Rubus blackberry rubhlh gene, expression protein and application thereof

Abstract

The application discloses a blackberry RubHLH gene, an expression protein thereof and application, and relates to the technical field of plant genetic engineering.The blackberry RubHLH 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 blackberry RubHLH gene, transforms the expression vector into tomatoes, and cultivates, screens and obtains transgenic tomato plants with significantly reduced anthocyanin substances.The RubHLH gene disclosed by the application inhibits the synthesis of anthocyanin substances such as pelargonidin, proanthocyanidin, naringenin, kaempferol-3-O-rutinoside, delphinidin-3-O-rhamnoside and petunidin-3-O-acetylglucoside.The leaf of the transgenic strain is larger, darker, wrinkled and has a shape significantly different from that of CK;the number of seeds of the fruit is reduced, and the fruit matures in advance.

Description

TECHNICAL FIELD

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

[0002] Blackberry is a characteristic small berry fruit tree of Rubus in Rosaceae, and the fruit has rich nutritional, health care and medicinal values and is widely welcomed by consumers in the market. The blackberry fruit is also a main research and utilization part, and the separated compounds have the effects of anti-tumor, anti-inflammatory, blood lipid-lowering and blood pressure-lowering. Anthocyanins, as a kind of natural pigment, are safe, non-toxic and have various health care functions on human body, and have been applied to food, health care, cosmetics, medicine and other industries, and are also a kind of compounds rich in blackberry fruit.

[0003] bHLH transcription factors, also known as MYCs, are the second largest transcription factor family in plants (Feller A, 2011). Currently, 162, 95, 167 and 152 bHLH genes have been identified in Arabidopsis (Bailey et al., 2003), grape (Wang et al., 2018), rice (Li et al., 2006) and tomato (Wang et al., 2015), respectively. The bHLH domain is composed of about 60 conserved amino acid residues, consisting of two conserved motifs, namely the base region and the helix-loop-helix region (HLH region) (Hao, Y., 2021). bHLHs can act as repressors or activators of gene transcription and play an important role in various physiological processes in plants (Feller A, 2011), and regulate various metabolic processes in plants, such as light morphogenesis (Leivar P, 2008), metal homeostasis (Li H, 2007), flavonoid biosynthesis pathway (Hichri I, 2011) and anthocyanin synthesis process (Albert NW, 2014; Xu W, 2015), but the specific role and function of bHLH in blackberry are not clear at present. 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 blackberry RubHLH gene. Another technical problem to be solved by the present application is to provide an expression protein of the blackberry RubHLH gene and application thereof. The present application also solves the technical problem of providing an application of the blackberry RubHLH gene for regulating the synthesis and accumulation of anthocyanins in plants.

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

[0006] A blackberry RubHLH gene, the nucleotide sequence of which is shown as SEQ ID NO. 1.

[0007] An expression protein of the blackberry RubHLH gene, the amino acid sequence of which is shown as SEQ ID NO. 2.

[0008] A vector, a recombinant bacterium or a host cell containing the blackberry RubHLH gene.

[0009] Application of the blackberry RubHLH gene in regulating anthocyanin synthesis.

[0010] The anthocyanin synthesis is inhibited.

[0011] The anthocyanins include pelargonidin, proanthocyanidin, naringenin, kaempferol-3-O-rutinoside, delphinidin-3-O-rhamnoside and petunidin-3-O-acetylglucoside.

[0012] Application of the blackberry RubHLH gene in regulating leaf area and color of tomato plants.

[0013] Application of the blackberry RubHLH gene in regulating seed number of tomato fruits.

[0014] Application of the blackberry RubHLH gene in regulating early ripening of tomato fruits.

[0015] Application of the blackberry RubHLH gene in regulating chlorophyll content of plant leaves.

[0016] Compared with the prior art, the application has the following beneficial effects:

[0017] 1) The blackberry RubHLH gene disclosed by the application has the nucleotide sequence shown as SEQ ID NO. 1, and the amino acid sequence of the expression protein thereof is shown as SEQ ID NO. 2. The application constructs an expression vector of the blackberry RubHLH gene, transforms the expression vector into tomatoes, and cultivates and screens to obtain transgenic tomato plants with significantly reduced anthocyanins.

[0018] 2) The RubHLH gene disclosed by the application inhibits the synthesis of anthocyanins such as pelargonidin, proanthocyanidin, naringenin, kaempferol-3-O-rutinoside, delphinidin-3-O-rhamnoside and petunidin-3-O-acetylglucoside.

[0019] 3) The bHLH transgenic strain constructed by the application has larger, darker and wrinkled leaves, and the leaf shape is significantly different from that of CK.

