技术领域Technical Field
本发明涉及农业植物保护技术领域，尤其涉及一种作为核酸载体递送dsRNA的碳基纳米材料及其制备方法与应用。The present invention relates to the technical field of agricultural plant protection, and in particular to a carbon-based nanomaterial used as a nucleic acid carrier to deliver dsRNA, and a preparation method and application thereof.
背景技术Background technique
由病毒引起的烟草花叶病是烟草生产上重要的病害之一，对烟草病毒病的防治农药品种少、效果差，使病毒病成为烟草栽培中的“绝症”，其造成的产量损失可达30-50％，严重时甚至造成绝产。自然状态下能侵染烟草的病毒有近20种，其中烟草普通花叶病毒(Tobacco mosaic virus，TMV)以及烟蚜传播的黄瓜花叶病毒(Cucumber mosaic virus，CMV)和芜菁花叶病毒(Potato virus Y，TuMV)分布广、危害大，是我国烟区的主要病毒病病原。同时，由于病毒病传播快、难防治，目前尚无有效单一的防治措施，以“预防为主，综合防治”为主，但植物一旦感染病毒，则很难采取有效措施进行治疗。Tobacco mosaic disease caused by virus is one of the important diseases in tobacco production. There are few varieties of pesticides for the prevention and treatment of tobacco virus disease and poor effect, which makes virus disease become a "terminal disease" in tobacco cultivation. The yield loss caused by it can reach 30-50%, and even causes a complete loss of production in severe cases. There are nearly 20 viruses that can infect tobacco under the natural state, among which tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) and turnip mosaic virus (TuMV) spread by tobacco aphids are widely distributed and have great harm, and are the main virus disease pathogens in my country's tobacco areas. At the same time, due to the fast spread and difficulty in prevention and treatment of virus disease, there is no effective single prevention and treatment measure at present, and "prevention is the main, comprehensive prevention and treatment" is the main one. However, once the plant is infected with the virus, it is difficult to take effective measures to treat it.
RNA干扰(RNAinterference，RNAi)是指内源或外源性的双链RNA分子诱导细胞内特定基因沉默的现象。RNAi是核酸药物研发的机制，高度特异性靶向目标基因，从而实现在基因水平上进行病理治疗。当下该技术已在肿瘤、癌症和糖尿病等医学领域研究成为热门之一，但与哺乳动物细胞不同，植物细胞具有细胞壁，构成了外源生物大分子递送的主要屏障，同时因RNA在环境中易受到RNA酶的攻击，导致RNA降解等因素限制，在植物领域研究仍处于初始阶段，尤其是在抗病毒方面的研究。RNA interference (RNAi) refers to the phenomenon that endogenous or exogenous double-stranded RNA molecules induce the silencing of specific genes in cells. RNAi is the mechanism of nucleic acid drug development, which targets target genes with high specificity, thereby achieving pathological treatment at the genetic level. At present, this technology has become one of the hot research topics in medical fields such as tumors, cancer and diabetes. However, unlike mammalian cells, plant cells have cell walls, which constitute the main barrier to the delivery of exogenous biomacromolecules. At the same time, because RNA is susceptible to attack by RNases in the environment, RNA degradation and other factors limit research in the plant field, especially in the field of antiviral research.
目前常见的基因载体包括天然高分子载体如壳聚糖、淀粉，或纳米载体如纳米管、纳米金、聚乙烯亚胺，但是壳聚糖类天然高分子在植物细胞内载核酸量低、转化效率低，而纳米载体如纳米金，金作为重金属在植物体内存在富集作用，导致安全性低，同时纳米金获取成本高，难以在农业生产中应用。纳米管的制备流程繁杂，存在分散性差易团聚沉降等缺点，也限制了其进一步应用，聚乙烯亚胺是否能穿透细胞壁进入植物细胞内也需要进一步研究。纳米石墨氮化碳(g-C3N4)属于重要的光催化的碳基纳米材料，具有带隙窄(2.7eV)、结构稳定等优点，在癌细胞成像和药物缓释领域有一定的研究，但是该碳基纳米材料也存在难以透过细胞壁和载核酸量低的问题，因此，扩宽纳米石墨氮化碳的应用途径，进一步发掘可运输RNA的纳米材料，研究其在抗病毒方面的功能，为我国绿色农业发展提供新的技术支持。At present, common gene carriers include natural polymer carriers such as chitosan, starch, or nanocarriers such as nanotubes, nanogold, and polyethyleneimine. However, chitosan-based natural polymers have low nucleic acid loading and low conversion efficiency in plant cells, while nanocarriers such as nanogold, as a heavy metal, have an enrichment effect in plants, resulting in low safety. At the same time, the cost of obtaining nanogold is high, making it difficult to use in agricultural production. The preparation process of nanotubes is complicated, and there are disadvantages such as poor dispersibility and easy agglomeration and sedimentation, which also limits its further application. Whether polyethyleneimine can penetrate the cell wall and enter the plant cell also needs further research. Nanographite carbon nitride (g-C3N4) is an important carbon-based nanomaterial for photocatalysis. It has the advantages of narrow band gap (2.7eV) and stable structure. It has been studied in the field of cancer cell imaging and drug sustained release. However, this carbon-based nanomaterial also has the problem of difficulty in penetrating the cell wall and low nucleic acid loading. Therefore, it is necessary to expand the application path of nanographite carbon nitride, further explore nanomaterials that can transport RNA, and study its antiviral function to provide new technical support for the development of green agriculture in my country.
