Transcription factor gene BrRAV8 for delaying senescence of flowering cabbage leaves and application of transcription factor gene BrRAV8

Abstract

The invention provides a BrRAV8 gene for delaying flowering cabbage leaf senescence and application of the BrRAV8 gene. A nucleotide sequence of the BrRAV8 gene is shown as SEQ ID NO.1, or has a nucleotide sequence complementary with the sequence shown as SEQ ID NO.1, or has 75% or more homology with the nucleotide sequence shown as SEQ ID NO.1, and the BrRAV8 gene can also be coded to participate in plant senescence regulation and control, and can be applied to plant senescence regulation and control. The invention particularly relates to a DNA molecule for inhibiting leaf senescence protein. The gene BrRAV8 can participate in the senescence regulation and control process, the senescence of leaves of the flowering cabbage is inhibited, and the shelf life of the harvested flowering cabbage is prolonged.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology, specifically to a gene BrRAV8 that delays leaf senescence in Chinese cabbage and its applications. Background Technology

[0002] RELATED TO ABI3 AND VP1 (RAV) is a plant-specific transcription factor containing B3 and AP2 domains. It can specifically bind to a specific sequence upstream of the 5′ end of a eukaryotic gene, thereby ensuring that the target gene is expressed as a protein molecule with a specific intensity at a specific time and space.

[0003] Chinese choy sum (Brassica campestris L.ssp.chinensis var. utilis Tsen et Lee), also known as flowering cabbage, belongs to the genus Brassica in the family Brassicaceae. It is a specialty vegetable of my country, widely cultivated in southern my country and loved by consumers. Because the edible parts of Chinese choy sum are its tender stems and leaves, which have a high water content, it is highly susceptible to dehydration, yellowing, rotting, and rapid quality decline after harvest, posing significant challenges to its storage, transportation, and sales. Therefore, inhibiting chlorophyll degradation is crucial for maintaining its nutritional quality and extending its shelf life. Summary of the Invention

[0004] The purpose of this invention is to provide a gene related to inhibiting leaf senescence in Chinese cabbage. Knocking out this gene can significantly inhibit leaf senescence in Chinese cabbage, while overexpressing this gene can promote leaf senescence, thus providing an effective means and tool for transgenic breeding of Chinese cabbage. To achieve the above objective, this invention provides the following technical solution:

[0005] The first objective of this invention is to provide the BrRAV8 gene for Chinese cabbage, the CDS sequence of which is shown in SEQ ID NO. 1.

[0006] In this invention, the gene BrRAV8 refers to the nucleotide sequence shown in SEQ ID NO.1, or a nucleotide sequence complementary to the sequence shown in SEQ ID NO.1, or having more than 75% homology with the nucleotide sequence shown in SEQ ID NO.1, and also encoding a DNA molecule involved in plant senescence regulation, particularly inhibiting leaf senescence proteins.

[0007] Furthermore, the amino acid sequence of the protein encoded by the BrRAV8 gene is shown in SEQ ID NO.2.

[0008] The protein encoded by the gene BrRAV8, whose sequence is shown in SEQ ID NO.2, or the amino acid sequence of SEQ ID NO.2 with substitution and / or deletion and / or addition of one or more amino acid residues, is a protein derived from SEQ ID NO.2 that is associated with the regulation of plant senescence, particularly the inhibition of leaf senescence.

[0009] A second objective of this invention is to provide a recombinant expression vector comprising the aforementioned Chinese cabbage BrRAV8 gene.

[0010] The plant expression vector can be used to constitutively express the BrRAV8 gene using the constitutive binary expression vector pFGC1008-HA containing the tobacco constitutive promoter CaM35S. For example, in this embodiment of the invention, overexpressed Chinese cabbage plants were obtained by transgenic Chinese cabbage.

[0011] The third objective of this invention is to provide a knockout vector for the BrRAV8 gene in Chinese cabbage.

