Red raspberry rusai gene, expression protein and application thereof

Abstract

The application discloses a red raspberry RuSA1 gene, an expression protein thereof and application, and relates to the technical field of plant genetic engineering.The red raspberry RuSA1 gene disclosed by the application has a nucleotide sequence as shown in SEQ ID NO.1, and the amino acid sequence of the expression protein thereof is as shown in SEQ ID NO.2.The application constructs an expression vector of the red raspberry RuSA1 gene, and then transforms the expression vector into tomatoes;transgenic tomato plants with significantly increased anthocyanin content are obtained through cultivation and screening.The RuSA1 gene promotes the synthesis of anthocyanins in the fruits of the transgenic lines.The expression amounts of the genes SlABA, SlNCED1, SlACS2 and SlACO1 related to the secretion of ABA hormone, the gene SlIAA36 related to the secretion of IAA hormone, and the genes Sl2ox4 and Sl20x2 related to the secretion of GA hormone are all significantly higher than those of CK.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and more particularly relates to a red raspberry RuSA1 gene, an expression protein thereof and application thereof. BACKGROUND

[0002] Red raspberry, also known as Rubus idaeus L., is a shrub plant of Rosaceae family and Rubus genus, with a height of 1-2 meters; young branches are covered with soft hair and spines, leaves are long ovate or elliptic; petal is spoon-shaped, covered with short soft hair or no hair, white; fruit is nearly spherical, aggregate fruit, red or orange yellow or black when mature, densely covered with short fluff. The fruit contains rich secondary metabolites, has the effects of benefiting kidney, solidifying essence, shrinking urine, nourishing liver and eyesight, anti-cancer, anti-oxidation, anti-aging, antibacterial and anti-inflammatory.

[0003] In the field of cell biology, signal transduction pathway is one of the important ways of interaction between cells and outside. In this signal transduction network, SA pathway is considered as an important pathway, which plays a key regulatory role in the life process and function of cells. SA pathway affects important physiological processes such as cell proliferation, differentiation and apoptosis, and stress response to external environment by regulating the expression and activity of a series of genes. In addition, SA can promote the accumulation of fruit polyphenols and the expression of flavonoid key pathway genes (Wei Ying, 2021). SA gene and its pathway may affect the main nutritional components and antioxidant capacity of plants, thereby improving the disease resistance of plants and being beneficial to the growth and development of plants. In order to explore the regulation mechanism of SA pathway and the function of key genes, improve the antioxidant components and growth potential of red raspberry plants, and provide strong support for production, utilization and breeding. Therefore, it has important scientific and application value to analyze the expression of red raspberry RuSA1 gene and its application research. SUMMARY

[0004] In view of the above problems existing in the prior art, the technical problems to be solved by the present application are to provide a red raspberry RuSA1 gene. Another technical problem to be solved by the present application is to provide an expression protein of the red raspberry RuSA1 gene. The present application also solves the technical problem of providing an application of the red raspberry RuSA1 gene for regulating the synthesis and accumulation of hormones in plants.

[0005] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0006] A red raspberry RuSA1 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0007] The expression protein of the red raspberry RuSA1 gene, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0008] The vector containing the red raspberry RuSA1 gene, the recombinant bacteria or the host cell.

[0009] The application of the red raspberry RuSA1 gene in regulating anthocyanin synthesis in fruits.

[0010] The anthocyanin synthesis is promoted.

[0011] The application comprises:

[0012] 1) constructing an expression vector of the red raspberry RuSA1 gene;

[0013] 2) transforming the expression vector into tomatoes;

[0014] 3) cultivating, screening and obtaining transgenic tomato plants with significantly increased anthocyanin content.

[0015] The application of the red raspberry RuSA1 gene in improving the total antioxidant capacity of tomato fruits.

[0016] The application of the red raspberry RuSA1 gene in promoting the expression of hormone-related genes.

[0017] The hormone-related genes comprise genes related to ABA hormone secretion, genes related to IAA hormone secretion and genes related to GA hormone secretion.

[0018] The genes related to ABA hormone secretion are SlABA, SlNCED1, SlACS2 and SlACO1; the gene related to IAA hormone secretion is SlIAA36; and the genes related to GA hormone secretion are Sl2ox4 and Sl20x2.

