WO2024099366A1 - SlPIF4作为负调控因子在提升番茄果实褪黑素含量中的应用 - Google Patents

SlPIF4作为负调控因子在提升番茄果实褪黑素含量中的应用 Download PDF

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WO2024099366A1
WO2024099366A1 PCT/CN2023/130497 CN2023130497W WO2024099366A1 WO 2024099366 A1 WO2024099366 A1 WO 2024099366A1 CN 2023130497 W CN2023130497 W CN 2023130497W WO 2024099366 A1 WO2024099366 A1 WO 2024099366A1
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slpif4
gene
tomato
melatonin
seq
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张阳
张子昕
张昕
张静
江文倩
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四川大学
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  • the invention relates to the fields of plant molecular biology and plant genetic engineering, and to the application of SlPIF4 as a negative regulatory factor in improving the melatonin content of tomato fruits.
  • Melatonin N-acetyl-5-methoxytryptamine, Melatonin
  • Melatonin is an essential indoleamine compound. It is a broad-spectrum physiological regulator with conservative functions that exists in most biological organisms. It has been proven to be the most powerful endogenous free radical scavenger with antioxidant effects known so far, and has important physiological significance for the life processes of animals and plants. In animals, melatonin has the functions of improving sleep, delaying aging, relieving allergic symptoms, and regulating the immune system.
  • melatonin mainly functions as a growth promoter and antioxidant, with the activity of delaying aging, enhancing photosynthesis, regulating photoperiod, affecting seed germination and root morphology, regulating flowering and fruit ripening, scavenging free radicals, and reducing stress damage. It can give plants the ability to resist adverse environments and is conducive to plant survival and prosperity.
  • TDC Tryptophan decarboxylase
  • T5H tryptamine-5-hydroxylase
  • Serotonin can be catalyzed by 5-hydroxytryptamine-N-acetyltransferase (SNAT) to produce N-acetylserotonin (N-acetylserotonin), and then catalyzed by N-acetyl-5-hydroxytryptamine-methyltransferase (ASMT)/caffeic acid-O-methyltransferase (COMT) to produce melatonin.
  • ASMT N-acetyl-5-hydroxytryptamine-methyltransferase
  • CAT caffeic acid-O-methyltransferase
  • serotonin is first catalyzed by ASMT/COMT to produce 5-methoxytryptamine (5-methoxytryptamine), and then catalyzed by SNAT to produce melatonin.
  • Tomato melatonin is closely related to the formation of fruit quality, but the specific molecular mechanism of its regulation is not clear, and the regulatory network formed has always been one of the hot spots and difficulties in domestic and foreign research.
  • the study of the synthesis and metabolic pathways of melatonin, an important growth regulator, based on tomatoes will help to deeply understand its dynamic accumulation and various physiological activities, and deeply reveal the molecular mechanism of melatonin's participation in the regulation of different growth stages of plants.
  • Increasing the content of melatonin in tomatoes by modern biotechnology has broad market value and important production application value for the development of high-quality and efficient modern agriculture.
  • one of the objects of the present invention is to provide the use of knocking out or interfering with the expression of the tomato SlPIF4 gene in improving the melatonin content of tomato fruit;
  • the second object of the present invention is to provide the use of a fragment, a recombinant vector or a transformant for specifically interfering with the tomato SlPIF4 gene in improving the melatonin content of tomato fruit;
  • the third object of the present invention is to provide the use of a fragment, a recombinant vector or a transformant for specifically knocking out the tomato SlPIF4 gene in improving the melatonin content of tomato fruit;
  • the fourth object of the present invention is to provide a method for increasing the melatonin content of tomato fruit.
  • the present invention provides the following technical solutions:
  • the method for knocking out the tomato SlPIF4 gene of the present invention is as follows: using the CRISPR/Cas9 gene editing system to edit the tomato SlPIF4 gene.
  • the sgRNA target sequence of CRISPR/Cas9 is shown in SEQ ID NO.11 and SEQ ID NO.12.
  • the sgRNA sequence of CRISPR/Cas9 is obtained by amplifying the primers shown in SEQ ID NO.15 and SEQ ID NO.16.
  • the method for interfering with the expression of the tomato SlPIF4 gene is as follows: the interference target sequence of the tomato SlPIF4 gene is reversely connected to a vector to construct an interference vector.
  • the interference target sequence of the tomato SlPIF4 gene is shown in SEQ ID NO.49.
  • the tomato SlPIF4 gene action site is the second G-Box of proSlCOMT2, and the domain sequence is CACCTG.
  • a method for increasing the melatonin content in tomato fruit comprising knocking out/interfering with the expression of the SlPIF4 gene in tomatoes or knocking out the action site of the tomato SlPIF4 gene, and the resulting transgenic tomatoes are tomatoes with high melatonin content in the fruits.
  • the nucleotide sequence of the SlPIF4 gene is shown in SEQ ID NO.44.