[0012] Furthermore, the knockout vector was constructed based on the CRISPR-Cas9 system. Specifically, it was constructed by ligating a fragment containing the target site of the Chinese cabbage BrRAV8 gene into the PTX041 vector.

[0013] Preferably, the knockout vector is constructed by inserting a fragment containing the target site of the Chinese cabbage BrRAV8 gene shown in SEQ ID NO.3 or SEQ ID NO.8 into the BsaI restriction enzyme site of the PTX041 vector.

[0014] The construction of BrRAV8 mutant materials was based on the CRISPR-Cas9 system. The BrRAV8 genome sequence was imported into the online CRISPR-2.0 website (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR), and a suitable target site was selected, namely 5'-TGACCCGCATCGGTTTCCGT-3' (SEQ ID NO.3). By adding adapter sequences before and after the target site (5' end adapter sequence: 5'-AATCTAACAGTGTAGTTTG-3' (SEQ ID NO.6); 3' end adapter sequence: 5'-GTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO.7)), PCR amplification was performed to obtain the PCR product containing the target site, namely 5'-AATCTAACAGTGTAGTTTGTGACCCGCATCGGTTTCCGTGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO.8). The purified product was then ligated into the PTX041 vector by simultaneously digesting it with BsaI-HF restriction enzyme and T4 ligase. The successfully constructed vector was obtained after sequencing.

[0015] A fourth object of the present invention is to provide a host cell containing the aforementioned recombinant expression vector or the aforementioned knockout vector. The host cell can be used for genetic engineering transformation; the host cell is preferably *Escherichia coli* cells or *Agrobacterium* cells, more preferably *Agrobacterium tumefaciens* cells.

[0016] The fifth objective of this invention is to provide the application of the aforementioned Chinese cabbage BrRAV8 gene, or the aforementioned recombinant expression vector, or the aforementioned Chinese cabbage BrRAV8 gene knockout vector in regulating the chlorophyll content and / or the Fv / Fm ratio of Chinese cabbage leaves.

[0017] Furthermore, overexpression of the BrRAV8 gene in Chinese cabbage can reduce the chlorophyll content and / or reduce the Fv / Fm ratio in Chinese cabbage leaves; knocking out the BrRAV8 gene in Chinese cabbage can increase the chlorophyll content and / or increase the Fv / Fm ratio in Chinese cabbage leaves.

[0018] The sixth objective of this invention is to provide the application of the aforementioned Chinese cabbage BrRAV8 gene, or the aforementioned recombinant expression vector, or the aforementioned Chinese cabbage BrRAV8 gene knockout vector in regulating the senescence of Chinese cabbage leaves.

[0019] Furthermore, overexpression of the BrRAV8 gene in Chinese cabbage can accelerate leaf senescence; knockout of the BrRAV8 gene in Chinese cabbage can inhibit leaf senescence.

[0020] Beneficial effects:

[0021] This invention is the first to discover, locate, and clone a gene, BrRAV8, closely associated with leaf senescence in Chinese cabbage. The protein encoded by this gene can participate in the senescence regulation process, inhibiting leaf senescence and thus improving plant quality. It can be used to breed transgenic plants resistant to senescence, particularly leaf senescence. Knocking out the BrRAV8 gene in Chinese cabbage can cultivate plant varieties resistant to leaf senescence, which can be applied to plant genetic improvement. Attached Figure Description

[0022] Figure 1 Identification of BrRAV8 overexpression lines in Chinese cabbage.

[0023] Figure 2 Identification of BrRAV8 mutant plants of Chinese cabbage, among which, Figure 2 A represents sequencing of the mutation site. Figure 2 B represents the expression level of the BrRAV8 gene in the mutant plant.

[0024] Figure 3 To demonstrate the dark-induced leaf senescence phenotype in BrRAV8 overexpression lines of Chinese cabbage, among which... Figure 3 A represents the chlorophyll content of the leaves. Figure 3 B represents the leaf Fv / Fm ratio.