[0019] Compared with the prior art, the application has the following beneficial effects:

[0020] 1) The red raspberry RuSA1 gene disclosed in the application has a nucleotide sequence as shown in SEQ ID NO. 1, and the amino acid sequence of the expressed protein is as shown in SEQ ID NO. 2. The application constructs an expression vector of the red raspberry RuSA1 gene, transforms the expression vector into tomatoes, and cultivates, screens and obtains transgenic tomato plants with significantly increased anthocyanin content.

[0021] 2) The results of the examples of the application show that the RuSA1 gene promotes the growth of tomato fruits and reduces the hardness at maturity. With the growth and development of the fruits, the mass, transverse diameter and longitudinal diameter of the fruits of the strains RuSA1-1 and RuSA1-8 gradually increase, and the hardness gradually decreases. Compared with the control group, the mass, transverse diameter and longitudinal diameter of the fruits of the strains RuSA1-1 and RuSA1-8 are significantly increased, and the difference in hardness is not significant.

[0022] 3) The results of the embodiments of the present application show that the RuSA1 gene promotes the accumulation of anthocyanins and total polyphenols and other substances in tomato fruits, and improves the total antioxidant capacity.

[0023] 4) The results of the embodiments of the present application show that the expression levels of the genes SlABA, SlNCED1, SlACS2 and SlACO1 related to the secretion of ABA hormone, the gene SlIAA36 related to the secretion of IAA hormone, and the genes Sl2ox4 and Sl20x2 related to the secretion of GA hormone in the fruits of the lines RuSA1-1 and RuSA1-8 are significantly higher than those of CK. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Fig. 1 is a graph showing the expression levels of the red raspberry RuSA1 gene at different development stages of red raspberry fruits (S1 is the green fruit period; S2 is the green-yellow fruit period; S3 is the yellow fruit period; S4 is the yellow-red color-changing fruit period; and S5 is the red fruit period);

[0025] Figure 2 Fig. 4 is a graph showing the expression level analysis of the RuSA1 gene in transgenic tomato leaves;

[0026] Figure 3 Fig. 5 is a graph showing the expression level analysis of the RuSA1 gene in transgenic tomato fruits at different development stages (S1 is the green fruit period; S2 is the yellow fruit period; S3 is the orange fruit period; S4 is the orange-red fruit period; and S5 is the red fruit period);

[0027] Figure 4 Fig. 6 is a graph showing the appearance of non-transgenic and transgenic tomato fruits of the line RuSA1-1;

[0028] Figure 5 Fig. 7 is a graph showing the determination of physiological indexes of transgenic tomato fruits;

[0029] Figure 6 Fig. 8 is a graph showing the expression level analysis of hormone-related genes in transgenic tomato fruits. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described below in combination with specific embodiments. If no detailed description is given in the following embodiments, the technical means used are all conventional means well known to those skilled in the art.

[0031] The red raspberry material selected in the present application was planted in the experimental base of the Jiangsu Institute of Botany of the Chinese Academy of Sciences in Baima Town, Lishui District, Nanjing City, Jiangsu Province; green fruits, green-yellow fruits, yellow fruits, yellow-red color-changing fruits and red fruits of red raspberries at different periods were picked respectively, immediately frozen in liquid nitrogen after fresh picking, and then stored at -80℃ for RNA extraction.

[0032] Tomato infection materials selected in this application were planted in the tissue culture room of the Science Building of the Jiangsu Province, Chinese Academy of Sciences, Institute of Botany (North Garden of Nanjing Zhongshan Botanical Garden); tomato leaves and fruits of different periods, green fruit, yellow fruit, orange fruit, orange-red fruit and red fruit were picked, immediately placed in liquid nitrogen after picking, then stored at -80°C for RNA extraction and subsequent physiological index determination.