[0020] 4) The bHLH transgenic line constructed in this invention has a reduced number of seeds in its fruit and the fruit matures earlier. Attached Figure Description

[0021] Figure 1 The expression level of the RubHLH gene in blackberry at different developmental stages (S1 is the green fruit stage; S2 is the green-red fruit stage; S3 is the red fruit stage; S4 is the red-purple fruit stage; S5 is the purple fruit stage).

[0022] Figure 2 Figure showing the expression level analysis of the RubHLH gene in transgenic tomato leaves;

[0023] Figure 3 Figure 1 shows the expression level analysis of the RubHLH gene at different developmental stages of transgenic tomato fruit (S1 is the green fruit stage; S2 is the green-red fruit stage; S3 is the red fruit stage; S4 is the red-purple fruit stage; S5 is the purple fruit stage).

[0024] Figure 4 A comparison image of leaves from genetically modified tomatoes and control (CK) tomatoes;

[0025] Figure 5 A graph showing the physiological indicators of genetically modified tomato fruits;

[0026] Figure 6 Cluster diagram of anthocyanins in mature transgenic tomato fruit (a) and violin diagram of differentially expressed components (b);

[0027] Figure 7 This diagram shows the expression levels of key genes in the anthocyanin pathway in transgenic tomato fruits. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0029] The blackberry material used in this application was grown at the blackberry experimental base in Baima Town, Lishui District, Nanjing City, Jiangsu Province. Different tissue parts of blackberries, including roots, stems, leaves, fruiting branches, flowers and fruits, as well as fruits at different stages, such as green fruits, green-red fruits, red fruits, purple-red fruits and purple fruits, were harvested. After fresh harvesting, the fruits were immediately frozen in liquid nitrogen and then stored at -80℃ for RNA extraction.

[0030] The tomatoes used in this application were grown in the nursery of the Institute of Botany, Chinese Academy of Sciences in Jiangsu Province. Tomato leaves and fruits at different stages, including green, yellow, orange, orange-red and red fruits, were harvested and immediately frozen in liquid nitrogen, and then stored at -80℃ for RNA extraction and subsequent physiological index determination.

[0031] Example 1

[0032] 1. Total RNA extraction and cDNA acquisition

[0033] Total RNA of blackberry was extracted using Plant Polysaccharide and Polyphenol Total RNA Extraction Kit (BioTeKe, Beijing, China) and the integrity of RNA was detected by agarose gel electrophoresis. The concentration and purity of RNA were detected by Nanodrop 2000c spectrophotometer (Thermo Fisher, Waltham, MA, USA). Reverse transcription into cDNA was performed using PrimeScript RT Master Mix Kit (TaKaRa, Otsu, Japan).

[0034] 2. Cloning of blackberry RubHLH gene

[0035] According to the blackberry genome sequence, the primers were designed using Oligo 7.0 software, 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:

[0036] RubHLH ORF-F: 5'-ATGAAAATTGAGATGGGTTTAG-3',

[0037] RubHLH ORF-R: 5'-ACCAACTGATGACAATGACTG-3'.

[0038] The gel cutting and purification of the target fragment were performed using the Quick Agarose Gel DNA Recovery Kit (BioTeKe, Beijing, China), and then the product after cutting and purification was connected with the T vector using the pClone007 Blunt Vector Kit reagent kit (TSINGKE, Beijing, China). After being transferred into the competent E. coli, it was briefly recovered and coated on the culture medium with resistance to ampicillin. 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.

[0039] The nucleotide sequence of the blackberry RubHLH gene obtained by final sequencing is shown in SEQ ID NO. 1, and the amino acid sequence of the expressed protein is shown in SEQ ID NO. 2.

[0040] 3. Analysis of RubHLH gene expression pattern in blackberry

[0041] RT-qPCR was performed using TB Green Premix Taq II reagent (Takara, Dalian, China) with cDNA from different tissues and fruit stages of blackberry. qRT-PCR was performed using qTOWER 2.2 real-time PCR system (Analytik, Jena, Germany) according to the manufacturer's instructions. The relative expression levels of candidate blackberry gene bHLH in different tissues and fruit stages were detected according to 2 -ΔΔCT method with 18S as the internal reference gene. The primer sequences are as follows:

[0042] RubHLH qRT-F: 5'-CAAGTGCTTGGCGTCTGGGA-3',

[0043] RubHLH qRT-R: 5'-ACCACACCCACATCCGTTGG-3'.

[0044] The results are shown in Figure 1 The expression level of RubHLH gene in the growth and development of blackberry fruit showed a trend of first rising and then falling, and the expression level at S4 stage was significantly higher than that at other stages, reaching 3.01, while the expression level at S1 stage was the lowest.