  • the beneficial effect of the present invention is that the present invention discloses the application of the tomato SlPIF4 gene in regulating the melatonin content of tomato fruit.
  • SlPIF4 can inhibit the expression of genes related to the melatonin synthesis pathway, and interfering with or knocking out the gene can significantly increase the content of melatonin in the downstream pathway, indicating that the SlPIF4 transcription factor has important guiding significance for synthetic biology application research and genetic breeding quality improvement, and has important guiding significance and broad market prospects for synthetic biology research and genetic breeding quality improvement of tomato melatonin.
  • Figure 1 is a diagram of the structure of the recombinant plasmid zmpl-IF4-Cas9 vector.
  • Figure 2 is the real-time fluorescence quantitative PCR detection of SlPIF4 gene in tomato fruit of SlPIF4 overexpression and RNAi interference strains and SlCOMT2 gene expression (A: SlPIF4 gene; B: SlCOMT2 gene).
  • FIG. 3 shows the expression of the SlCOMT2 gene in tomato fruits of SlCOMT2 overexpression and RNAi interference strains detected by real-time fluorescence quantitative PCR.
  • FIG. 4 shows the melatonin content in SlPIF4 overexpression, RNAi interference strains and knockout strains determined by LC-MS/MS.
  • FIG. 5 shows the melatonin content in the overexpression, RNAi interference and knockout strains of SlCOMT2 determined by LC-MS/MS.
  • Figure 6 shows the verification of SlPIF4 binding to the second G-Box domain P2 of proSlCOMT2 through yeast one-hybrid, and the verification of SlPIF4 binding to proSlCOMT2 through tobacco Dual-LUC, thereby transcriptionally activating the expression of SlCOMT2 (A: proSlCOMT2; B: yeast one-hybrid verification; C: tobacco Dual-LUC verification results).
  • Figure 7 shows EMSA and tomato protoplast Dual-LUC verification that SlPIF4 binds to the second G-Box domain P2 of proSlCOMT2, thereby transcriptionally activating the expression of SlCOMT2 (A: EMSA detection results; B: LUC to REN ratio).
  • Figure 8 shows the melatonin content in the Cas9-proSlCOMT2 strain measured by LC-MS/MS.
  • RNA from tomato fruit was extracted according to the method of Plant RNA Extraction Kit v1.5 (BIOFIT), followed by agarose gel electrophoresis to ensure RNA quality, and then the RNA concentration was determined by NanoDrop 2,000. Further, total RNA (1 ⁇ g) from tomato fruit was reverse transcribed into first-strand cDNA according to the method described by the reverse transcription kit (PrimeScriptTM RT regent kit, TaKaRa). PCR amplification primers were designed according to the SlPIF4 and SlCOMT2 gene sequences, and the primer sequences are shown in Table 1.
  • the obtained cDNA was used as a template for PCR amplification.
  • the procedure was as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 20 s, 52°C annealing for 20 s, 72°C extension for 30 s, repeated for 34 cycles; 72°C final extension for 5 min; stored at 4°C.
  • PCR amplification was performed using the primers for SlPIF4 gene and SlCOMT2 gene in Table 1.
  • the PCR amplification product was gel-purified and sequenced to obtain the tomato SlPIF4 gene. and SlCOMT2 gene sequences.
  • the sequencing results showed that the obtained SlPIF4 gene was 1,488 bp in length, and its nucleotide sequence was shown in SEQ ID NO.44; the obtained SlPIF4 gene encoded 495 amino acids, and its amino acid sequence was shown in SEQ ID NO.45.
  • the obtained SlCOMT2 gene was 1,086 bp in length, and its nucleotide sequence was shown in SEQ ID NO.46; the SlCOMT2 gene encoded 361 amino acids, and its amino acid sequence was shown in SEQ ID NO.47.
  • Example 2 Amplification of 2000 bp of the promoter sequence of tomato proS1COMT2
  • PCR amplification primers were designed according to the 2000bp sequence of the proSlCOMT2 promoter. The primer sequences are shown in Table 2.
  • the obtained DNA was used as a template for PCR amplification, and the procedure was: 95°C pre-denaturation for 3min; 95°C denaturation for 20s, 52°C annealing for 20s, 72°C extension for 30s, repeated 34 cycles; 72°C final extension for 5min; stored at 4°C.
  • the PCR amplification product was gel-recovered, purified and sequenced to obtain the promoter sequence of tomato proSlCOMT2.
  • the sequencing results showed that the obtained proSlCOMT2 promoter sequence was 2,000bp in length, and its sequence is shown in SEQ ID NO.32.
  • the vector construction vector for plant overexpression was pCAMBIA1306, and the double enzyme digestion method was used to construct the vector.
