BcC3H33 gene of flowering cabbage and application of encoded protein of BcC3H33 gene in resistance to cadmium stress

Abstract

The invention discloses a BcC3H33 gene of flowering cabbage and an application of a protein encoded by the BcC3H33 gene in resisting cadmium stress. The invention provides a new application of the BcC3H33 gene of the flowering cabbage and the encoded protein thereof in regulation and control of growth and development of the flowering cabbage and cadmium stress response, and research shows that the BcC3H33 gene has very strong response to cadmium stress in roots and leaves of the flowering cabbage and can regulate and control the growth and development of the flowering cabbage and promote the growth of the flowering cabbage; after the BcC3H33 gene is over-expressed in the flowering cabbage, the biological accumulation amount of the flowering cabbage is increased, the morphological index is improved, the photosynthetic ability is enhanced, the stress resistance is enhanced, the influence of cadmium stress on the growth and development of the flowering cabbage can be relieved, and the growth of the flowering cabbage can also be promoted under the cadmium stress. Therefore, the BcC3H33 gene can improve the cadmium stress tolerance of the Chinese flowering cabbage, improve the crop adaptability, provide a basis and a method for stress-resistant breeding of the Chinese flowering cabbage, provide key gene resources for the Chinese flowering cabbage and other crops, and meet different cultivation environment requirements.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and breeding technology, and more specifically, to Chinese cabbage. BcC3H33 Application of genes and their encoded proteins in cadmium stress resistance. Background Technology

[0002] With the rapid advancement of industrialization and urbanization, as well as the irrational use of chemical fertilizers and pesticides in agricultural production, soil cadmium pollution has become increasingly serious. Because cadmium has high mobility and bioavailability in soil, it is easily absorbed by crops and accumulates within them. This not only severely interferes with the normal growth and development of crops, leading to hindered seed germination, abnormal root and leaf growth, and imbalances in the antioxidant defense system, but it can also enter the human body through the food chain, posing a serious threat to human health.

[0003] Chinese cabbage (Cheyale chinensis) is the vegetable with the largest cultivation area and demand in South China. During its growth and development, it is highly susceptible to environmental stressors such as drought, salinity, and heavy metals. These stresses severely restrict the yield and quality of Chinese cabbage, causing significant economic losses to the vegetable industry. With the development of molecular biology, the molecular mechanisms of Chinese cabbage quality and stress resistance have received increasing attention. The C3H gene family (a family of transcription factors containing zinc finger domains) plays a crucial role in plant growth and development, stress response, and hormone signal transduction. Existing studies, through genome-wide analysis, have identified multiple C3H genes in Chinese cabbage (such as...). BcC3H1 - 30 ),part BcC3H Gene regulation of flowering time (e.g., Arabidopsis thaliana) AtC3H14 It is involved in homologous genes, seed development, and leaf morphogenesis, and participates in drought, salt stress (e.g., through the ABA signaling pathway) and pathogen defense responses (e.g., regulating PR gene expression). For example, existing studies have reported the genome-wide identification of the C3H gene in Chinese cabbage and its response expression to ABA, high temperature, drought, and salt stress.

[0004] However, current research on the C3H gene family of Chinese cabbage is still in its early stages. There has been no systematic study and identification of its members, nor is there an in-depth analysis of its molecular mechanisms in response to environmental stress. Therefore, in-depth research into the molecular mechanisms and functions of C3H-type zinc finger proteins in regulating the response of Chinese cabbage to abiotic stress, and systematically revealing the mysteries of the C3H gene family, will provide a solid theoretical basis and abundant genetic resources for stress-resistant breeding of Chinese cabbage, helping to improve the yield and quality of Chinese cabbage under complex environmental stress, and promoting the sustainable development of the vegetable industry in South China. Summary of the Invention

[0005] The technical problem this invention aims to solve is to overcome the shortcomings of existing research on the C3H gene in Chinese cabbage in cadmium stress resistance. This invention provides a method for addressing this issue.BcC3H33 Gene and its encoded protein in anti-cadmium stress.

[0006] The first object of the present application is to provide Brassica parachinensis BcC3H33 Gene and its encoded protein.

[0007] The second object of the present application is to provide overexpression BcC3H33 Gene or its encoded protein preparation.

[0008] The third object of the present application is to provide a method for improving the cadmium stress tolerance of Brassica parachinensis.

[0009] The fourth object of the present application is to provide a method for promoting the growth of Brassica parachinensis and / or promoting the growth of Brassica parachinensis under cadmium stress.