[0025] Figure 4 To induce leaf senescence phenotype in BrRAV8 knockout plants of Chinese cabbage under darkness, among other things... Figure 4 A represents the chlorophyll content of the leaves. Figure 4 B represents the leaf Fv / Fm ratio. Detailed Implementation

[0026] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0027] Example 1: Cloning of the BrRAV8 gene and construction of an overexpression vector

[0028] 1. Materials

[0029] 1.1 Plant materials

[0030] The leaves of Chinese cabbage were taken from the Vegetable Molecular Physiology Laboratory of the College of Horticulture, South China Agricultural University.

[0031] 2 methods

[0032] 2.1 Cloning of the BrRAV8 gene sequence

[0033] Based on the results of previous experimental analysis, the transcript sequence information of the BrRAV8 gene was obtained. Using the Chinese cabbage variety 'Youqing Sijiu' as a template, specific primers 004RAV8-F and 004RAV8-R were designed in the CDS region using PCR to amplify the target gene. The CDS sequence of BrRAV8 was obtained, as shown in SEQ ID NO.1.

[0034] Primer sequences:

[0035] 004RAV8-F:TTACAATTACCATGGGGCGCGCCATGGTGCCAACAACAACAA (SEQ ID NO. 4);

[0036] 004RAV8-R:AACATCGTATGGGTAGGTACCTCATCGAATTAATGGACCA(SEQ ID NO.5)

[0037] RT-programmed PCR:

[0038]

[0039] PCR reaction system:

[0040]

[0041] After the PCR reaction was completed, the PCR products of each round were detected by 1% agarose gel electrophoresis. The PCR products were recovered, digested with enzymes, and ligated into pFGC1008-HA to obtain the overexpression vector pFGC1008-BrRAV8-HA-OE. The sequencing results are shown in SEQ TD NO: 1, and the encoded protein sequence is shown in SEQ TD NO: 2. The results show that the cloned sequence is consistent with the BrRAV8 sequence.

[0042] 3. Construction of engineered Agrobacterium tumefaciens strain

[0043] The pFGC1008-BrRAV8-HA-OE vector was transformed into Agrobacterium tumefaciens EHA105 to obtain Agrobacterium tumefaciens engineered strain A containing the overexpression vector of the BrRAV8 gene in Chinese cabbage, namely pFGC1008-BrRAV8.

[0044] Example 2: Vector construction of BrRAV8 gene mutant

[0045] The BrRAV8 mutant material of Chinese cabbage was constructed using the CRISPR-Cas9 system. The BrRAV8 genome sequence was imported into the online CRISPR-2.0 website (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR), and a suitable target site was selected, namely 5'-TGACCCGCATCGGTTTCCGT-3' (SEQ ID NO.3). By adding adapter sequences (5' end adapter sequence: 5'-AATCTAACAGTGTAGTTTG-3' (SEQ ID NO.6); 3' end adapter sequence: 5'-GTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO.7)) before the target site, PCR amplification was performed to obtain the PCR product containing the target site, namely 5'-AATCTAACAGTGTAGTTTGTGACCCGCATCGGTTTCCGTGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO.8). The purified product was ligated into the PTX041 vector (purchased from Beijing Huayueyang Biotechnology Co., Ltd.) by simultaneous digestion and ligation with BsaI-HF restriction enzyme and T4 ligase. After sequencing, the successfully constructed mutant vector PTX041-BrRAV8 was obtained.

[0046] Example 3: Construction of BrRAV8 mutant plants and overexpression plants of Chinese cabbage

[0047] The methods for constructing mutant plants and overexpression plants are as follows:

[0048] First, the positive clones pFGC1008-BrRAV8 and PTX041-BrRAV8 obtained in Examples 1 and 2 were sequenced correctly and then used for transformation. That is, the vectors were introduced into Chinese cabbage through the Agrobacterium-mediated tomato genetic transformation system. After pre-culture, infection, co-culture, screening for resistant callus, differentiation, rooting, transplanting, and identification, transgenic plants were obtained.