  • the SlPIF4 overexpression vector connected the SlPIF4 gene to the pCAMBIA1306 vector with BamHI and AccI; the SlCOMT2 overexpression vector connected the SlCOMT2 gene to the pCAMBIA1306 vector with Sac I and AccI, and the SlPIF4 gene and SlCOMT2 gene were amplified with the primers shown in Sequence Table 3. After the connection vector was transformed, sequencing was performed, and the correct plasmid was compared for subsequent transformation. Both the overexpression SlPIF4 and SlCOMT2 genes were 35S constitutive promoters.
  • RNAi plant interference
  • Knockout plant gene knockout
  • the plant interference vector was constructed using the Gateway homologous recombination technology developed by Invitrogen TM . Fragments with higher gene specificity were selected for constructing the interference vector to obtain interference plasmids RNAi-SlPIF4 and RNAi-SlCOMT2.
  • the interference sequences used in the construction were SEQ ID NO.49 and SEQ ID NO.50.
  • Cas9-SlPIF4 is a sequence amplified from SEQ ID NO.15 and SEQ ID NO.16 and inserted into a plant transformation plasmid containing CRISPR/Cas9 gene editing function to obtain the recombinant plasmid zmpl-IF4-Cas9 (this vector was constructed by Wuhan Boyuan Biotechnology Co., Ltd., and the vector structure is shown in Figure 1);
  • Cas9-SlCOMT2 is a sequence amplified from SEQ ID NO.17 and SEQ ID NO.18 and inserted into a plant transformation plasmid containing CRISPR/Cas9 gene editing function to obtain the recombinant plasmid Zmpl-SlCOMT2-Cas9 (this vector was constructed by Wuhan Boyuan Biotechnology Co., Ltd., with IF4 in zmpl-IF4-Cas9 replaced by SlCOMT2).
  • Example 3 Stable genetic transformation of tomato mediated by Agrobacterium tumefaciens
  • the obtained plant transformation plasmids of the overexpression vectors of SlPIF4 and SlCOMT2, the interference SlPIF4 (RNAi-SIPIF4) expression vector and the knockout vector were respectively introduced into Agrobacterium tumefaciens EHA105 for infecting tomatoes.
  • the specific steps are as follows:
  • Preparation and detection of Agrobacterium Take 1 ⁇ L plasmid and add it to 50 ⁇ L EHA105 Agrobacterium tumefaciens competent cells, mix thoroughly and then transfer to the electroporation cup, add 1mL YEB liquid medium after electroporation, mix thoroughly and then transfer to a 1.5mL centrifuge tube, shake and culture on a shaker at 30°C and 180rpm for 30min, take 50 ⁇ L of the activated Agrobacterium liquid and inoculate it on the YEB solid medium, and culture it in the dark at 28°C for 48h.
  • the primers are shown in Table 1; after amplification, prepare 1% agarose gel for gel electrophoresis detection, the electrophoresis bands of the positive control and the sample are clear and of the correct size, and there are no bands in the negative control, indicating that the sample can enter the next step.
  • the positive plants of the T 0 generation were identified by PCR (identification primers are shown in Table 7 ), and the positive plants of the T 1 generation were obtained by self-pollination for subsequent experiments and determination of related compound contents.
  • plasmids containing SlCOMT2 (OX-SICOMT2) and interfering SlCOMT2 (RNAi-SlCOMT2) were transformed at the same time, and then the expression of SlCOMT2 gene was detected, and the results are shown in Figure 3.
  • OX-SICOMT2 strain the expression level of SlCOMT2 gene increased, while in the RNAi-SlCOMT2 strain, the expression level of SlCOMT2 gene decreased.
  • Example 4 Determination of melatonin content in tomato fruit by LC-MS/MS
  • Liquid chromatography tandem mass spectrometry can meet the requirements of accurate qualitative and quantitative determination of melatonin. The process is briefly described as follows:
  • the melatonin detection and data acquisition system mainly includes: Ultra Performance Liquid Chromatography (UPLC) (ExionLC TM AD, https://sciex.com.cn/) and Tandem Mass Spectrometry (MS/MS) ( 6500+,https://sciex.com.cn/).
  • UPLC Ultra Performance Liquid Chromatography
  • MS/MS Tandem Mass Spectrometry
  • the test results are shown in Figures 4 and 5.
  • the results showed that the melatonin content of the overexpression strain of SlPIF4 was lower than that of the wild type, but the melatonin content of the plant interference (RNAi) and gene knockout (Knockout) strains of SlPIF4 was significantly increased.
  • the melatonin content of the overexpression strain of SlCOMT2 was significantly increased, but the melatonin content of the plant interference (RNAi) and gene knockout (Knockout) strains of SlCOMT2 was significantly reduced.
  • SlPIF4 can inhibit the synthesis of melatonin, and its loss has a significant effect on melatonin synthesis
  • SlCOMT2 is an effective melatonin synthase gene that promotes the synthesis of melatonin.
  • the SlPIF4 obtained by the above cloning and the upstream 2000bp proSlCOMT2 sequence were constructed into the vector.