发明内容Summary of the invention
有鉴于此，本发明的目的是提供一种作为核酸载体递送dsRNA的碳基纳米材料及其制备方法与应用，解决目前递送载体制备过程中载核酸量低、安全性差，同时制备的递送载体难以穿透植物细胞壁的缺点。In view of this, the purpose of the present invention is to provide a carbon-based nanomaterial for delivering dsRNA as a nucleic acid carrier and a preparation method and application thereof, so as to solve the shortcomings of low nucleic acid loading and poor safety in the current preparation process of delivery vectors, and the difficulty of the prepared delivery vectors in penetrating plant cell walls.
本发明通过以下技术手段解决上述技术问题：The present invention solves the above technical problems by the following technical means:
一种作为核酸载体递送dsRNA的碳基纳米材料，所述碳基纳米材料是由碳点氮化碳经聚乙烯亚胺修饰后得到具有表面突刺的纳米材料。A carbon-based nanomaterial used as a nucleic acid carrier to deliver dsRNA. The carbon-based nanomaterial is a nanomaterial with surface spikes obtained by modifying carbon dot carbon nitride with polyethyleneimine.
进一步，所述碳基纳米材料还可通过静电吸附负载来源于植物病毒的双链RNA(dSrna)。Furthermore, the carbon-based nanomaterial can also load double-stranded RNA (dSRNA) derived from plant viruses through electrostatic adsorption.
进一步，所述RNA病毒为烟草普通花叶病毒、黄瓜花叶病毒、芜菁花叶病毒中的一种或多种。Furthermore, the RNA virus is one or more of tobacco common mosaic virus, cucumber mosaic virus, and turnip mosaic virus.
常见的碳基纳米材料在递送核酸的时候载核酸量低、且较动物细胞相比植物细胞具有难以突破细胞壁，因此本发明制备的纳米材料表面具有突刺结构，不仅具有较高的比表面积，更容易突破植物细胞壁，方便载有病毒的纳米材料进入。同时纳米材料表面具有突刺结构，可以防止酶与植物核酸接触，免其受到酶的攻击，导致核酸降解。Common carbon-based nanomaterials have low nucleic acid loading when delivering nucleic acids, and are more difficult to break through the cell walls of plant cells than animal cells. Therefore, the nanomaterial prepared by the present invention has a thorn structure on the surface, which not only has a higher specific surface area, but also makes it easier to break through the plant cell wall, facilitating the entry of nanomaterials carrying viruses. At the same time, the thorn structure on the surface of the nanomaterial can prevent enzymes from contacting plant nucleic acids, preventing them from being attacked by enzymes and causing nucleic acid degradation.
进一步，所述纳米材料的制备方法包括以下步骤：Furthermore, the method for preparing the nanomaterial comprises the following steps:
(1)碳点氮化碳制备：将尿素与柠檬酸钠按10：8的质量比混合后研磨充分，随后以5℃/min的升温速率加热至175-190℃，保温50min后加入草酸和氢氧化钙，继续保温反应30min后，降温至120℃，加入枸橼酸钠保持反应10min，经无水乙醇溶解洗涤后，于12000rpm离心条件下离心10min，透析后即得到碳点氮化碳溶液，50℃干燥得到粉末状碳点氮化碳，待用；(1) Preparation of carbon dot carbon nitride: urea and sodium citrate were mixed in a mass ratio of 10:8 and then ground thoroughly. The mixture was then heated to 175-190°C at a heating rate of 5°C/min. After being kept warm for 50 minutes, oxalic acid and calcium hydroxide were added. The mixture was kept warm for 30 minutes, then cooled to 120°C, sodium citrate was added, and the reaction was continued for 10 minutes. After being dissolved and washed with anhydrous ethanol, the mixture was centrifuged at 12000 rpm for 10 minutes. After dialysis, a carbon dot carbon nitride solution was obtained. The solution was dried at 50°C to obtain a powdered carbon dot carbon nitride for later use.
