Camel hoof valve gene ZbBCAT2 and application thereof in improving salt tolerance of plants

Abstract

The invention discloses a Camel hoof valve gene ZbBCAT2 and application thereof to improvement of salt tolerance of plants. The CDS sequence of the Camel hoof valve ZbBCAT2 gene is as shown in SEQ ID NO.1, and the coded amino acid sequence is as shown in SEQ ID NO.2. The invention further discloses a preparation method of the Camel hoof valve gene ZbBCAT2. The invention also discloses an application of the Camel hoof valve gene ZbBCAT2 in improving the salt tolerance of plants. The overexpression of the Camel hoof valve ZbBCAT2 gene plays a negative regulation role in the salt tolerance. The Camel hoof valve ZbBCAT2 gene provided by the invention can be used as a candidate gene for breeding the salt-resistant new variety of the plant, and has important significance for cultivating the salt-resistant new variety of the plant by adopting a genetic engineering method, accelerating the breeding process and reducing the breeding workload.

Description

ZbBCAT2 gene for slender-stemmed camel-hoof petals and its application in improving plant salt tolerance Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the ZbBCAT2 gene of the thin-stemmed camel hoof and its application in improving the salt tolerance of plants. Background Technology

[0002] Soil salinization is one of the core constraints facing global agricultural production and ecological environment restoration. In arid and semi-arid regions of Northwest my country (such as the Tarim Basin), the widespread distribution of saline-alkali land and high levels of salt stress due to drought, intense evaporation, and high salt stress restrict plant growth and reduce vegetation cover, impacting crop yields and exacerbating the risk of desertification. Developing salt-tolerant plant varieties, elucidating the molecular mechanisms of plant salt tolerance, and identifying key salt-tolerant genes are crucial pathways to achieving efficient utilization and ecological restoration of saline-alkali land, possessing significant agricultural and ecological value.

[0003] When plants cope with salt stress, they establish adaptation mechanisms at multiple levels, including morphological and structural adjustments, physiological and metabolic regulation, and molecular signal transduction.

[0004] In terms of morphology, structural changes such as fleshy leaves, increased root cortex area, and reduced stomatal opening rate reduce water loss and salt absorption.

[0005] Physiologically, cells maintain osmotic balance and redox homeostasis by accumulating osmotic regulators such as proline and enhancing the activity of antioxidant enzymes such as superoxide dismutase (SOD) and peroxidase (POD). Simultaneously, they maintain cellular osmotic balance and redox homeostasis through the vacuolar membrane's Na+. + (K) + ) / H + Antitransporter (NHX), H + -ATPase (V-ATPase) and other enzymes enable Na+ production. + Separation to maintain ion balance;

[0006] At the molecular level, the SOS signaling pathway, ABA signaling pathway, and other pathways mediate plant responses to salt stress through gene expression regulation.

[0007] However, the regulatory efficiency and specificity of these mechanisms vary significantly across different plant species, and most studies focus on model plants, while the discovery and mechanism analysis of salt tolerance genes in native salt-tolerant plants in saline-alkali lands remain relatively scarce.

[0008] Therefore, in order to solve the above-mentioned technical problems, it is an urgent technical problem for those skilled in the art to provide a gene and its application that relies on high-quality salt-tolerant germplasm of plants of the genus *Hylocereus* to explore key salt-tolerant genes, which has important theoretical and practical significance for enriching plant salt-tolerant gene resources and promoting the breeding of salt-tolerant crops and the ecological restoration of saline-alkali land. Summary of the Invention

[0009] In view of this, the present invention provides the ZbBCAT2 gene for slender stem camel hoof petals and its application in improving plant salt tolerance.

[0010] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0011] The ZbBCAT2 gene for the slender-stemmed camel hoof is shown in SEQ ID NO.1.

[0012] Preferably, the amino acid sequence encoded by the ZbBCAT2 gene of the thin-stemmed camel hoof petal is shown in SEQ ID NO.2.