  • the construction of pB42AD, pGREENII0800-LUC, and pGEXT4 vectors refers to the seamless cloning kit (NO.B632219) of Sangon Biotech. As long as the ends of the inserted DNA fragments and the ends of the vector have 15-20 homologous base sequences, cloning and recombination can be completed at any site of the vector.
  • the vector is linearized by enzyme digestion or PCR amplification, and the target DNA fragment is PCR amplified.
  • the target DNA fragment and the linearized vector are added to the PCR tube at a certain molar ratio for recombination reaction.
  • the plasmid that is successfully transformed and sequenced correctly will be used for subsequent experiments.
  • the construction of pCAMBI1302 vector adopts the construction method of double enzyme digestion. The corresponding enzyme sites are added to the upstream and downstream primers, and the target gene and fragment are double-digested respectively. After connection and transformation, sequencing is performed, and the correct plasmid is compared for subsequent transformation. The other mentioned fragment sequences and their vector constructions are all completed by artificial synthesis. The interaction was verified using the mature yeast one-hybrid system, Dual-LUC and EMSA.
  • the yeast one-hybrid system uses the EGY48 yeast competent system, and the vectors used are pLacZi and pB42AD.
  • the blue plaques on the three-deficient plate coated with X-gal can confirm the interaction between DNA and protein. Dual-LUC was verified in tobacco leaves and tomato protoplasts, and the binding of DNA and protein was determined by fluorescent signals.
  • EMSA uses the EMSA probe biotin labeling kit of Beyotime Company and the chemiluminescent nucleic acid detection module kit of Thermo Scientific Company.
  • biotin-labeling EMSA probes and verifying the binding of DNA probes to proteins are mainly based on the different mobility of DNA-protein complexes in polyacrylamide gel electrophoresis to determine whether DNA interacts with proteins. By designing competition and mutation probe binding experiments, it is also possible to distinguish whether the interaction between DNA and protein is specific. See Table 6 for vector and primer information.