[0033] Example 1

[0034] 1. Total RNA extraction and cDNA acquisition

[0035] Total RNA of red raspberry and tomato was extracted using a plant polysaccharide polyphenol total RNA extraction kit (BioTeke, Beijing, China), and the integrity of the RNA was detected by agarose gel electrophoresis. The concentration and purity of the RNA were detected using a Nanodrop 2000c spectrophotometer (Thermo Fisher, Waltham, MA, USA). Reverse transcription into cDNA was performed using a PrimeScript RT Master Mix kit (TaKaRa, Otsu, Japan).

[0036] 2. Cloning of red raspberry RuSA1 gene

[0037] According to the red raspberry genome sequence, the Oligo 7.0 software was used to design primers, and the open reading frame ORF sequence of the candidate gene was cloned by high-speed high-fidelity PCR enzyme PrimeSTAR Max DNA Polymerase. The primer sequences are as follows:

[0038] RuSA1 ORF-F: 5'-ATGGAGAATATAAGACTGAAGC-3',

[0039] RuSA1 ORF-R: 5'-AGAATATTTCTGAGCAACCTTG-3'.

[0040] The gel cutting recovery and purification of the target fragment were performed using a fast agarose gel DNA recovery kit (BioTeKe, Beijing, China), and then the product after gel cutting recovery and purification was connected with a T vector using a pClone007 Blunt Vector Kit reagent kit (TSINGKE, Beijing, China), and was transferred into E. coli competent cells, and after short recovery, was coated on an ampicillin-resistant medium, and after overnight culture at 37°C, single colonies were selected. The 2xT5 Super PCR Mix (Colony) was used for bacterial liquid PCR verification (TSINGKE, Beijing, China), and the positive bacterial liquid was sent to Nanjing Qikexin Biotechnology Co., Ltd. for first-generation Sanger sequencing verification.

[0041] The nucleotide sequence of the red raspberry RuSA1 gene obtained by final sequencing is shown as SEQ ID NO. 1, and the amino acid sequence of the expressed protein is shown as SEQ ID NO. 2.

[0042] 3, Expression pattern analysis of red raspberry RuSA1 gene

[0043] The cDNA of different fruit periods of red raspberry was subjected to RT-qPCR experiment using TB Green Premix TaqⅡreagent (Takara, Dalian, China). The qRT-PCR reaction was performed using a qTOWER2.2 real-time PCR system (Analytik, Jena, Germany), and the reaction system and steps were performed according to the manufacturer's instructions. According to the 2 -ΔΔCT method, the relative expression level of the candidate red raspberry gene RuSA1 in different fruit periods was detected with 18S as the internal reference gene. The primer sequences are as follows:

[0044] RuSA1 qRT-F: 5'-TGGAGCTGGTCATGGAGCGT-3',

[0045] RuSA1 qRT-R: 5'-TCGGGTTGATCCCGGATGCT-3'.

[0046] The results are shown as Figure 1 follows: the expression amount of the RuSA1 gene in the growth and development process of red raspberry first decreased and then increased, and the expression amount was the lowest at S2 stage, which was 0.09, and the expression amount was the highest at S5 stage, which was 5.10.

[0047] Example 2

[0048] 1, Construction of red raspberry RuSA1 gene overexpression vector and transformation of agrobacterium

[0049] Gateway technology was used to construct a red raspberry overexpression vector. The target fragment from the entry vector (Linker-7) was transferred to the overexpression vector Pbi121-des-3HA, driven by the strong 35S promoter, via LR reaction. Successful bacterial cultures were used to extract plasmids. 100 ng of the plasmid to be transformed was added to 100 μL of competent Agrobacterium tumefaciens GV3101 for transformation. The plasmids were then plated on a kanamycin- and rifampicin-resistant culture medium and incubated at 28°C for 2 days. Single colonies were randomly selected, verified by PCR, and stored at 4°C for later use.