[0010] The above objects of the present application are achieved by the following technical solutions: The present application provides a gene as shown in SEQ ID NO. 1 BcC3H33 Gene or its encoded protein in improving the cadmium stress tolerance of Brassica parachinensis, overexpression BcC3H33 Gene in Brassica parachinensis.

[0011] The present application provides Brassica parachinensis BcC3H33 Gene and its encoded protein in regulating the growth and development of Brassica parachinensis and the response to cadmium stress, research shows BcC3H33 Gene has a very strong response to cadmium stress in the roots and leaves of Brassica parachinensis, and can regulate the growth and development of Brassica parachinensis and promote the growth of Brassica parachinensis; by constructing a yeast fusion expression vector and heterologous expression in yeast, it is verified that BcC3H33 Gene can enhance the tolerance of yeast to Cd stress; by constructing a plant expression vector to overexpress BcC3H33 Gene in Brassica parachinensis, it is shown that overexpression BcC3H33 Gene can increase the biomass accumulation of Brassica parachinensis, improve morphological indicators, enhance photosynthetic capacity and stress resistance, improve the cadmium stress tolerance of Brassica parachinensis, and improve the stress environment adaptability of crops; and after cadmium stress treatment, overexpression BcC3H33 Gene can alleviate the impact of cadmium stress on the growth and development of Brassica parachinensis, break the growth restriction of cadmium on Brassica parachinensis, and promote the development of Brassica parachinensis, the present application provides a basis and method for Brassica parachinensis stress resistance breeding, and provides more gene resources for cadmium-polluted environments and improving the cadmium stress tolerance of plants, to meet the needs of different cultivation environments.

[0012] Therefore, the present application provides a gene as shown in SEQ ID NO. 1 BcC3H33 Gene or its encoded protein is applied: In promoting the growth of Brassica parachinensis and / or promoting the growth of Brassica parachinensis under cadmium stress.

[0013] In cultivating Brassica parachinensis plants resistant to cadmium stress.

[0014] Further, the gene is over-expressed in the flowering Chinese cabbage. BcC3H33 The gene is preferably over-expressed in the flowering Chinese cabbage.

[0015] Preferably, the gene or the encoded protein is used for promoting root growth of the flowering Chinese cabbage.

[0016] Meanwhile, the application also provides use of the preparation of the over-expressed gene or the encoded protein in improving cadmium stress tolerance of the flowering Chinese cabbage. BcC3H33 The application provides use of the preparation of the over-expressed gene or the encoded protein in promoting growth of the flowering Chinese cabbage and / or promoting growth of the flowering Chinese cabbage under cadmium stress.

[0017] The application provides use of the preparation of the over-expressed gene or the encoded protein in cultivating the flowering Chinese cabbage plant with cadmium stress tolerance. BcC3H33 The application provides use of the preparation of the over-expressed gene or the encoded protein in cultivating the flowering Chinese cabbage plant with cadmium stress tolerance.

[0018] Preferably, the preparation of the over-expressed gene or the encoded protein is used for promoting root growth of the flowering Chinese cabbage. BcC3H33 The application provides use of the preparation of the over-expressed gene or the encoded protein in cultivating the flowering Chinese cabbage plant with cadmium stress tolerance.

[0019] The application provides use of the preparation of the over-expressed gene or the encoded protein in cultivating the flowering Chinese cabbage plant with cadmium stress tolerance. BcC3H33 The application provides a method for improving cadmium stress tolerance of the flowering Chinese cabbage, by over-expressing the gene or the encoded protein in the flowering Chinese cabbage, so as to improve cadmium stress tolerance of the flowering Chinese cabbage, increase plant biomass accumulation, improve morphological indexes, enhance photosynthetic capacity and stress resistance.

[0020] The application also provides a method for promoting growth of the flowering Chinese cabbage and / or promoting growth of the flowering Chinese cabbage under cadmium stress, by transferring the preparation of the over-expressed gene or the encoded protein into the plant body, so as to promote growth of the flowering Chinese cabbage. BcC3H33 The application also provides a method for promoting growth of the flowering Chinese cabbage and / or promoting growth of the flowering Chinese cabbage under cadmium stress, by transferring the preparation of the over-expressed gene or the encoded protein into the plant body, so as to promote growth of the flowering Chinese cabbage.

[0021] The application provides a method for improving cadmium stress tolerance of the flowering Chinese cabbage, by over-expressing the gene or the encoded protein in the flowering Chinese cabbage, so as to improve cadmium stress tolerance of the flowering Chinese cabbage, increase plant biomass accumulation, improve morphological indexes, enhance photosynthetic capacity and stress resistance. BcC3H33 The application provides a method for improving cadmium stress tolerance of the flowering Chinese cabbage, by over-expressing the gene or the encoded protein in the flowering Chinese cabbage, so as to improve cadmium stress tolerance of the flowering Chinese cabbage, increase plant biomass accumulation, improve morphological indexes, enhance photosynthetic capacity and stress resistance.

