
转化后的拟南芥收获T0代种子后，取约100μL的种子进行繁种鉴定。具体步骤为：整个操作均在超净工作台中完成，先用70％乙醇漂洗1min，离心吸除乙醇，再用已灭菌的二级水清洗1次；再用10％的次氯酸钠预洗1次，离心吸除次氯酸钠，再用1mL的10％的次氯酸钠震动漂洗5min，离心吸除次氯酸钠，用无菌水漂洗5～6次；最后用无菌水重悬种子，并将种子均匀撒播在含除草剂的MS培养基上，4℃低温处理1d后打破休眠，移入光周期16h/8h(光照/黑暗)，温度为22℃/20℃(白天/夜晚)的植物光照培养箱；约2周后，将正常存活的T1代幼苗移入基质中继续生长，待植株长大后摘取少许叶片进行DNA的提取，并进行PCR鉴定阳性植株。After harvesting the T0 generation seeds from the transformed Arabidopsis thaliana, take about 100 μL of seeds for breeding identification. The specific steps are: the entire operation is completed in a ultra-clean workbench, first rinse with 70% ethanol for 1 minute, centrifuge to remove the ethanol, then wash once with sterilized secondary water; then pre-wash once with 10% sodium hypochlorite , centrifuge to remove sodium hypochlorite, and then use 1 mL of 10% sodium hypochlorite to vibrate and rinse for 5 minutes, centrifuge to remove sodium hypochlorite, and rinse with sterile water 5 to 6 times; finally resuspend the seeds in sterile water, and spread the seeds evenly on a bed containing weeding. On the MS medium of the agent, the dormancy was broken after low temperature treatment at 4°C for 1 day, and then moved into a plant light incubator with a photoperiod of 16h/8h (light/dark) and a temperature of 22°C/20°C (day/night); about 2 weeks later , move the normally surviving T1 generation seedlings into the substrate to continue growing. When the plants grow up, pick a few leaves for DNA extraction, and perform PCR to identify positive plants.
3、转基因拟南芥阳性鉴定3. Positive identification of transgenic Arabidopsis thaliana
将上述构建好的超量表达载体(OX-GmCXIP1-1-pTF101s)质粒转入农杆菌GV3101，再采用上述花序浸染法以及除草剂筛选获取T3代转基因拟南芥种子，最后利用定量PCR确认得到GmCXIP1-1高表达量的不同转基因拟南芥株系用于后续基因功能研究。拟南芥GmCXIP1-1定量引物为：GmCXIP1-1-RT-F和GmCXIP1-1-RT-R。以拟南芥看家基因EF1-α为内参基因，拟南芥的看家基因采用AtEF定量引物，具体引物序列如下所示：The above-constructed overexpression vector (OX-GmCXIP1-1-pTF101s) plasmid was transferred into Agrobacterium GV3101, and then the above-mentioned inflorescence dyeing method and herbicide screening were used to obtain T3 generation transgenic Arabidopsis seeds, and finally confirmed by quantitative PCR. Different transgenic Arabidopsis lines with high GmCXIP1-1 expression were used for subsequent gene function studies. The Arabidopsis thaliana GmCXIP1-1 quantitative primers are: GmCXIP1-1-RT-F and GmCXIP1-1-RT-R. Taking the Arabidopsis thaliana housekeeping gene EF1-α as the internal reference gene, the Arabidopsis housekeeping gene uses AtEF quantitative primers. The specific primer sequences are as follows:
GmCXIP1-1-RT-F：5’-TTGCCGTTGCAATCAAGAGC-3’；GmCXIP1-1-RT-F: 5’-TTGCCGTTGCAATCAAGAGC-3’;
GmCXIP1-1-RT-R：5’-CCTTGGCGCAACAAGTCATT-3’；GmCXIP1-1-RT-R: 5’-CCTTGGCGCAACAAGTCATT-3’;
AtEF1-α-F：5’-GTCGATTCTGGAAAGTCGACC-3’；AtEF1-α-F: 5’-GTCGATTCTGGAAAGTCGACC-3’;
AtEF1-α-R：5’-AATGTCAATGGTGATACCACGC-3’。AtEF1-α-R: 5’-AATGTCAATGGTGATACCACGC-3’.