[0050] 2. Genetic transformation of tomatoes and screening of positive plants

[0051] When the two cotyledons of the tomato seedlings are fully expanded, cut the cotyledons into small pieces with a scalpel and transfer them to the pre-culture medium for 2 days of dark culture. 600 =0.5-1), centrifuge the bacterial solution at 4000rpm at room temperature for 10min, collect the bacteria, resuspend the bacteria in MS medium with acetosyringone, and mix immediately. Place the cut leaves in the infection for 10-20min. After infection, dry the surface moisture and place them on the co-culture medium for dark culture for 2-3d. Move the explants in the co-culture medium into sterile water containing 100mg / L Ti (Timentin) and soak for 1h, then soak them in sterile water containing 300mg / L Ti for 15min, dry the surface moisture and place them in the screening medium (containing Kan screening pressure: 100mg / L; Ti inhibits Agrobacterium: 300mg / L), culture at 28℃, and then change the culture medium every half month. After 3 weeks, transfer the explants to the budding medium. When the explants differentiate into seedlings, cut the seedlings with a scalpel and insert them into the rooting medium (containing Kan screening pressure: 50mg / L; Ti inhibits Agrobacterium: 300mg / L) for culture. Once the seedlings developed a well-developed root system, they were transplanted into pots and cultured in a greenhouse at 25°C with 16 hours of light per day. Leaf RNA from transgenic tomato lines and blank controls was extracted and reverse-transcribed into cDNA. Quantitative PCR was then used to detect overexpression of the target gene, thereby identifying positive transgenic lines with high expression levels.

[0052] 3. Analysis of gene expression patterns in transgenic tomatoes

[0053] The gene expression level of the leaves of positive transgenic tomatoes was analyzed, and the lines RuSA1-1 and RuSA1-8 ( Figure 2 Subsequently, gene expression levels were analyzed in fruits of strains RuSA1-1 and RuSA1-8 at different stages.

[0054] The results are as follows Figure 3As shown in the figure, the expression levels of the fruits of different periods of the strains RuSA1-1 and RuSA1-8 were significantly higher than those of CK, and the expression level of the strain RuSA1-1 was the highest.

[0055] 4. Appearance index and physiological index of the transgenic tomato plants

[0056] The appearance index (plant height, inflorescence, crown width, flower diameter) of the strains RuSA1-1 and RuSA1-8 was determined, and the SPAD value and N content were determined by using a SPAD instrument (TYS-4N type, Zhejiang Topu Yun Agricultural Science and Technology Co., Ltd.).

[0057] As shown in Table 1, the inflorescences of the transgenic strains were less than those of CK at 45d, and the SPAD value and N content of the strains RuSA1-1 and RuSA1-8 were significantly higher than those of CK; at 90d, the plant height of the strains RuSA1-1 and RuSA1-8 was higher than that of CK; at 165d, the SPAD value and N content of the strains RuSA1-1 and RuSA1-8 were not significantly different from those of CK. The results showed that the SPAD value and N content gradually decreased with the growth and development of the tomato plants. In summary, the RuSA1 gene promotes the growth of the tomato strains and increases the chlorophyll content and N content of the leaves.

[0058] Table 1 Determination of appearance index of transgenic tomato plants

[0059]

[0060] 5. Appearance index of transgenic tomato fruits

[0061] The longitudinal diameter (length from fruit base to fruit top) and transverse diameter (diameter of the thickest part of the fruit) of the fruits of the strains RuSA1-1 and RuSA1-8 were measured by using a vernier caliper. The mass of a single tomato fruit was measured by using a balance with an accuracy of 0.001 grams. The fruit hardness was determined by using a KM-5 type hardness tester.

[0062] As shown in Table 2, with the growth and development of the fruits, the mass, transverse diameter and longitudinal diameter of the fruits of the strains RuSA1-1 and RuSA1-8 gradually increased, while the hardness gradually decreased. Compared with the control group, the mass, transverse diameter and longitudinal diameter of the fruits of the strains RuSA1-1 and RuSA1-8 were significantly increased, and the hardness difference was not significant. Figure 4 The above results showed that the RuSA1 gene promotes the growth of tomato fruits and reduces the hardness at maturity.