[0013] Application of the ZbBCAT2 gene in improving salt tolerance of *Ceratophyllum demersum*: Overexpression of the ZbBCAT2 gene in *Ceratophyllum demersum* plays a negative regulatory role in salt tolerance.

[0014] Application of the vector of the ZbBCAT2 gene in the thin-stemmed camel hoof petal in improving plant salt tolerance, wherein the vector is an overexpression vector.

[0015] Preferably, the vector is the overexpression vector pCAMBIA3301-ZbBCAT2.

[0016] Application of strains containing the ZbBCAT2 gene of *Z. slender stem camel hoof* in improving plant salt tolerance, wherein the strains are *E. coli* and / or *Agrobacterium* containing a recombinant overexpression vector of the *Z. slender stem camel hoof* gene.

[0017] The present invention achieves the following technical effects compared to the prior art:

[0018] (1) The CDS sequence of the ZbBCAT2 gene of the slender stem camel hoof petal is disclosed for the first time in this invention, as shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2;

[0019] (2) This invention discloses for the first time the role of the ZbBCAT2 gene of *Ceratophyllum demersum* in improving salt tolerance of plants, and that overexpression of the ZbBCAT2 gene of *Ceratophyllum demersum* plays a negative regulatory role in salt tolerance.

[0020] (3) The ZbBCAT2 gene of the thin-stemmed camel hoof petal provided in this invention can be used as a candidate gene for the breeding of salt-tolerant new plant varieties. It is of great significance for the use of genetic engineering methods to breed salt-tolerant new plant varieties, accelerate the breeding process, and reduce the workload of breeding. Attached Figure Description

[0021] Figure 1 is a diagram showing the salt tolerance phenotype of heterologous ZbBCAT2 transgenic plants in Arabidopsis thaliana in Example 2 of the present invention.

[0022] Among them, A represents the phenotype of two-week-old transgenic Arabidopsis seedlings treated with 500 mM NaCl for 3 weeks; B represents the relative expression level of ZbBCAT2 in transgenic Arabidopsis; and C represents the leaf whitening rate statistics of transgenic Arabidopsis.

[0023] Figure 2 is a diagram showing the salt stress phenotype analysis of the heterologous ZbBCAT2 transgenic rice lines in Example 3 of the present invention.

[0024] Wherein, A represents the phenotype of two-week-old transgenic rice seedlings treated with 500 mM NaCl for 3 weeks; B represents the relative expression level of ZbBCAT2 in transgenic rice; and C represents the survival rate statistics of transgenic rice. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] This invention discloses the ZbBCAT2 gene for the slender-stemmed camel hoof, and the CDS sequence of the ZbBCAT2 gene is shown in SEQ ID NO.1.

[0027] The amino acid sequence encoded by the ZbBCAT2 gene of the slender-stemmed camel hoof petal is shown in SEQ ID NO.2.

[0028] This invention also discloses the application of the ZbBCAT2 gene in improving the salt tolerance of plants, and that overexpression of the ZbBCAT2 gene plays a negative regulatory role in salt tolerance.

[0029] This invention also discloses the application of a vector for the ZbBCAT2 gene of the slender stem camel hoof petal in improving the salt tolerance of plants, wherein the vector is an overexpression vector.

[0030] The vector was the overexpression vector pCAMBIA3301-ZbBCAT2.

[0031] This invention also discloses the application of strains containing the ZbBCAT2 gene of *Z. slender stem camel hoof* in improving plant salt tolerance. The strains are *E. coli* and / or *Agrobacterium* containing a recombinant overexpression vector of the *Z. slender stem camel hoof* gene.

[0032] The pCAMBIA3301-ZbBCAT2 recombinant expression vector product consists of:

[0033] ZbBCAT2 gene CDS clone (dissolved in TE buffer, concentration 100 ng / μL), pCAMBIA3301-ZbBCAT2 recombinant expression vector (plasmid concentration 500 ng / μL, dissolved in ultrapure water).