  • the second G-Box knockout vector of proSlCOMT2 was constructed, and then transformed into tomatoes, and the content of melatonin in the Cas9-proSlCOMT2 strain was determined by LC-MS/MS, and the results are shown in Figure 8. The results showed that the content of melatonin in the Cas9-proSlCOMT2 strain was increased, indicating that the content of melatonin can be increased by knocking out the second G-Box of proSlCOMT2.

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Abstract

本发明公开了SlPIF4作为负调控因子在提升番茄果实褪黑素含量中的应用，通过在番茄中以组成型启动子35S驱动的SlPIF4基因过量表达，降低了番茄果实中褪黑素的含量；而干扰和敲除株系的褪黑素含量提高，表明SlPIF4作为负调控因子抑制褪黑素的合成，因此可以通过干扰和敲除SlPIF4基因使褪黑素合成显著提高，可应用于番茄褪黑素的合成生物学研究和遗传育种品质改良。

Description

SlPIF4作为负调控因子在提升番茄果实褪黑素含量中的应用 技术领域
本发明涉及植物分子生物学与植物遗传工程领域，涉及SlPIF4作为负调控因子在提升番茄果实褪黑素含量中的应用。
背景技术
褪黑素(N-乙酰-5-甲氧基色胺，Melatonin)是一种生命必需的吲哚胺类化合物，是存在于绝大多数生物有机体中的一种功能保守的广谱生理调节剂，且已被证实是目前已知的抗氧化作用最强的内源自由基清除剂，对动植物生命进程均有重要的生理学意义。在动物中，褪黑素具有改善睡眠、延缓衰老、缓解过敏症状、调节免疫系统等功能。在植物中，褪黑素主要作为生长促进剂和抗氧化剂行使功能，具有延缓衰老、增强光合作用、调节光周期、影响种子萌发和根形态建成、调控开花及果实成熟、清除自由基、减轻胁迫损伤的活性，能够赋予植物抵抗不良环境的能力，有利于植物生存繁荣。
植物褪黑素的合成起始于自身合成前体色氨酸(Tryptophan)，需要经过四个连续的酶促反应。色氨酸脱羧酶(TDC)和色胺-5-羟化酶(T5H)是褪黑素合成前两步的关键酶，催化产生血清素(5-羟基色胺，serotonin)，血清素能够被5-羟色胺-N-乙酰转移酶(SNAT)催化生成N-乙酰血清素(N-acetylserotonin)，继而被N-乙酰基-5-羟色胺-甲基转移酶(ASMT)/咖啡酸-O-甲基转移酶(COMT)催化生成褪黑素。也存在另一条支路即血清素先被ASMT/COMT催化生成5-甲氧基色胺(5-methoxytryptamine)，再被SNAT催化生成褪黑素。
番茄褪黑素与果实品质形成密切相关，但其调控的具体分子机制并不清楚，所形成的调控网络也一直是国内外研究的热点和难点之一。以番茄为底盘对重要生长调节物质褪黑素合成和代谢途径的研究，有助于深入理解其动态积累和多种生理活性，深入揭示褪黑素参与植物不同生长期调控的分子机理。以现代生物技术手段提高番茄中褪黑素的含量，具有广泛市场价值，对优质、高效现代化农业的发展也具有重要的生产应用价值。
发明内容
有鉴于此，本发明的目的之一在于提供敲除或干扰番茄SlPIF4基因表达在提升番茄果实褪黑素含量中的应用；本发明的目的之二在于提供用于特异性干扰番茄SlPIF4基因的片段、重组载体或转化子在提升番茄果实褪黑素含量中的应用；本发明的目的之三在于提供用于特异性敲除番茄SlPIF4基因的片段、重组载体或转化子在提升番茄果实褪黑素含量中的应用；本发明的目的之四在于提供一种提高番茄果实褪黑素含量的方法。
为达到上述目的，本发明提供如下技术方案：
1、敲除/干扰番茄SlPIF4基因或番茄SlPIF4基因作用位点表达在提升番茄果实褪黑素含量中的应用，所述SlPIF4基因的核苷酸序列如SEQ ID NO.44所示。
本发明优选的，敲除番茄SlPIF4基因的方法如下：使用CRISPR/Cas9基因编辑系统编辑番茄SlPIF4基因。
本发明优选的，所述CRISPR/Cas9的sgRNA靶序列如SEQ ID NO.11和SEQ ID NO.12所示。
本发明优选的，所述CRISPR/Cas9的sgRNA序列由SEQ ID NO.15和SEQ ID NO.16所示引物扩增获得。
本发明优选的，干扰番茄SlPIF4基因表达的方法如下：将番茄SlPIF4基因的干扰靶序列反向连接于载体中构建得到干扰载体。
本发明优选的，所述番茄SlPIF4基因的干扰靶序列如SEQ ID NO.49所示。
本发明优选的，所述番茄SlPIF4基因作用位点为proSlCOMT2的第二个G-Box，结构域序列为CACCTG。
2、用于特异性干扰番茄SlPIF4基因的片段、重组载体或转化子在提升番茄果实褪黑素含量中的应用，所述SlPIF4基因的核苷酸序列如SEQ ID NO.44所示。
3、用于特异性敲除番茄SlPIF4基因的片段、重组载体或转化子在提升番茄果实褪黑素含量中的应用，所述SlPIF4基因的核苷酸序列如SEQ ID NO.44所示。