(2)聚乙烯亚胺修饰：取制备的5mg碳点氮化碳溶解于10mL 1mM MES(pH＝7.5)缓冲液中，震荡混匀30-40min后，加入24mg N-羟基琥珀酰亚胺和72mg 1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐充分混匀溶解，随后再加入60μL聚乙烯亚胺充分混匀后孵育8h，过夜透析制备得到具有表面突刺的纳米材料。(2) Polyethylenimine modification: 5 mg of the prepared carbon dot carbon nitride was dissolved in 10 mL of 1 mM MES (pH = 7.5) buffer and shaken for 30-40 min. Then, 24 mg of N-hydroxysuccinimide and 72 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride were added and mixed thoroughly to dissolve. Subsequently, 60 μL of polyethyleneimine was added and mixed thoroughly, incubated for 8 h, and dialyzed overnight to prepare a nanomaterial with surface spikes.
进一步，所述草酸、氢氧化钙、枸橼酸钠的质量比为1：(0.9-1.1)：1。Furthermore, the mass ratio of oxalic acid, calcium hydroxide and sodium citrate is 1:(0.9-1.1):1.
进一步，所述尿素和柠檬酸钠总质量与草酸的质量比为1：(0.3-0.5)。Furthermore, the mass ratio of the total mass of the urea and sodium citrate to the mass of oxalic acid is 1:(0.3-0.5).
通过尿素和柠檬酸钠合成过程中加入草酸和氢氧化钙，于纳米材料的表面生成草酸钙针状晶体，草酸钙针状晶体引导纳米材料合成过程中球形表面生成不规则的突刺结构，但是由于该纳米材料的用于植物病毒核酸递送，因此需要控制纳米材料表面的突刺数目以及突刺长度，避免植物病毒核酸难以负载以及突刺过长造成的载核酸量降低和对防治植物造成损伤的问题，因此保温反应后加入枸橼酸钠，抑制草酸钙晶体过多聚集于纳米材料表面，进而控制突刺长度和数量。By adding oxalic acid and calcium hydroxide during the synthesis of urea and sodium citrate, calcium oxalate needle-shaped crystals are generated on the surface of the nanomaterial. The calcium oxalate needle-shaped crystals guide the spherical surface to form irregular thorn structures during the synthesis of the nanomaterial. However, since the nanomaterial is used for plant viral nucleic acid delivery, it is necessary to control the number and length of thorns on the surface of the nanomaterial to avoid the problem of difficulty in loading plant viral nucleic acids and reduced nucleic acid loading and damage to the control plants due to excessive length of the thorns. Therefore, sodium citrate is added after the insulation reaction to inhibit excessive aggregation of calcium oxalate crystals on the surface of the nanomaterial, thereby controlling the length and number of the thorns.
进一步，所述纳米材料负载植物病毒的双链RNA的步骤如下：Furthermore, the steps of loading the double-stranded RNA of plant viruses onto the nanomaterial are as follows:
(1)dSRNA表达载体的构建：选取长度为400bp的植物病毒RNA病毒CP基因，引物序列如SEQ ID NO：1-2所示，将PCR扩增获得大产物采用无缝克隆技术插入PstI和XbaI限制性内切酶消化后的L440载体中，同时转入HT115感受态细胞中，得到L440-CP重组载体；(1) Construction of dSRNA expression vector: A plant viral RNA virus CP gene with a length of 400 bp was selected, and the primer sequences were shown in SEQ ID NO: 1-2. The large product obtained by PCR amplification was inserted into the L440 vector digested with PstI and XbaI restriction endonucleases using seamless cloning technology, and then transformed into HT115 competent cells to obtain the L440-CP recombinant vector;
(2)dsRNA提取：将上述获得的L440-CP重组载体转化到HT115大肠杆菌中，挑取鉴定获得阳性克隆，对阳性菌株在37℃、200rpm的恒温摇床培养获得种子液，次日扩大培养，按照1：100的体积比将种子液加入到LB培养基中，使菌液初始OD600＝0.1，继续在37℃、200rpm的恒温摇床中培养直至OD600＝0.5,加入适量IPTG(IPTG终浓度为8mM)，继续诱导培养7h，采用TRIZOL提取总RNA；(2) dsRNA extraction: The L440-CP recombinant vector obtained above was transformed into HT115 Escherichia coli, and positive clones were selected and identified. The positive strains were cultured in a constant temperature shaker at 37°C and 200 rpm to obtain seed liquid. The next day, the culture was expanded and the seed liquid was added to LB medium at a volume ratio of 1:100 to make the initialOD600 of the bacterial liquid = 0.1. The culture was continued in a constant temperature shaker at 37°C and 200 rpm untilOD600 = 0.5. An appropriate amount of IPTG (the final concentration of IPTG was 8 mM) was added, and the induction culture was continued for 7 h. Total RNA was extracted using TRIZOL;
(3)纳米材料负载dsRNA：将纳米材料与总RNA按照质量比(4-10)：1的比例混合后超声10min，得到负载有植物RNA病毒的纳米材料。(3) Nanomaterials loaded with dsRNA: The nanomaterials were mixed with total RNA at a mass ratio of (4-10):1 and then sonicated for 10 min to obtain nanomaterials loaded with plant RNA viruses.