[0034] Structural features:

[0035] ZbBCAT2 CDS and protein structure: 1176bp in length, encoding 391 amino acids, containing a conserved "Aminotran_4" domain, which contains a PLP binding site;

[0036] Recombinant vector structure: pCAMBIA3301 is used as the backbone. The inserted fragment contains the CaMV35S promoter, ZbBCAT2CDS, and NOS terminator. The selection marker gene is the hygromycin resistance gene, which contains BamHI and SacⅠ double restriction sites.

[0037] Composition of ZbBCAT2 transgenic plant material product:

[0038] Transgenic Arabidopsis thaliana: 3 T3 generation homozygous lines (OE2, OE8, OE27), each line containing 100 seedlings (grown in a peat moss: perlite: vermiculite = 1:1:1 substrate);

[0039] Transgenic rice: 2 T3 generation homozygous lines (OE1, OE7), each line containing 50 seedlings (grown on MS medium + 0.8% agar solid medium).

[0040] Structural features:

[0041] Gene integration method: The ZbBCAT2 gene is randomly integrated into the recipient genome via Agrobacterium-mediated T-DNA. The average number of integrated copies in Arabidopsis is 1-2, while the average number of integrated copies in rice is 1.

[0042] Example 1:

[0043] A recombinant overexpression vector containing the ZbBCAT2 gene of the slender-stemmed camel hoof was constructed, as follows:

[0044] S1: Based on the known ZbBCAT2 gene of *Ceratophyllum demersum*, PCR amplification was performed using ZbBCAT2-F (sequence shown in SEQ ID NO.3) and ZbBCAT2-R (sequence shown in SEQ ID NO.4) as primers and cDNA from young leaves of *Ceratophyllum demersum* seedlings as a template. The amplification system and procedure followed the instructions accompanying the Phanta Max high-fidelity DNA polymerase. A portion of the amplified product was sent to the company for sequencing. The sequencing results showed that the nucleotide sequence of the *Ceratophyllum demersum* ZbBCAT2 gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0045] SEQ ID NO.1:

[0046]

[0047] SEQ ID NO.2:

[0048] MTSRINFSRGFLQSVRAGTFCKLGACHGYTSQAASTLQKLSEPSDVRSASGLDDYADFDWDNLGFALVPTDFMYIMKSTQDNRFEEGRLNRFGNIEISPSAGVLNYGQGVYEGTKAYRREDGRLLLFRPDQNAIRLRIGAERMCMPAPSSEQFIDAVKHTVLANKRWVPPPGKGSLYIRPLLIGSGPILGLAPAP EYTFLVYASPVRNYFKEGSKPLTLYVEQEYDRAARRGTGGVKTIANYAPVLKAQLRAKSRGFSDVLYLDSVNKKNLEEVSSCNIFIVKGNLISTPLSNGTILEGITRKSVMEIARDRGYEVKERVIPVDDLFDTDEVFCTGTAVGVAPVGSITYQDKRIDFKTDVHSVCGELLSALVGIQTGRCEDKNGWVVVID

[0049] SEQ ID NO.3:

[0050] GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGACTTCAAGAATCAATTTTTCTAGA

[0051] SEQ ID NO.4:

[0052] GGGGACCACTTTGTACAAGAAAGCTGGGTAATCTATCACCACAACCCAACC

[0053] Using the Gateway method, the CDS of ZbBCAT2 was ligated into the expression vector pCAMBIA3301 via BP and LR cloning reactions, driven by the CaMV35S promoter, to obtain the recombinant plasmid.

[0054] S2. Following the instructions attached to the Top10 competent cells of E. coli, ligate the recombinant plasmid obtained in S1 and transform it into E. coli.