[0045] Example 2

[0046] 1. Construction of blackberry RubHLH gene overexpression vector and transformation of Agrobacterium

[0047] Gateway technology was used to construct blackberry overexpression vector, and the target fragment in the linker-7 vector was transferred to the 35S strong promoter overexpression vector Pbi121-des-3HA by LR reaction, and the successful bacterial liquid was used for plasmid extraction. Take 150 ng of the transformed plasmid and add 100 μL of Agrobacterium GV3101 competent cells for transformation, and spread on kanamycin and rifampicin double-antibiotic medium, 28°C for 2 days, randomly select single colonies, bacterial liquid PCR verification, 4°C storage for standby.

[0048] 2. Genetic transformation of tomato and screening of positive plants

[0049] When the cotyledon of tomato seedling was fully expanded, the cotyledon was cut into small pieces and moved to the pre-culture medium, and dark culture for 2d. The prepared Agrobacterium containing the transgene was expanded (OD 600=0.5-1.0), centrifuge the bacterial suspension at 4000 rpm for 10 min at room temperature, collect the bacterial cells, resuspend the cells in MS medium containing acetylsuccinyl syringone, and mix immediately. Place the cut leaves into the inoculation medium for 10-20 min. After infection, blot dry the surface moisture and place them on co-culture medium for dark incubation for 2-3 days. Transfer the explants from the co-culture medium into sterile water containing 100 mg / L Ti (termethin) and soak for 1 h, then soak in sterile water containing 300 mg / L Ti for 15 min, blot dry the surface moisture, and place them in selection medium. Incubate at 28℃, and then change the medium every half month (100 mg / L). -1 Kan (MS + 100 mg / L Ti) was used. After 3 weeks, the explants were transferred to shoot-forming medium (Kan resistance selection). When the explants differentiated into seedlings, the seedlings were surgically cut and inserted into rooting medium for culture. After the seedlings developed a root system, they were transferred to pots and cultured in a greenhouse at 25°C with 16 hours of light per day. RNA was extracted from the leaves of the tomato transgenic lines and blank control, reverse transcribed into cDNA, and the overexpression of the target gene was detected by real-time PCR to screen for positive transgenic lines with high expression levels.

[0050] 3. Analysis of gene expression patterns in transgenic tomatoes

[0051] To further clarify the expression patterns of different transgenic lines, gene expression levels were analyzed in the leaves of positive transgenic tomatoes. Figure 2 The lines bHLH-11, bHLH-13 and bHLH-14 with high RubHLH gene expression were selected, with bHLH-14 showing the highest RubHLH gene expression at 15441.46.

[0052] Gene expression levels were analyzed in the fruits of the bHLH-11, bHLH-13, and bHLH-14 lines.

[0053] The results are as follows Figure 3 As shown, the expression level of transgenic tomato fruits was significantly higher than that of CK. During the fruit growth and development process, the expression level of bHLH-11 first increased and then decreased, with the highest expression level in stage S3. The expression level of bHLH-13 showed a trend of "increase-decrease-increase", with the highest expression level in stage S4. The expression level of bHLH-14 showed a gradual increasing trend, with the highest expression level in stage S4.

[0054] 4. Appearance and physiological indicators of transgenic tomato plants

[0055] The appearance indicators (plant height, inflorescence, crown width, and flower diameter) of the transgenic tomato plants were measured. The chlorophyll content (SPAD) and nitrogen content were measured using a SPAD instrument (TYS-4N type, Zhejiang Top Cloud Agriculture Technology Co., Ltd.).

[0056] The results are shown in Table 1. The plant height of the transgenic tomatoes was lower than that of CK at 45d, and the inflorescences were less than those of CK. The SPAD value and N content of the bHLH-11, bHLH-13 and bHLH-14 lines were significantly higher than those of CK. At 90d, the crown width of the bHLH-13 line was the largest, and the inflorescences were the most. The flower diameter of the bHLH-14 line was extremely significantly larger than that of CK, and the SPAD value of the bHLH-11 line was extremely significantly higher than that of CK. At 165d, the SPAD value and N content of the transgenic tomatoes were not significantly different from those of CK, and gradually decreased with the growth and development of the tomato plants. The leaves of the bHLH transgenic lines were larger, darker, wrinkled, and the leaf shape was significantly different from that of CK. Figure 4

[0057] Table 1. Appearance index determination of transgenic tomato plants

[0058]

[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 were measured using a vernier caliper. The mass of a single tomato fruit was measured using a balance with an accuracy of 0.001 g. The fruit hardness was measured using a KM-5 type hardness tester.