进一步，所述植物病毒为烟草普通花叶病毒(TMV)、黄瓜花叶病毒(CMV)、芜菁花叶病毒(TuMV)中的一种或多种。Furthermore, the plant virus is one or more of tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and turnip mosaic virus (TuMV).
当然的，在dSRNA表达载体的构建时，可获得含L440-TMV CP、L440-TuMVCP、L440-CMV CP重组载体的菌株。Of course, when constructing the dSRNA expression vector, strains containing L440-TMV CP, L440-TuMVCP, and L440-CMV CP recombinant vectors can be obtained.
进一步，所述纳米材料用于植物病毒防治领域。Furthermore, the nanomaterial is used in the field of plant virus prevention and control.
进一步，所述植物病毒防治为烟草普通花叶病毒、黄瓜花叶病毒、芜菁花叶病毒防治。Furthermore, the plant virus control is control of tobacco common mosaic virus, cucumber mosaic virus, and turnip mosaic virus.
本发明制备的材料可携带大量靶向病毒dsRNA并快速递送进植株，产生相关siRNA，进一步提升抗病毒效果。The material prepared by the present invention can carry a large amount of targeted virus dsRNA and quickly deliver it into plants to produce related siRNA, further improving the antiviral effect.
有益效果：Beneficial effects:
本发明制备出了一种用于植物病毒防治领域且防治效果显著的纳米材料，同时解决了目前递送载体在制备过程中载核酸量低且安全性差的问题，将该材料运用于TMV病毒的本氏烟防治上，该材料明显的穿透了植物细胞壁，显著降低了病毒的积累量，达到了防治目的。除此之外，制备的材料具有较好的水溶性和植物细胞成像能力，因此可以在植物防治领域有较宽的应用范围。The present invention prepares a nano material for plant virus control with significant control effect, and solves the problem of low nucleic acid loading and poor safety of the current delivery carrier in the preparation process. The material is applied to the control of TMV virus in Nicotiana benthamiana. The material obviously penetrates the plant cell wall, significantly reduces the accumulation of the virus, and achieves the purpose of control. In addition, the prepared material has good water solubility and plant cell imaging ability, so it can have a wide range of applications in the field of plant control.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1：L440-CP载体示意图；Figure 1: Schematic diagram of L440-CP vector;
图2：L440-TMV CP重组载体构建及IPTG诱导表达dsRNA，其中a为TMV CP PCR扩增片段，b为TMV CP诱导产生的dsRNA；c为IPTG不同浓度的筛选，浓度依次是0、1、2、4、6、8mM；Figure 2: Construction of L440-TMV CP recombinant vector and IPTG-induced expression of dsRNA, where a is the TMV CP PCR amplification fragment, b is the dsRNA induced by TMV CP; c is the screening of different concentrations of IPTG, the concentrations are 0, 1, 2, 4, 6, 8 mM in sequence;
图3：纳米材料TEM，SEM表征图；Figure 3: TEM and SEM characterization images of nanomaterials;
图4：纳米材FT-IR表征图；Figure 4: FT-IR characterization of nanomaterials;
图5：TMV CP-纳米材料不同负载比例结果图(1：8，1：4，1：2，1：0，0：1，1：1，2：1，4：1，6：1，8：1，10：1)；Figure 5: Results of different loading ratios of TMV CP-nanomaterials (1:8, 1:4, 1:2, 1:0, 0:1, 1:1, 2:1, 4:1, 6:1, 8:1, 10:1);
图6：病毒防治效果图。Figure 6: Virus prevention and control effect diagram.
具体实施方式Detailed ways
以下将结合具体实施例和附图对本发明进行详细说明：The present invention will be described in detail below with reference to specific embodiments and accompanying drawings:
实施例1：提取植物烟草普通花叶病毒(TMV)的dsRNAExample 1: Extraction of dsRNA from tobacco mosaic virus (TMV)
(1)dSRNA表达载体的构建：选取长度为400bp的TMV CP基因，引物序列SEQ ID NO1(L440-TMV CP-F)为：GCCTTATACAATCAACTCTCC；引物序列SEQ ID NO 2(L440-TMV CP-R)为：GAACCAGTTCATTAGCCAAA；将PCR扩增获得的产物采用无缝克隆技术插入经PstI和XbaI限制性内切酶消化后的L440载体中，同时转入HT115感受态细胞中，获得含L440-TMV CP重组载体；(1) Construction of dsRNA expression vector: A 400 bp TMV CP gene was selected, and the primer sequence SEQ ID NO 1 (L440-TMV CP-F) was GCCTTATACAATCAACTCTCC; the primer sequence SEQ ID NO 2 (L440-TMV CP-R) was GAACCAGTTCATTAGCCAAA; the product obtained by PCR amplification was inserted into the L440 vector digested with PstI and XbaI restriction endonucleases using seamless cloning technology, and then transferred into HT115 competent cells to obtain a recombinant vector containing L440-TMV CP;
(2)dsRNA提取：将上述步骤(1)中获得的L440-TMV CP重组载体转化到HT115大肠杆菌中，挑取鉴定获得阳性克隆，对阳性菌株在37℃、200rpm的恒温摇床培养获得种子液，次日扩大培养，按照1：100体积比将种子液加入到LB培养基中，使菌液初始OD600＝0.1，继续在37℃、200rpm的恒温摇床中培养直至OD600＝0.5,加入适量IPTG(IPTG浓度依次是0、1、2、4、6、8mM)，继续诱导培养7h，采用TRizol提取总RNA。(2) dsRNA extraction: The L440-TMV CP recombinant vector obtained in step (1) above was transformed into HT115 Escherichia coli, and positive clones were selected and identified. The positive strains were cultured in a constant temperature shaker at 37°C and 200 rpm to obtain seed liquid. The culture was expanded the next day, and the seed liquid was added to LB medium at a volume ratio of 1:100 to make the initialOD600 of the bacterial liquid = 0.1. The culture was continued in a constant temperature shaker at 37°C and 200 rpm untilOD600 = 0.5, and an appropriate amount of IPTG was added (the IPTG concentrations were 0, 1, 2, 4, 6, and 8 mM, respectively). The induction culture was continued for 7 h, and total RNA was extracted using TRizol.