[0055] First, thaw E. coli Top10 competent cells on ice, add recombinant plasmid to the competent cells, and incubate on ice for 20 min. Then, heat shock in a 42℃ water bath for 42 s, incubate on ice for 2 min, and add 500 μL of recovery solution. Spread the recovered bacterial solution evenly on LB solid medium containing 0.1% kanamycin and incubate upside down in a 37℃ incubator for 12-16 h until single colonies grow.

[0056] S3, identification of positive bacteria.

[0057] First, a single colony was picked and placed in LB liquid medium. The bacterial suspension was cultured in a shaker at 37°C. Using the bacterial suspension as a template, PCR amplification was performed using primers ZbBCAT2-F (SEQ ID NO.3) and ZbBCAT2-R (SEQ ID NO.4). The PCR products were verified by agarose gel electrophoresis. Then, the positive bacterial suspension with the same size as the target gene fragment was sent to the company for sequencing. Plasmids were extracted from the bacterial suspension with the correct sequencing results. The bacterial suspension with the correct sequencing results was mixed with 50% glycerol at a volume ratio of 1:1. After mixing by inverting, it was stored in a freezer at -80°C.

[0058] Example 2:

[0059] The recombinant overexpression vector constructed in Example 1 was transformed into Arabidopsis thaliana. The specific steps are as follows:

[0060] S1. Preparation of Agrobacterium infection solution.

[0061] The plasmid extracted in step S3 of Example 1 was electroporated into Agrobacterium competent cells GV3101, spread on LB plates, and cultured in an incubator at 28°C for 2 days. The grown colonies were scraped and cultured in LB liquid medium containing 0.1% kanamycin and rifampin at 28°C and 200 rpm in a shaker until OD=1. The Agrobacterium culture was resuspended in 250 mL of 5% sucrose solution. Before infection, 100 μL of silwet-L77 solution was added and mixed well to complete the preparation of the infection solution.

[0062] S2, Arabidopsis thaliana culture.

[0063] First, the Arabidopsis thaliana Col-0 seeds were shaken and washed for 5 minutes with 75% ethanol, and then shaken and washed for 1 minute with 95% ethanol. The Arabidopsis thaliana seeds were then dried in a clean bench to complete the sterilization treatment. After that, the sterilized Arabidopsis thaliana seeds were evenly scattered on 1 / 2 MS medium and vernalized at 4℃ in the dark for 2-3 days before being transferred to a growth chamber for cultivation. After germination for 7 days in the growth chamber, seedlings with uniform growth were selected and transplanted into soil for further cultivation. The cultivation temperature was 21℃, the relative humidity was 60%, the light intensity was 4400 lx, and the light duration was 16 hours of light followed by 8 hours of darkness. When the Arabidopsis thaliana reached the full flowering stage, the infection solution prepared by S1 was transformed into Arabidopsis thaliana through Agrobacterium inflorescence infection.

[0064] S3, genetic transformation of Arabidopsis thaliana.

[0065] First, cut off the pods of Arabidopsis thaliana plants in full bloom, allowing the inflorescences to be completely immersed in the infection solution prepared by S1 for 45 seconds, then treated in the dark for 12 hours, and finally the infected Arabidopsis thaliana plants were taken out and cultured in a conventional manner.

[0066] S4. Identification of salt tolerance in Arabidopsis thaliana overexpression.

[0067] First, seeds of the infected Arabidopsis thaliana were harvested to obtain T0 generation seeds. These seeds were then dried in a 30℃ drying oven for 2-3 days to ensure thorough sterilization. Afterward, the seeds were evenly scattered onto 1 / 2 MS medium containing 0.1% hygromycin using sterilized filter paper. After vernalization at 4℃ for 2 days, the seeds were placed in a constant temperature incubator for 2 weeks. Seedlings that successfully developed true leaves and roots were identified as positive seedlings. These positive seedlings were then planted in mixed soil. Once the seeds matured, individual seedlings were harvested to obtain T1 generation seeds. Subsequently, T2 generation seeds were selected using the same method on 1 / 2 MS medium containing 25 mg / L hygromycin. Seeds with a genetic segregation ratio of 3:1 were selected from the T2 generation. The same method was continued until no segregation ratio was observed in the offspring, which were designated as T3 generation. Finally, the transgenic positive seedlings were verified by PCR amplification. The primers used for PCR amplification were ZbBCAT2-F (SEQ ID NO. 3) and ZbBCAT2-R (SEQ ID NO. 3). NO.4), resulting in 3 T3 generation homozygous transgenic materials.