[0049] Two independent overexpressing Chinese cabbage plants were obtained after transformation of the pFGC1008-BrRAV8 vector, numbered BrRAV8-OE-1 and BrRAV8-OE-2. Figure 1 After transformation with the PTX041-BrRAV8 vector, two independent mutant plants were obtained, numbered brrav8-1 and brrav8-2. Figure 2 ).

[0050] Specific steps:

[0051] 1. Sowing

[0052] Select plump seeds using the floating method - wrap them in gauze (good permeability, easy to thoroughly disinfect and sterilize) - disinfect with 75% alcohol for 40 seconds - disinfect with 2% sodium hypochlorite for 8 minutes (the time can be appropriately reduced in summer) - rinse with sterile water 4 times, 2 minutes each time - place them on sterile filter paper to absorb the moisture - sow them on 1 / 2 MS medium (1 / 2 MS + 30g sucrose + 8g / L agar, pH = 5.8, use bottles to hold the sowing medium, about 30ml per bottle), about 60 seeds per bottle - place them in a tissue culture room (temperature range between 22-25℃) - place them for 5 days (e.g., October 1st to October 6th, sow on October 1st, cut seedlings on October 6th).

[0053] 2. Pre-culture

[0054] After cutting the seedlings, place the cut seedlings on a pre-culture medium plate (MS + 2 mg / L 6-BA + 0.08 mg / L NAA + 0.01 mg / LABA + 4 mg / L AgNO3 + 30 g / L sucrose + 8 g / L agar, pH = 5.8) and keep them in the tissue culture room for 3 days (e.g., seedlings cut on October 6th will be used for infection on October 9th). Place 15 explants (cut seedlings) in each plate.

[0055] 3. Shaking and infection

[0056] Before infection, shake gently and vigorously. Prepare sterile filter paper, sterile tubes, sterile water, sterile gauze, sterile forceps, sterile empty bottles, and other sterile supplies. Pour the well-shaken bacterial solution into a sterilized 50mL centrifuge tube (approximately 45mL for centrifugation, 4℃, 4000-6000g, 10min) in a laminar flow hood. Discard the supernatant after centrifugation and resuspend the solution in 10mL of MS liquid medium (pH 5.8). To prepare the resuspension in MS liquid medium, add 100μmol / L AS. Culture Agrobacterium using pFGC1008-BrRAV8 or PTX041-BrRAV8 to OD. 600 At a concentration of around 0.4, the incubation time is 15-20 minutes. After incubation, wash 3-4 times with sterile water, then place it on sterile filter paper and blot away any excess bacterial solution with sterile filter paper (failure to blot away the solution will result in severe browning).

[0057] 4. Co-cultivation and screening

[0058] The explants were transferred to co-culture plates (MS + 2 mg / L 6-BA + 0.08 mg / L NAA + 0.01 mg / L LABA + 4 mg / L AgNO3 + 100 μmol / L AS 30 g / L sucrose + 8 g / L agar, pH 5.8). They were co-cultured in the dark for 3 days. The co-cultured explants were then placed on selection medium to screen for resistant shoots.

[0059] 5. Rooting Culture

[0060] The cut buds are placed in rooting medium (MS + 0.5 mg / L NAA + 0.02 mg / L GA3 + 300 mg / L TMT). When a large number of roots have grown, the cap of the tissue culture bottle is opened (first a small opening, then a large opening). After about 3-5 days, the seedlings can be transplanted using sterilized substrate and nutrient pots. The transplanted seedlings can be placed in a suitable environment to grow and survive before DNA is extracted for identification.