上述过程的示意图如图1所示，L440-TMV CP重组载体构建及IPTG诱导表达dsRNA如图2所示。The schematic diagram of the above process is shown in FIG1 , and the construction of L440-TMV CP recombinant vector and IPTG-induced expression of dsRNA are shown in FIG2 .
实施例2：纳米材料制备Example 2: Nanomaterial Preparation
(1)碳点氮化碳制备：将10g尿素与8g柠檬酸钠混合后研磨充分，随后以5℃/min的升温速率加热至190℃，保温50min后，加入5.4g草酸和4.86g氢氧化钙，继续190℃保温反应30min后，降温至120℃，加入5.4g枸橼酸钠保持反应10min，经无水乙醇溶解洗涤后，于12000rpm离心条件下离心10min，透析后即得到碳点氮化碳溶液，50℃干燥得到粉末状碳点氮化碳，待用；(1) Preparation of carbon dot carbon nitride: 10 g urea and 8 g sodium citrate were mixed and ground thoroughly, and then heated to 190°C at a heating rate of 5°C/min. After keeping the temperature for 50 min, 5.4 g oxalic acid and 4.86 g calcium hydroxide were added, and the temperature was kept at 190°C for 30 min. Then, the temperature was lowered to 120°C, 5.4 g sodium citrate was added, and the reaction was continued for 10 min. After being dissolved and washed with anhydrous ethanol, the mixture was centrifuged at 12000 rpm for 10 min. After dialysis, a carbon dot carbon nitride solution was obtained, which was dried at 50°C to obtain a powdered carbon dot carbon nitride for standby use.
(2)聚乙烯亚胺修饰：分别取5mg上述碳基纳米材料溶解于10mL pH＝7.5的1mMMES缓冲液中，震荡混匀30min后加入24mg N-羟基琥珀酰亚胺和72mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐充分混匀溶解，加入60μL聚乙烯亚胺充分混匀后孵育8h，过夜透析制备得到具有表面突刺的纳米材料CDs。(2) Polyethyleneimine modification: 5 mg of the above carbon-based nanomaterials were dissolved in 10 mL of 1 mM MES buffer (pH = 7.5), and 24 mg of N-hydroxysuccinimide and 72 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added and mixed thoroughly for 30 min. 60 μL of polyethyleneimine was added and mixed thoroughly, and then incubated for 8 h. The nanomaterial CDs with surface spikes were prepared by overnight dialysis.
对制备的纳米材料采用TEM、SEM、FT-IR进行表征，得到的结果如图3和图4所示。The prepared nanomaterials were characterized by TEM, SEM and FT-IR, and the results are shown in Figures 3 and 4.
分析图3-4的结果可知：The results of analyzing Figure 3-4 show that:
1、对CDs进行表征，根据TEM结果合成制备CDs为直径约为20nm的表面突刺的纳米材料。同时FT-IR结果表明在1713cm-1处的吸收峰为C＝O键伸缩振动峰，1350cm-1处的吸收峰为C-O键伸缩振动峰，说明制备的CDs存在羧基；1448cm-1和1585cm-1处的吸收峰分别对应C-N伸缩振动吸收峰和N-H弯曲振动吸收峰，表明所制备的CDs存在氨基，位于2901cm-1处的吸收峰为C-H伸缩振动吸收峰。红外表征结果表明，合成的荧光CDs带有-OH、-NH2、-COOH等官能团，使得CDs具有较好的水溶性，因此在植物防治领域具有较宽的应用场景。1. Characterize CDs. According to the TEM results, CDs synthesized and prepared are nanomaterials with surface spikes of about 20 nm in diameter. At the same time, the FT-IR results show that the absorption peak at 1713cm-1 is the C=O bond stretching vibration peak, and the absorption peak at 1350cm-1 is the CO bond stretching vibration peak, indicating that the prepared CDs have carboxyl groups; the absorption peaks at 1448cm-1 and 1585cm-1 correspond to the CN stretching vibration absorption peak and the NH bending vibration absorption peak, respectively, indicating that the prepared CDs have amino groups, and the absorption peak at 2901cm-1 is the CH stretching vibration absorption peak. The infrared characterization results show that the synthesized fluorescent CDs have functional groups such as -OH, -NH2, and -COOH, which makes CDs have good water solubility, and therefore have a wide range of application scenarios in the field of plant control.