[0068] The expression level of the ZbBCAT2 gene in the three T3 generation transgenic positive seedlings (OE2, OE8, and OE27) was detected by real-time quantitative PCR (Real-time PCR) with Actin as the internal reference gene. The forward primers included (sequence shown in SEQ ID NO.7) and ZbBCAT2-qF (sequence shown in SEQ ID NO.5), and the reverse primers included (sequence shown in SEQ ID NO.8) and ZbBCAT2-qR (sequence shown in SEQ ID NO.6). The Bio-Rad CFX96 Real-Time System was used with the following reaction program: 95℃ for 15 sec, 60℃ for 15 sec, 72℃ for 30 sec, for 40 cycles. Finally, the data were processed using the Comparative CT method. Using wild-type Arabidopsis thaliana (Col-0) as a control, the results are shown in Figure 1(B). It shows that ZbBCAT2 is not expressed in wild-type Arabidopsis thaliana and the loss-of-function mutant bcat2, but the expression level is significantly increased by 7-13 times in the three positive lines that overexpress ZbBCAT2, indicating that ZbBCAT2 is successfully heterologously expressed in Arabidopsis thaliana.

[0069] Phenotypic differences were observed after treating two-week-old ZbBCAT2 transgenic Arabidopsis seedlings grown in nutrient soil with 500 mM NaCl for three weeks. The results showed that before the stress treatment, the growth of the Arabidopsis T-DNA mutant bcat2 and the three overexpressing lines was consistent with that of wild-type Arabidopsis. After three weeks of 500 mM NaCl treatment, the leaves of wild-type plants turned chlorotic and white, while only some leaves of bcat2 showed chlorosis and most turned red. In contrast, most leaves of the overexpressing plants showed severe chlorosis, gradually turning white and dying (Figure 1A). The whitening rate of Arabidopsis leaves was statistically analyzed, revealing a whitening rate of 22.23% for Col-0, only 4.91% for bcat2, and over 30.47% for the transgenic materials, showing a highly significant difference (Figure 1C). Therefore, we conclude that ZbBCAT2 negatively regulates the salt tolerance of Arabidopsis seedlings.

[0070] SEQ ID NO.5: TGCTTCTGGCTTGGACGACTA

[0071] SEQ ID NO.6: GCCGACGGGCTAATCTCAATA

[0072] SEQ ID NO.7: TAGATGGCTGGAACAGAACTT

[0073] SEQ ID NO.8: GTTGCTATTGATTATGAGAAGG

[0074] Example 3:

[0075] The recombinant overexpression vector constructed in Example 1 was transformed into rice, and the specific steps are as follows:

[0076] S1, Induction.

[0077] Select seeds of the Zhonghua 11 strain that are free of mold and have normal bud openings. Disinfect with 75% alcohol for 1 min and rinse with sterile water for 1 min; disinfect with 15% sodium hypochlorite for 20 min and rinse with sterile water 3 times, 1 min each time; inoculate the disinfected seeds into induction medium and culture at 26℃ under light for 20 days.

[0078] S2, Agrobacterium infection.

[0079] The plasmid extracted in S3 of Example 1 was electroporated into Agrobacterium competent cells EHA105. Callus grown in the induction medium in step S1 was picked and placed in an Erlenmeyer flask. The infection solution of Agrobacterium EHA105 was added and the bacterial solution was discarded after 10-15 min of infection. The callus was then inoculated into a co-culture medium and co-cultured at 20°C for 48-72 h.