4、一种提高番茄果实褪黑素含量的方法，将在番茄中SlPIF4基因敲除/干扰表达或敲出番茄SlPIF4基因作用位点，得到的转基因番茄为果实褪黑素含量高的番茄，所述SlPIF4基因的核苷酸序列如SEQ ID NO.44所示。
本发明的有益效果在于：本发明公开了番茄SlPIF4基因在调控番茄果实褪黑素含量的应用。研究发现SlPIF4能够抑制褪黑素合成途径相关基因表达，干扰或敲除该基因能够显著提升下游途径褪黑素的含量，表明SlPIF4转录因子对于合成生物学应用研究和遗传育种品质改良具有重要的指导意义，对于番茄褪黑素的合成生物学研究和遗传育种品质改良具有重要的指导意义和广阔的市场前景。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为重组质粒zmpl-IF4-Cas9载体结构图。
图2为实时荧光定量PCR检测SlPIF4过表达和RNAi干扰株系番茄果实中SlPIF4基因 和SlCOMT2基因的表达情况(A：SlPIF4基因；B:SlCOMT2基因)。
图3为通过实时荧光定量PCR检测SlCOMT2过表达和RNAi干扰株系番茄果实中SlCOMT2基因的表达情况。
图4为通过LC-MS/MS测定的SlPIF4过表达、RNAi干扰株系和敲除株系中褪黑素含量。
图5为通过LC-MS/MS测定的SlCOMT2的过表达、RNAi干扰株系和敲除株系中褪黑素含量。
图6为通过酵母单杂验证SlPIF4结合proSlCOMT2的第二个G-Box结构域P2，及通过烟草Dual-LUC验证SlPIF4结合proSlCOMT2，从而转录激活SlCOMT2的表达(A:proSlCOMT2；B:酵母单杂验证验证；C:烟草Dual-LUC验证结果)。
图7为EMSA及番茄原生质体Dual-LUC验证SlPIF4结合proSlCOMT2的第二个G-Box结构域P2，从而转录激活SlCOMT2的表达(A:EMSA检测结果；B：LUC与REN比值)。
图8为通过LC-MS/MS测定的Cas9-proSlCOMT2株系中褪黑素的含量。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好的理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1、番茄SlPIF4和SlCOMT2的基因克隆
参考植物RNA提取试剂盒v1.5(BIOFIT)的方法，提取番茄果实的总RNA，随后进行琼脂糖凝胶电泳检测，以确保RNA质量，然后通过NanoDrop 2,000测定RNA浓度。进一步，参考反转录试剂盒(PrimeScriptTM RT regent kit,TaKaRa)描述的方法，将番茄果实总RNA(1μg)反转录成第一链cDNA。根据SlPIF4和SlCOMT2基因序列设计PCR扩增引物，引物序列见表1。
表1、番茄SlPIF4和SlCOMT2的克隆引物
以获得的cDNA为模板进行PCR扩增，程序为：95℃预变性3min；95℃变性20s，52℃退火20s，72℃延伸30s，重复34个循环；72℃终延伸5min；保存4℃。以表1中的SlPIF4基因和SlCOMT2基因引物进行PCR扩增，PCR扩增产物进行胶回收纯化并测序，获得番茄SlPIF4 和SlCOMT2基因序列。测序结果显示，获得的SlPIF4基因全长1,488bp，其核苷酸序列如SEQ ID NO.44所示；所述获得的SlPIF4基因编码495个氨基酸，其氨基酸序列如SEQ ID NO.45所示。获得的SlCOMT2基因全长1,086bp，其核苷酸序列如SEQ ID NO.46所示；SlCOMT2基因编码361个氨基酸，其氨基酸序列如SEQ ID NO.47所示。
实施例2、番茄proSlCOMT2的启动子序列2000bp的扩增
参考植物基因组的SDS提取方法，提取番茄果实DNA。具体如下，将新鲜样品置于1.5mL离心管中，加入400μL DNA提取缓冲液(0.2M Tris-HCl pH8.0，0.4M LiCl，25mM EDTA和1％SDS)，研磨后65℃水浴10min，13000rpm离心10min。取上清液300μL转移到新的离心管中，加入300μL异丙醇混合均匀，-20℃静置1h。13000rpm离心10min，弃上清。加入200μL75％的乙醇，13000rpm离心5min，弃废液。加入200μL无水乙醇重复离心一次，弃废液。晾干后，加入65℃预热的ddH2O 30μL以溶解基因组DNA。根据proSlCOMT2启动子2000bp序列设计PCR扩增引物，引物序列见表2。
表2、番茄proSlCOMT2的扩增引物
以获得的DNA为模板进行PCR扩增，程序为：95℃预变性3min；95℃变性20s，52℃退火20s，72℃延伸30s，重复34个循环；72℃终延伸5min；保存4℃。将PCR扩增产物进行胶回收纯化并测序，获得番茄proSlCOMT2的启动子序列。测序结果显示，获得的proSlCOMT2启动子序列全长2,000bp，其序列如SEQ ID NO.32所示。
实施例2、植物过表达的载体构建
植物过表达的载体构建载体为pCAMBIA1306，采用双酶切的构建方法，过表达SlPIF4载体将SlPIF4基因用BamHⅠ和AccⅠ连入pCAMBIA1306载体上；过表达SlCOMT2载体将SlCOMT2基因用Sac I和AccⅠ连入pCAMBIA1306载体上，SlPIF4基因和SlCOMT2基因用序列表3所示引物扩增。连接载体转化后进行测序，比对正确的质粒进行后续转化。过表达SlPIF4和SlCOMT2基因均为35S组成型启动子。