[0063] Table 2 Determination of appearance index of transgenic tomato fruits

[0064]

[0065]

[0066] 6. Physiological indexes of transgenic tomato fruits

[0067] Soluble solids were determined using a PAL-1 refractometer. Titration acid content (TA) was determined by sodium hydroxide titration. The detection method of anthocyanin content: the fruit sample was ground into powder with liquid nitrogen, 1 g was weighed, extraction solution (50% ethanol containing 0.1% formic acid) was added, and it was shaken and ultrasonically treated at 60 Hz for 20 min at 35°C, centrifuged at 5000 rpm for 5 min, 0.3 mL of supernatant was taken, 2.7 mL of PBS with pH = 1 was added, shaken, and placed at room temperature for 20 min in the dark, and the absorbance was measured at 510 nm to calculate the anthocyanin content FW (mg / g). The total polyphenol content was determined using a total polyphenol detection kit (A143-1-1) from Nanjing Jiancheng Biological Engineering Institute. The total antioxidant capacity (T-AOC) of the transgenic plant fruits was determined using the FRAP method and a total antioxidant capacity (T-AOC) determination kit (A015-3-1).

[0068] The results are shown in Figure 5 Fig. 1. The soluble solids content of the fruits of lines RuSA1-1 and RuSA1-8 at the S5 stage was not significantly different from that of CK, but the soluble solids content of the transgenic fruits at the S3 stage was significantly higher than that of CK Figure 5 a). The titratable acid content of the transgenic fruits at the S3 and S5 stages was significantly higher than that of CK, and the titratable acid content of RuSA1-1 was the highest at 2.36% at the S5 stage Figure 5 b). The anthocyanin content of the fruits of lines RuSA1-1 and RuSA1-8 at the S5 stage was significantly higher than that of CK Figure 5 c). The total polyphenol content of the SA transgenic fruits at the S3 stage was significantly higher than that of CK, and the total polyphenol content of the transgenic fruits at the S5 stage was slightly higher than that of CK, and the content of RuSA1-1 was the highest at 6.18 mg·g -1 FW Figure 5 d). The total antioxidant capacity of the tomato was the highest at the S5 stage, and the total antioxidant capacity of the transgenic fruits was significantly higher than that of CK, and the total antioxidant capacity of RuSA1-8 was the highest at 251.14 mmol·g -1 FW Figure 5 e). The results show that the RuSA1 gene promotes the accumulation of anthocyanins and total polyphenols and improves the total antioxidant capacity of tomato fruits.

[0069] 7. Analysis of the expression level of hormone-related genes in transgenic tomato fruits

[0070] The results are shown in Figure 6As shown, the expression levels of genes SlABA, SlNCED1, SlACS2 and SlACO1 related to ABA hormone secretion, the gene SlIAA36 related to IAA hormone secretion, and the genes Sl2ox4 and Sl20x2 related to GA hormone secretion in the fruit of the strains RuSA1-1 and RuSA1-8 were all significantly higher than those in CK. The results show that the RuSA1 gene can promote the expression of related genes in the IAA, ABA and GA hormone synthesis pathways.

[0071] The above description is only illustrative and is not restrictive, and those skilled in the art understand that many modifications, changes or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them will fall within the protection scope of the present application.

Claims

1. A red raspberry RuSA1 gene, the nucleotide sequence of which is shown as SEQ ID NO.

1.

2. An expression protein of the red raspberry RuSA1 gene of claim 1, the amino acid sequence of which is shown as SEQ ID NO.

2.

3. A vector containing the red raspberry RuSA1 gene of claim 1, a recombinant bacterium.

4. Use of the red raspberry RuSA1 gene of claim 1 in increasing the anthocyanin content of tomato fruits.

5. Use according to claim 4, characterized in that, Comprising: 1) constructing an expression vector of the red raspberry RuSA1 gene; 2) transforming the expression vector into tomato; 3) cultivating, screening and obtaining transgenic tomato plants with significantly increased anthocyanin content.

6. Use of the red raspberry RuSA1 gene of claim 1 in increasing the total antioxidant capacity of tomato fruits.

7. Use of the mangrove raspberry RuSA1 gene of claim 1 to promote expression of genes associated with ABA hormone secretion, genes associated with IAA hormone secretion, and genes associated with GA hormone secretion in tomato fruits; wherein, The genes related to ABA hormone secretion are SlABA, SlNCED1, SlACS2 and SlACO1; the gene related to IAA hormone secretion is SlIAA36; the genes related to GA hormone secretion are Sl2ox4 and Sl20x2.

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