[0080] S3, callus screening.

[0081] Positive callus was screened 2-3 times on selective media containing carbenicillin or hygromycin (the first and second screenings were performed with 400 ppm and 250 ppm carbenicillin, respectively, and the subsequent screenings were performed with 250 ppm hygromycin) to obtain homozygous transgenic rice plants for phenotypic analysis.

[0082] S4, differentiation and rooting.

[0083] Positive callus was inoculated onto differentiation medium and cultured under light at 25-27℃ for about 15 days. After the shoots differentiated into 2-5cm buds, they were inoculated onto rooting medium.

[0084] S5, positive vaccine detection.

[0085] Genomic DNA was extracted from rice seedlings using the CTAB method and then subjected to PCR detection. The primers used for PCR amplification were ZbBCAT2-F (SEQ ID NO.3) and ZbBCAT2-R (SEQ ID NO.4).

[0086] S6. Identification of salt tolerance phenotype in rice with overexpression.

[0087] Uniform, plump, and healthy transgenic rice seedlings and wild-type (ZH11) rice seeds were selected and dried in a 37℃ oven for 3 days. The seeds were then soaked in distilled water in 90mm round dishes and incubated in a 37℃ constant temperature incubator for 36 hours, with the water changed every 12 hours. Seeds with uniform germination were then spread evenly in 96-well black germination boxes and cultured in a 28℃ tissue culture room, with the water changed every 2 days. In the first stage, the seedlings were cultured in nutrient solution for 14 days, and photographs were taken. In the second stage, for each family of rice seedlings cultured for 14 days, a control group and a 500 mM NaCl stress group were established. The control group received sterile water, while the stress group received an equal amount of 500 mM NaCl to simulate salt stress. After 3 weeks of stress, photographs were taken, and the survival rate of the transgenic plants was recorded.

[0088] The expression level of ZbBCAT2 was detected by qPCR in homozygous positive transgenic rice plants. The results showed that the expression level increased by 2-3 times in the overexpression lines OE1 and OE7, confirming that ZbBCAT2 was successfully heterologously expressed in rice (Figure 2B).

[0089] Phenotypic analysis was performed on transgenic rice plants grown in soil for two weeks after treatment with 500 mM NaCl. The results showed that the overexpressing plants exhibited a more salt-intolerant phenotype (Figure 2A) and had a significantly lower survival rate than the wild-type ZH11 (Figure 2C). This indicates that ZbBCAT2 plays a negative regulatory role in salt tolerance.

[0090] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. The ZbBCAT2 gene for thin-stemmed camel-hoof petals is characterized by, The CDS sequence of the ZbBCAT2 gene of the slender stem camel hoof petal is shown in SEQ ID NO.

1.

2. The ZbBCAT2 gene for thin-stemmed camel hoof petals according to claim 1, characterized in that, The amino acid sequence encoded by the ZbBCAT2 gene of the slender stem camel hoof petal is shown in SEQ ID NO.

2.

3. Application of the ZbBCAT2 gene in improving plant salt tolerance, characterized in that... Overexpression of the ZbBCAT2 gene in the slender stem camel hoof petals plays a negative regulatory role in salt tolerance.

4. Application of the vector of the thin-stemmed camel-hoof petal gene ZbBCAT2 in improving plant salt tolerance, characterized in that, The vector is an overexpression vector.

5. The application according to claim 3, characterized in that, The vector is the overexpression vector pCAMBIA3301-ZbBCAT2.

6. Application of strains containing the ZbBCAT2 gene in improving plant salt tolerance, characterized in that... The strains are *Escherichia coli* and / or *Agrobacterium* containing a recombinant overexpression vector of the *ZbBCAT2* gene of the camel hoof.

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