表3、番茄过表达载体的扩增引物

实施例3、植物干扰(RNAi)和植物基因敲除(Knockout)的载体构建
植物干扰载体的构建采用InvitrogenTM开发的Gateway同源重组技术，选择基因特异性较高的片段用于构建干扰载体，得到干扰质粒RNAi-SlPIF4，RNAi-SlCOMT2，构建使用的干扰序列如SEQ ID NO.49和SEQ ID NO.50。
植物基因敲除载体的构建首先通过在线软件CRISPR-P2.0(http://crispr.hzau.edu.cn/CRISPR2)设计基因编辑靶位点，采用双靶位点策略。利用50μL的PCR扩增体系，酶切连接后进行转化和菌斑PCR鉴定。SlPIF4和SlCOMT2的靶位点序列见表4，扩增引物见表5，构建成功的菌液PCR鉴定引物见表6。其中Cas9-SlPIF4是由SEQ ID NO.15和SEQ ID NO.16扩增获得的序列插入到含有CRISPR/Cas9基因编辑功能的植物转化质粒，获得重组质粒zmpl-IF4-Cas9(该载体以由武汉伯远生物科技有限公司构建，载体结构如图1所示)；Cas9-SlCOMT2由SEQ ID NO.17和SEQ ID NO.18扩增获得的序列插入到含有CRISPR/Cas9基因编辑功能的植物转化质粒，获得重组质粒Zmpl-SlCOMT2-Cas9(该载体以由武汉伯远生物科技有限公司构建，由zmpl-IF4-Cas9中的IF4替换为SlCOMT2)。
表4、靶位点设计信息
表5、Cas9构建的扩增引物

表6、菌液PCR鉴定引物
实施例3、根癌农杆菌介导的番茄稳定遗传转化
将所述获得的分别SlPIF4和SlCOMT2的过表达载体、干扰SlPIF4(RNAi-SIPIF4)表达载体和敲除载体的植物转化质粒分别导入根癌农杆菌EHA105，用于浸染番茄。具体步骤如下：
a.农杆菌准备和检测:取1μL质粒加入50μL EHA105根癌农杆菌感受态细胞中，充分混匀后吸取至电转杯中，电转后加1mL YEB液体培养基，充分混匀后吸取至1.5mL离心管中，于摇床30℃、180rpm振荡培养30min，将活化好的农杆菌菌液吸取50μL接种于YEB固体培养基上，28℃暗培养48h。配制PCR扩增体系，完成配制后充分混匀，使用PCR仪进行扩增，扩增程序根据引物信息等进行相应设置，引物见表1；扩增后,制备1％琼脂糖凝胶进行凝胶电泳检测，阳性对照及样品的电泳条带清晰、大小正确，且阴性对照均无条带的情况下，表明该样品可进入下一步骤。
b.番茄遗传转化:萌发的番茄组培苗，待子叶完全展开，用手术刀切为约0.5cm小段接种于预培养基，23℃预培养1～2d。挑取农杆菌于侵染液中，制备OD600＝0.5的农杆菌重悬液；侵染10min，将晾干的外植体接于共培培养基中，23℃暗培养2d。将共培后的外植体用1g/L的头孢水清洗两次，15min/次，接种于恢复培养基中，23℃、16h/8h光照/黑暗培养3～5d。将恢复培养的愈伤接种于筛选培养基，23℃、16h/8h光照/黑暗培养15-30d；筛选出的愈伤接种于分化培养基，23℃、16h/8h光照/黑暗培养30-40d。待分化的丛生芽生长至2-3cm左右，将其从愈伤上切除，接种于含有1mg/L IAA的生根培养基上，23℃、16h/8h光照/黑暗培养10-15d。待番茄幼苗完成生根，用镊子夹住轻轻取出，小心洗去根部残留的培养基，在1/3 开口的组培瓶中炼苗1-2天，用ddH2O覆盖幼苗根部防止失水，随后移栽至营养土中，待植物长势健壮之后，揭开保鲜膜正常培养。
通过PCR鉴定T0代阳性植株(鉴定引物见表7)，并继续自交一代获得T1代阳性植物，用于后续实验和相关化合物含量测定。
表7、阳性植株PCR鉴定引物
然后通过荧光定量PCR番茄植物中SlPIF4和SlCOMT2基因表达情况，检测引物如表8。
表8、qPCR鉴定引物
检测结果如图2所示。结果显示，RNAi-SlPIF4干扰株系的番茄果实中SlPIF4基因表达量显著降低，而SlCOMT2基因的表达量显著上调，说明SlPIF4能够抑制SlCOMT2基因的表达。
按照相同的方法，同时转化含SlCOMT2(OX-SICOMT2)和干扰SlCOMT2(RNAi-SlCOMT2)的质粒，然后检测SlCOMT2基因的表达情况，结果如图3所示。结果显示，在OX-SICOMT2株系中，SlCOMT2基因表达量上升，而RNAi-SlCOMT2株系，SlCOMT2基因表达量下降。
实施例4、通过LC-MS/MS测定番茄果实褪黑素的含量
液相色谱串联质谱(LC-MS/MS)能够满足褪黑素测定准确定性和定量的要求。其分析 流程简单描述如下：
(1)褪黑素的提取
a.磨样(液氮速冻的新鲜样品或存储在负80摄氏度的冷冻干燥的样品)，用低温研磨仪(50Hz,磨3次，每次1min)至粉末状；
b.用1mL含0.1％甲酸的80％甲醇/水(V/V)提取1g新鲜样品或100mg冻干样品，将混合物涡旋30s，4℃超声30min，再涡旋30s；20000g离心10min，过0.22μm有机滤膜后于棕色瓶中保存，用于LC-MS/MS分析。
(2)色谱质谱采集
褪黑素检测与数据采集系统主要包括：超高效液相色谱(Ultra Performance Liquid Chromatography,UPLC)(ExionLCTM AD,https://sciex.com.cn/)和串联质谱(Tandem Mass Spectrometry,MS/MS)(6500+,https://sciex.com.cn/)。