对实施例2制备的纳米材料进行细胞成像测试，得到的结果如图5所示。The nanomaterial prepared in Example 2 was subjected to a cell imaging test, and the obtained results are shown in FIG5 .
1、实验方法：称取5mg纳米材料溶解于10mL的水中，配制浓度为0.5mg/mL，的溶液，取0.2mL溶液采用无头注射器对16C转基因进行注射，次日采用共聚焦进行观察。1. Experimental method: Weigh 5 mg of nanomaterials and dissolve them in 10 mL of water to prepare a solution with a concentration of 0.5 mg/mL. Take 0.2 mL of the solution and inject it into the 16C transgene using a headless syringe. Use confocal microscopy for observation the next day.
2、结果分析：由图5的结果可知，发现纳米次材料发射红色荧光且与GFP(绿色荧光蛋白)重叠，表明纳米材料可穿透植物细胞壁，并定位于植物细胞质膜上，具有良好的细胞成像能力。2. Result analysis: From the results of Figure 5, it can be seen that the nanomaterials emit red fluorescence and overlap with GFP (green fluorescent protein), indicating that the nanomaterials can penetrate the plant cell wall and locate on the plant cell plasma membrane, and have good cell imaging capabilities.
实施例3：TMV CP-纳米材料制备Example 3: TMV CP-nanomaterial preparation
纳米材料负载dsRNA：将实施例2制备得到的纳米材料与实施例1制备的总RNA按照质量比4：1的比例混合，功率36W的超声条件下超声10min后得到TMV CP-纳米材料，即CDS-TMV CP。Nanomaterial loaded with dsRNA: The nanomaterial prepared in Example 2 was mixed with the total RNA prepared in Example 1 at a mass ratio of 4:1, and ultrasonicated for 10 min at a power of 36 W to obtain TMV CP-nanomaterial, namely CDS-TMV CP.
此外，还按照纳米材料：总RNA质量比分别为1：8，1：4，1：2，1：0，0：1，1：1，2：1，4：1，6：1，8：1，10：1的比例按照上述方法进行负载，得到的结果如图5所示。In addition, the above method was used to load the nanomaterials:total RNA at a mass ratio of 1:8, 1:4, 1:2, 1:0, 0:1, 1:1, 2:1, 4:1, 6:1, 8:1, and 10:1, respectively. The results are shown in FIG5 .
根据图5的结果可知，按照4：1、6：1，8：1，10：1的质量比可以完全负载RNA，从实际的成本和考虑节约材料出发，可以优选为按照4：1的比例进行负载。According to the results of FIG5 , RNA can be completely loaded at a mass ratio of 4:1, 6:1, 8:1, and 10:1. From the perspective of actual cost and material saving, it is preferred to load RNA at a ratio of 4:1.
实施例4：抗病毒应用Example 4: Antiviral Application
1)抗病毒处理，于4周龄本式烟叶片上分别喷施200uL等量的MES、CDs(实施例2制备纳米材料CDs)、CP(实施例1制备的总RNA)、CDS-CP(实施例3制备的CDS-TMV CP)，次日用毛笔蘸取涂抹的方式接种接种TMV-GFP，病毒接种量为100uL，观察接种病毒第3天、第5天的植株图，得到的结果如图6所示。1) Antiviral treatment: 200uL of MES, CDs (nanomaterial CDs prepared in Example 2), CP (total RNA prepared in Example 1), and CDS-CP (CDS-TMV CP prepared in Example 3) were sprayed on 4-week-old tobacco leaves respectively. The next day, TMV-GFP was inoculated with a brush in a smearing manner. The virus inoculation amount was 100uL. The plant pictures on the 3rd and 5th days after the virus inoculation were observed. The results are shown in Figure 6.