结果如图3所示，与野生型(WT)拟南芥相比，PCR结果表明转基因株系中有GmCXIP1-1基因表达(图3A)，实时荧光定量PCR结果表明，相比WT，超量表达株系OX1、OX2和OX3中均有GmCXIP1-1的表达量(图3B)，差异显著。The results are shown in Figure 3. Compared with wild-type (WT) Arabidopsis thaliana, the PCR results showed that the GmCXIP1-1 gene was expressed in the transgenic line (Figure 3A). The real-time fluorescence quantitative PCR results showed that compared with WT, excessive The expression levels of GmCXIP1-1 were all found in the expression lines OX1, OX2 and OX3 (Figure 3B), and the differences were significant.
4、转基因拟南芥的高低磷处理4. High and low phosphorus treatment of transgenic Arabidopsis thaliana
选取适量野生型和2个超量表达GmCXIP1-1阳性株系OX1和OX2的饱满种子。消毒处理后，先播于正常MS培养基，约3～4d后拟南芥根系长到约1cm，挑选整齐一致的幼苗，移到相应处理的1/2MS培养基的方皿中，设置两个磷水平处理：正常供磷处理(1250μM KH2PO4)和低磷处理(6.25μM KH2PO4)，每个方形皿为一个重复，每个处理5个重复。处理后第9天收样，测定植株地上部鲜重、地下部鲜重、侧根数和主根长。Select an appropriate amount of plump seeds of wild type and two overexpressing GmCXIP1-1 positive lines OX1 and OX2. After disinfection, first sow in normal MS medium. After about 3 to 4 days, the Arabidopsis root system will grow to about 1cm. Select the neat and consistent seedlings, move them to the square dish of the corresponding 1/2 MS medium, and set two Phosphorus level treatment: normal phosphorus supply treatment (1250 μM KH2 PO4 ) and low phosphorus treatment (6.25 μM KH2 PO4 ), each square dish is a replicate, and each treatment has 5 replicates. Samples were collected on the 9th day after treatment, and the fresh weight of the aboveground part, the fresh weight of the underground part, the number of lateral roots and the length of the main root of the plants were measured.
结果如图4所示，在低磷处理条件下，超量表达GmCXIP1-1明显促进了转基因拟南芥的根系生长(图4A)。低磷条件下，转基因株系OX1和OX2的侧根数与WT相比增加了50％和20％(图4B)。在正常磷处理条件下，以WT相比，超量表达GmCXIP1-1的转基因株系OX1和OX2的主根长均增加了9％左右，在低磷处理条件下，转基因株系OX1和OX2的主根长与WT相比分别增加了17％和23％(图4C)。同时，在正常磷处理条件下，以WT相比，超量表达GmCXIP1-1的转基因株系OX1和OX2的地上部和根系鲜重均降低了20％左右，而在低磷处理条件下，转基因株系OX1和OX2的地上部和根系鲜重与WT相比分别增加了20％和40％(图4D和图4E)。The results are shown in Figure 4. Under low phosphorus treatment conditions, overexpression of GmCXIP1-1 significantly promoted root growth of transgenic Arabidopsis (Figure 4A). Under low phosphorus conditions, the number of lateral roots of transgenic lines OX1 and OX2 increased by 50% and 20% compared with WT (Fig. 4B). Under normal phosphorus treatment conditions, compared with WT, the main root length of transgenic lines OX1 and OX2 overexpressing GmCXIP1-1 increased by about 9%. Under low phosphorus treatment conditions, the main root lengths of transgenic lines OX1 and OX2 increased by about 9%. The length increased by 17% and 23% respectively compared with WT (Fig. 4C). At the same time, under normal phosphorus treatment conditions, compared with WT, the fresh weight of the shoots and roots of the transgenic lines OX1 and OX2 overexpressing GmCXIP1-1 were reduced by about 20%, while under low phosphorus treatment conditions, the transgenic lines OX1 and OX2 The fresh weight of shoots and roots of lines OX1 and OX2 increased by 20% and 40% respectively compared with WT (Figure 4D and Figure 4E).
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.
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