检测结果如图4和图5所示。结果显示，SlPIF4的超表达株系褪黑素含量相对野生型有所降低，但SlPIF4的植物干扰(RNAi)和基因敲除(Knockout)株系褪黑素含量显著提高。而SlCOMT2的超表达株系褪黑素含量显著提高，但SlCOMT2的植物干扰(RNAi)和基因敲除(Knockout)株系褪黑素含量显著降低。这说明SlPIF4能够抑制褪黑素的合成，其缺失对褪黑素合成有显著影响；SlCOMT2是有效的褪黑素合成酶基因，促进褪黑素的合成。
实施例5、SlPIF4和proSlCOMT2互作情况及作用位点验证
将上述克隆获得的SlPIF4和上游2000bp的proSlCOMT2序列构建至载体上。pB42AD、pGREENⅡ0800-LUC、pGEXT4载体的构建参考Sangon Biotech的无缝克隆试剂盒(NO.B632219)，只需插入的DNA片段末端与载体末端具有15-20个同源碱基序列就可以在载体的任意位点完成克隆重组。首先采用酶切或PCR扩增的方法将载体线性化，进行目的DNA片段的PCR扩增，将目的DNA片段与线性化载体以一定的摩尔比加到PCR管中进行重组反应。将转化成功并测序正确的质粒进行后续实验。pCAMBI1302载体的构建是采用双酶切的构建方法，将相应的酶切位点加在上下游引物上，将目的基因和片段分别进行双酶切，连接转化后进行测序，比对正确的质粒进行后续转化。其它提到的片段序列及其载体构建均为人工合成构建完成。利用成熟的酵母单杂体系、Dual-LUC及EMSA验证其互作情况。其中酵母单杂交体系是利用EGY48的酵母感受态系统，用到的载体为pLacZi和pB42AD，能够在涂了X-gal的三缺板上变蓝的菌斑即能确定DNA与蛋白之间存在相互作用。Dual-LUC在烟草叶片和番茄原生质体中都进行了验证，通过荧光信号判定DNA与蛋白的结合情况。EMSA采用的是碧云天公司EMSA探针生物素标记试剂盒和Thermo Scientific公司的化学发光核酸检测模块试剂盒， 通过生物素标记EMSA探针、验证DNA探针与蛋白的结合等步骤进行，主要是依据DNA-蛋白质复合体在聚丙烯酰胺凝胶电泳中的不同迁移率，来判定DNA是否与蛋白质相互作用。通过设计竞争和突变探针的结合实验，还可以区分DNA与蛋白质的相互作用是否具有特异性。载体及引物信息见表6。
表9、载体及引物信息
结果如图6和图7所示，SlPIF4能够结合在SlCOMT2的启动子上，抑制其表达，继而进一步证明了SlPIF4是结合在proSlCOMT2的第二个G-Box(CACCTG)结构域上，突变该结构域番茄果实褪黑素含量显著升高。
按照实施例3的方法构建proSlCOMT2的第二个G-Box敲除(Knockout)载体，然后转化番茄，并通过LC-MS/MS测定的Cas9-proSlCOMT2株系中褪黑素的含量，结果如图8所示。结果显示，Cas9-proSlCOMT2株系中褪黑素的含量提高，表明可以通过敲除proSlCOMT2的第二个G-Box来提高褪黑素的含量。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (10)

  1. 敲除/干扰番茄SlPIF4基因或番茄SlPIF4基因作用位点表达在提升番茄果实褪黑素含量中的应用，其特征在于：所述SlPIF4基因的核苷酸序列如SEQ ID NO.44所示。
  2. 根据权利要求1所述的应用，其特征在于：敲除番茄SlPIF4基因的方法如下：使用CRISPR/Cas9基因编辑系统编辑番茄SlPIF4基因。
  3. 根据权利要求2所述的应用，其特征在于：所述CRISPR/Cas9的sgRNA靶序列如SEQ ID NO.11和SEQ ID NO.12所示。
  4. 根据权利要求2所述的应用，其特征在于：所述CRISPR/Cas9的sgRNA序列由SEQ ID NO.15和SEQ ID NO.16所示引物扩增获得。
  5. 根据权利要求1所述的应用，其特征在于：干扰番茄SlPIF4基因表达的方法如下：将番茄SlPIF4基因的干扰靶序列反向连接于载体中构建得到干扰载体。
  6. 根据权利要求5所述的应用，其特征在于：所述番茄SlPIF4基因的干扰靶序列如SEQ ID NO.49所示。
  7. 根据权利要求1所述的应用，其特征在于：所述番茄SlPIF4基因作用位点为proSlCOMT2的第二个G-Box，结构域序列为CACCTG。
  8. 用于特异性干扰番茄SlPIF4基因的片段、重组载体或转化子在提升番茄果实褪黑素含量中的应用，其特征在于：所述SlPIF4基因的核苷酸序列如SEQ ID NO.44所示。
  9. 用于特异性敲除番茄SlPIF4基因的片段、重组载体或转化子在提升番茄果实褪黑素含量中的应用，其特征在于：所述SlPIF4基因的核苷酸序列如SEQ ID NO.44所示。
  10. 一种提高番茄果实褪黑素含量的方法，其特征在于：将在番茄中SlPIF4基因敲除/干扰表达或敲出番茄SlPIF4基因作用位点，得到的转基因番茄为果实褪黑素含量高的番茄，所述SlPIF4基因的核苷酸序列如SEQ ID NO.44所示。
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