根据图6的结果可知：According to the results in Figure 6, we can see that:
本发明制备的TMV CP-纳米材料可以快速通过细胞壁进入植物细胞，该纳米材料进入细胞后会被降解为20nt左右的siRNA，当TMV侵染植物后其编码的CP基因会被大量siRNA靶向结合，从而被降解，而TMV则无法翻译外壳蛋白和组装病毒粒子，从而失活，进而防治TMV病毒。具体的，接种后第3天和第5天在紫外线灯下观察TMV-GFP浸染本氏烟后的植株情况，其中CDS-CP的病斑数量明显的少于MES、CDs、CP处理后的病斑数量，说明本发明制备的纳米材料负载RNA后可以有效的防治植物TMV病毒。The TMV CP-nanomaterial prepared by the present invention can quickly pass through the cell wall and enter the plant cell. After entering the cell, the nanomaterial will be degraded into siRNA of about 20 nt. When TMV infects the plant, the CP gene encoded by it will be targeted and bound by a large number of siRNAs, thereby being degraded, and TMV cannot translate the capsid protein and assemble virus particles, thereby being inactivated, thereby preventing and controlling the TMV virus. Specifically, the plants of Nicotiana benthamiana infected with TMV-GFP were observed under ultraviolet light on the 3rd and 5th days after inoculation, and the number of lesions of CDS-CP was significantly less than that of the lesions after MES, CDs, and CP treatment, indicating that the nanomaterial prepared by the present invention can effectively prevent and control the plant TMV virus after loading RNA.
为了验证制备的纳米材料对递送dsRNA后对植物的病毒的防治效果，本发明还制备了对比例，具体的制备方法如下：In order to verify the control effect of the prepared nanomaterial on plant viruses after delivering dsRNA, the present invention also prepared a comparative example, and the specific preparation method is as follows:
对比例1：Comparative Example 1:
(1)碳点氮化碳制备：将10g尿素与8g柠檬酸钠混合后研磨充分，随后以5℃/min的升温速率加热至190℃，保温1h，经无水乙醇溶解洗涤后，于12000rpm离心条件下离心10min，透析后即得到碳点氮化碳溶液，50℃干燥得到粉末状碳点氮化碳，待用；(1) Preparation of carbon dot carbon nitride: 10 g urea and 8 g sodium citrate were mixed and ground thoroughly, then heated to 190°C at a heating rate of 5°C/min, kept warm for 1 h, dissolved and washed with anhydrous ethanol, centrifuged at 12000 rpm for 10 min, and dialyzed to obtain a carbon dot carbon nitride solution, which was dried at 50°C to obtain a powdered carbon dot carbon nitride for later use;
(2)聚乙烯亚胺修饰：分别取5mg上述碳基纳米材料溶解于10mL pH＝7.5的1mMMES缓冲液中，震荡混匀30min后加入24mg N-羟基琥珀酰亚胺和72mg1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐充分混匀溶解，加入60μL聚乙烯亚胺充分混匀后孵育8h，过夜透析制备得到纳米材料。(2) Polyethyleneimine modification: 5 mg of the above carbon-based nanomaterials were dissolved in 10 mL of 1 mM MES buffer (pH = 7.5), and 24 mg of N-hydroxysuccinimide and 72 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added and mixed thoroughly for 30 min. 60 μL of polyethyleneimine was added and mixed thoroughly, and the mixture was incubated for 8 h. The nanomaterials were prepared by overnight dialysis.
对比例2：Comparative Example 2:
本对比例与实施例2的区别仅在于加入5.4g草酸和4.86g氢氧化钙后继续190℃保温反应30min后，随后不进行降温操作，仍采用190℃的温度加入5.4g枸橼酸钠保持反应10min，经无水乙醇溶解洗涤后，于12000rpm离心条件下离心10min，透析后即得到碳点氮化碳溶液，50℃干燥得到粉末状碳点氮化碳，待用；The difference between this comparative example and Example 2 is that after adding 5.4g of oxalic acid and 4.86g of calcium hydroxide, the reaction is continued at 190°C for 30min, and then the temperature is not lowered. 5.4g of sodium citrate is added at 190°C to keep the reaction for 10min, and after dissolving and washing with anhydrous ethanol, the mixture is centrifuged at 12000rpm for 10min, and a carbon point carbon nitride solution is obtained after dialysis, and the powdered carbon point carbon nitride is dried at 50°C for standby use;
后续的制备过程以及聚乙烯亚胺修饰步骤相同。The subsequent preparation process and polyethyleneimine modification steps are the same.
对比例3：Comparative Example 3:
本对比例与实施例2的区别仅在于，制备粉末状碳点氮化碳时，草酸、氢氧化钙、枸橼酸钠的质量比为1：0.5：1，而实施例2的质量比为1：0.9：1；The difference between this comparative example and Example 2 is that when preparing powdered carbon dot carbon nitride, the mass ratio of oxalic acid, calcium hydroxide and sodium citrate is 1:0.5:1, while the mass ratio of Example 2 is 1:0.9:1;
后续的制备过程以及聚乙烯亚胺修饰步骤相同。The subsequent preparation process and polyethyleneimine modification steps are the same.