[0061] Example 4: Genetic transformation and functional identification of Chinese cabbage

[0062] The overexpression lines BrRAV8-OE-1 and BrRAV8-OE-2 and the mutant lines brrav8-1 and brrav8-2 obtained in Example 3 were transplanted into the substrate. The first fully expanded leaf was subjected to in vitro dark treatment in a constant temperature incubator at 26°C without light. The results showed that after 72 hours of dark treatment, the chlorophyll content of the knockout lines was significantly higher than that of the wild type. Figure 4 A), the degradation rate was significantly lower than that of the wild type, and the Fv / Fm ratio was significantly higher than that of the wild type. Figure 4 B); however, the chlorophyll content of the overexpression lines was significantly lower than that of the wild type. Figure 3 A), the degradation rate was significantly faster than that of the wild type, and the Fv / Fm ratio was significantly lower than that of the wild type. Figure 3 B) indicates that BrRAV8 can effectively regulate the senescence of Chinese cabbage plants.

[0063] Note: WT: wild type; brrav8-1 and brrav8-2 represent gene-edited plants of Chinese cabbage; BrRAV8-OE-1 and BrRAV8-OE-2 represent BrRAV8 overexpression plants.

[0064] SEQ ID NO.1

[0065] BrRAV8 gene CDS sequence

[0066]

[0067] SEQ ID NO.2

[0068] Protein encoded by the BrRAV8 gene

[0069] MDNSCIDDSTSNTSGSLSTSTLSAKKKLSPPPPPPATMRLYRMGSGGSSVVLDSENGVETESRKLPSSKFKGVVPQPNGRWGAQIYEKHQRVWLGTFNEEEEAAASYDIAARRFRGRDAVTNFKSPALDGTDAESAFLEAHSKAEIVDMLRKHTYADELQQSKRKFLDGNGKRCGSGTAAMSKGND GVSRAREVLFEKAVTPSDVGKLNRLVIPKQHAEKHFPLPAMTTATVVTAMTPSPTKGVLINLEDRTGKVWRFRYSYWNSSQSYVLTKGWSRFVKEKNLRAGDVVCFERSTGPDRQLYIDWKVQSGKEEITPVQSVVRLFGVNIFNETTNAKPNDVAVECGGKKRSREVDLFALGCSKKQTMMINAL*

[0070] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The BrRAV8 gene of Chinese cabbage, characterized by, The BrRAV8 gene CDS sequence is shown in SEQ ID NO.

1.

2. The BrRAV8 gene according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the BrRAV8 gene is shown in SEQ ID NO.

2.

3. A recombinant expression vector comprising the BrRAV8 gene of Chinese cabbage as described in claim 1.

4. Knockout vector for the BrRAV8 gene in Chinese cabbage.

5. The knockout carrier according to claim 4, characterized in that, The knockout vector is constructed by ligating a fragment containing the target site of the Chinese cabbage BrRAV8 gene into the PTX41 vector; preferably, the knockout vector is constructed by ligating a fragment containing the target site of the Chinese cabbage BrRAV8 gene shown in SEQ ID NO.3 or SEQ ID NO.8 into the BsaI restriction enzyme site of the PTX041 vector.

6. A host cell containing the recombinant expression vector of claim 3 or the knockout vector of claim 4.

7. The application of the Chinese cabbage BrRAV8 gene as described in claim 1, or the recombinant expression vector as described in claim 3, or the Chinese cabbage BrRAV8 gene knockout vector as described in claim 4 in regulating the chlorophyll content and / or the Fv / Fm ratio of Chinese cabbage leaves.

8. The application according to claim 7, characterized in that, Overexpression of the BrRAV8 gene in Chinese cabbage can reduce the chlorophyll content and / or the Fv / Fm ratio in Chinese cabbage leaves; knocking out the BrRAV8 gene in Chinese cabbage can increase the chlorophyll content and / or the Fv / Fm ratio in Chinese cabbage leaves.

9. The application of the Chinese cabbage BrRAV8 gene as described in claim 1, or the recombinant expression vector as described in claim 3, or the Chinese cabbage BrRAV8 gene knockout vector as described in claim 4 in regulating the senescence of Chinese cabbage leaves.

10. The application according to claim 9, characterized in that, Overexpression of the BrRAV8 gene in Chinese cabbage accelerates leaf senescence; knockout of the BrRAV8 gene in Chinese cabbage inhibits leaf senescence.

Images