[0062] The results are shown in Table 2. The mass of a single fruit of the transgenic tomato lines was lower than that of CK. Significant differences between the transgenic lines and CK occurred from the S3 stage, and the mass of the bHLH-11 line was the smallest at 3.07 g at the S4 stage. The longitudinal diameter and transverse diameter of the bHLH-11 and bHLH-14 lines were smaller than those of CK and the bHLH-13 line. The hardness of the bHLH-13 line was significantly lower than that of CK at the S3 stage, and the hardness of the bHLH-14 line was significantly higher than that of CK. It was found that the number of seeds in the transgenic tomato fruits was significantly reduced, and the fruits matured early, at the S4 stage.

[0063] Table 2. Appearance index determination of transgenic tomato fruits

[0064]

[0065]

[0066] 6. Physiological index of transgenic tomato fruits

[0067] ​Soluble solids were determined using a PAL-1 saccharimeter. Titratable acid content (TA) was determined by sodium hydroxide titration. Plant protein detection kits (A045-2) and total phenols detection kits (A143-1-1) from Nanjing Jiancheng Bioengineering Institute were used for determination. For anthocyanin content determination, fruit samples were ground into powder under liquid nitrogen, 1 g was weighed, added to extraction solution (50% ethanol containing 0.1% formic acid), shaken well, and ultrasonically treated at 35℃ for 20 min at 60 Hz. After centrifugation at 5000 rpm for 5 min, 0.3 mL of the supernatant was collected, 2.7 mL of pH=1 PBS was added, shaken well, and allowed to stand at room temperature in the dark for 20 min. The absorbance was measured at 510 nm, and the anthocyanin content (FW) (mg / g) was calculated. The total antioxidant capacity of transgenic plant leaves and fruits was determined using the FRAP method and the Total Antioxidant Capacity (T-AOC) assay kit (A015-3-1).

[0068] The results are as follows Figure 5 As shown, the soluble solids content of the transgenic strain's fruit was lower than that of the control (CK). Figure 5 a) The titratable acid content of transgenic lines was significantly lower than that of the control (CK) at S1 and S3 stages. The anthocyanin content of tomatoes showed an increasing trend during fruit growth and development, and at the S5 stage, the content of transgenic lines was significantly lower than that of the CK, with the bHLH-14 line having the lowest content at 562.50 μg·g⁻¹. -1 FW( Figure 5 c). The total polyphenol content of the bHLH transgenic lines was significantly lower than that of the CK, with the bHLH-13 line having the lowest total polyphenol content at 6.08 mg / g during the S5 stage. -1 FW( Figure 5 d). Their total antioxidant capacity was also significantly lower than that of the control (CK). During the S5 period, bHLH-14 exhibited the weakest antioxidant capacity at 113.65 mmol·g⁻¹. -1 FW( Figure 5 e).

[0069] 7. Determination of anthocyanin components in transgenic tomato fruits

[0070] The results are as follows Figure 6 As shown, the contents of pelargonidin and proanthocyanidins in transgenic tomato fruits were lower than those in the control (CK). The contents of naringenin and kaempferol-3-O-rutin in flavonoids, delphinidin-3-O-rhamnoside in delphinidin, and petunidin-3-O-sophoroside in morning glory pigments were also lower than those in the control (CK).

[0071] from Figure 6 The violin plot of the differentially expressed components (b) concluded that the contents of delphinidin-3-O-rhamnoside, pelargonidin-3-O-sophoroside, and delphinidin in the control group (CK) were approximately 0.03 μg·g. -1and 0 in the transgenic tomato. -1 0.02 μg·g in the transgenic tomato -1 .

[0072] The above results show that the content of the above components decreases after overexpression of the RubHLH gene, resulting in lighter color of the transgenic tomato fruit.

[0073] 8. Expression level analysis of key genes in anthocyanin pathway in transgenic tomato fruit

[0074] The results are shown in Table 4. Figure 7 The expression levels of Sl4CL and SlANS in the transgenic tomato fruit are lower than those in CK at S3 and S4, while the expression level of SlF3'5'H is lower than that in CK at S4. The RubHLH gene regulates the synthesis of anthocyanin by regulating the gene expression of SlC4H, SlANS and SlF3'5'H in the anthocyanin pathway of tomato.

[0075] The above is only illustrative but not restrictive to the present application, 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 blackberry RubHLH gene, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The expression protein of the blackberry RubHLH gene according to claim 1, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. A vector containing the blackberry RubHLH gene as described in claim 1.

4. The application of the blackberry RubHLH gene as described in claim 1 in regulating the early ripening of tomato fruits.

5. A recombinant bacterium containing the blackberry RubHLH gene as described in claim 1.

6. A host cell containing the blackberry RubHLH gene as described in claim 1.

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