对比例4：Comparative Example 4:
本对比例与实施例2的区别仅在于，制备粉末状碳点氮化碳时，草酸、氢氧化钙、枸橼酸钠的质量比为1：1.5：1，而实施例2的质量比为1：0.9：1；The difference between this comparative example and Example 2 is that when preparing powdered carbon dot carbon nitride, the mass ratio of oxalic acid, calcium hydroxide and sodium citrate is 1:1.5:1, while the mass ratio of Example 2 is 1:0.9:1;
后续的制备过程以及聚乙烯亚胺修饰步骤相同。The subsequent preparation process and polyethyleneimine modification steps are the same.
对比例5：Comparative Example 5:
本对比例与实施例2的区别仅在于，制备粉末状碳点氮化碳时，尿素和柠檬酸钠总质量与草酸的质量比为1：0.1，而实施例2的质量比为1：0.5；The difference between this comparative example and Example 2 is that when preparing powdered carbon dot carbon nitride, the mass ratio of the total mass of urea and sodium citrate to oxalic acid is 1:0.1, while the mass ratio of Example 2 is 1:0.5;
后续的制备过程以及聚乙烯亚胺修饰步骤相同。The subsequent preparation process and polyethyleneimine modification steps are the same.
对比例6：Comparative Example 6:
本对比例与实施例2的区别仅在于，制备粉末状碳点氮化碳时，尿素和柠檬酸钠总质量与草酸的质量比为1：1，而实施例2的质量比为1：0.5；The difference between this comparative example and Example 2 is that when preparing powdered carbon dot carbon nitride, the mass ratio of the total mass of urea and sodium citrate to oxalic acid is 1:1, while the mass ratio of Example 2 is 1:0.5;
后续的制备过程以及聚乙烯亚胺修饰步骤相同。The subsequent preparation process and polyethyleneimine modification steps are the same.
将对比例1-6制备的纳米材料采用实施例3的方法负载植物烟草普通花叶病毒(TMV)的总RNA，负载方法与实施例3相同，分别得到TMV CP-纳米材料。The nanomaterials prepared in Comparative Examples 1-6 were loaded with total RNA of tobacco mosaic virus (TMV) using the method of Example 3, and the loading method was the same as that of Example 3, to obtain TMV CP-nanomaterials respectively.
对上述对比例制备的TMV CP-纳米材料对TMV进行防治实验，具体实验过程如下：The TMV CP-nanomaterial prepared in the above comparative example was subjected to a TMV prevention and treatment experiment, and the specific experimental process is as follows:
(1)植物培养：在温室(光周期L：D＝16：8h，25℃，相对湿度75-80％，光合有效辐射100μmol·m-2·s-1)内培养本氏烟植株至5叶期；(1) Plant cultivation: N. benthamiana plants were cultivated in a greenhouse (photoperiod L:D = 16:8 h, 25°C, relative humidity 75-80%, photosynthetically active radiation 100 μmol·m-2 ·s-1 ) until the 5-leaf stage;
(2)样品：(2) Samples:
实验组：实施例3制备的TMV CP-纳米材料；Experimental group: TMV CP-nanomaterial prepared in Example 3;
对照组：对比例1-6制备的TMV CP-纳米材料；Control group: TMV CP-nanomaterials prepared in Comparative Examples 1-6;
清水对照；Clear water control;
(3)实验过程：样品按照实施例4的抗病毒处理方法进行处理，用毛笔蘸取涂抹的方式接种TMV-GFP病毒，于第5天后观察植株病斑面积(将病斑面积占据整张叶片的比例统计：无病斑为-；病斑面积＜10％为+，10％≤病斑面积≤30％为++，30％＜病斑面积≤50％+++，50％＜病斑面积≤70％++++，病斑面积＞70％为+++++)，得到的结果如表1所示。(3) Experimental process: The samples were treated according to the antiviral treatment method of Example 4, and TMV-GFP virus was inoculated by smearing with a brush. The diseased spot area of the plants was observed after 5 days (the proportion of the diseased spot area to the entire leaf was calculated: no diseased spot was -; diseased spot area <10% was +, 10% ≤ diseased spot area ≤30% was ++, 30% < diseased spot area ≤50% +++, 50% < diseased spot area ≤70% ++++, and diseased spot area >70% was +++++). The results are shown in Table 1.
表1Table 1
根据表1的结果可知，本发明制备的TMV CP-纳米材料具有良好的防治植物病毒的效果，尤其与对比例1相比防治效果显著提升，且与对比例2-6相比可知，无论是更改原料用量或者是更改制备条件，均降低了制备的纳米材料的防治效果。According to the results in Table 1, the TMV CP-nanomaterial prepared by the present invention has a good effect on preventing and controlling plant viruses, especially compared with Comparative Example 1, the prevention and control effect is significantly improved, and compared with Comparative Examples 2-6, it can be seen that whether changing the amount of raw materials or changing the preparation conditions, the prevention and control effect of the prepared nanomaterial is reduced.
以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。本发明未详细描述的技术、形状、构造部分均为公知技术。The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the preferred embodiments, a person skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all well-known technologies.
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