[0022] The application also provides a method for promoting growth of the flowering Chinese cabbage and / or promoting growth of the flowering Chinese cabbage under cadmium stress, by transferring the preparation of the over-expressed gene or the encoded protein into the plant body, so as to promote growth of the flowering Chinese cabbage. BcC3H33 The application also provides a method for promoting growth of the flowering Chinese cabbage and / or promoting growth of the flowering Chinese cabbage under cadmium stress, by transferring the preparation of the over-expressed gene or the encoded protein into the plant body, so as to promote growth of the flowering Chinese cabbage.

[0023] Preferably, the preparation of the over-expressed gene or the encoded protein is an expression cassette of the over-expressed gene, a recombinant expression vector or a recombinant bacterium. BcC3H33 Preferably, the preparation of the over-expressed gene or the encoded protein is an expression cassette of the over-expressed gene, a recombinant expression vector or a recombinant bacterium. BcC3H33 Preferably, the preparation of the over-expressed gene or the encoded protein is an expression cassette of the over-expressed gene, a recombinant expression vector or a recombinant bacterium.

[0024] The application has the following beneficial effects: The application provides the flowering Chinese cabbage BcC3H33 The application provides the flowering Chinese cabbage BcC3H33 The application provides the flowering Chinese cabbage BcC3H33Following gene modification, the bioaccumulation of Chinese cabbage increased, morphological indicators improved, photosynthetic capacity enhanced, and resistance to stress strengthened. This can alleviate the impact of cadmium stress on the growth and development of Chinese cabbage, improve its tolerance to cadmium stress, and even promote its growth under cadmium stress. This makes it better suited for the cultivation of Chinese cabbage in cadmium-polluted environments, providing more effective methods for stress-resistant breeding of Chinese cabbage to reduce the impact of cadmium pollution on its growth and development. This invention has identified a cadmium-resistant C3H gene in Chinese cabbage. Further gene editing or transgenic technology can be used to cultivate more stress-resistant varieties to address the challenges of climate change, and also provide more genetic resources for cadmium-polluted environments and improving plant tolerance to cadmium stress. Attached Figure Description

[0025] Figure 1 This is a chromosomal mapping of the C3H gene family in Chinese cabbage.

[0026] Figure 2 for BcC3H33 A graph showing gene expression levels in different tissues.

[0027] Figure 3 for BcC3H33 Gene-based yeast resistance detection graph.

[0028] Figure 4 pCAMBIA-35S- BcC3H33 The detection results of the overexpression vector.

[0029] Figure 5 This refers to the tissue culture and screening process of Chinese cabbage.

[0030] Figure 6 for BcC3H33 Relative expression level of the OE gene overexpressing line in Chinese cabbage.

[0031] Figure 7 This is a diagram showing the growth status of seedlings of Chinese cabbage overexpressing OE.

[0032] Figure 8 For overexpression BcC3H33 Phenotypic diagram of Chinese cabbage seedlings treated with cadmium.

[0033] Figure 9 For overexpression BcC3H33 A graph showing the growth-related indicators of Chinese cabbage seedlings under cadmium treatment.

[0034] Figure 10 For overexpression BcC3H33 A graph showing the absorption activity of genes in the roots of Chinese cabbage under cadmium treatment.

[0035] Figure 11 For overexpression BcC3H33 Phenotypic diagram of Chinese cabbage leaves under cadmium treatment.

[0036] Figure 12 For overexpression BcC3H33 Figure of the influence of the gene on the leaf surface area of Brassica parachinensis under cadmium treatment.

[0037] Figure 13 For overexpression BcC3H33 Figure of the influence of the gene on the chlorophyll fluorescence characteristics of Brassica parachinensis under cadmium treatment.

[0038] Figure 14 For overexpression BcC3H33 group Figure of the influence of the gene on the MDA content and antioxidant enzyme activity of Brassica parachinensis under cadmium treatment.

[0039] Figure 15 For overexpression BcC3H33 Figure of the influence of the gene on the hormone content of Brassica parachinensis under cadmium treatment. DETAILED DESCRIPTION

[0040] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0041] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0042] The Brassica parachinensis materials used in the examples are all from South China Agricultural University and are planted in Room 704 of the Horticulture College of South China Agricultural University.

[0043] The primer sequences used in the examples are shown in Table 1 below.

[0044] Table 1 Primer sequence table

[0045] Example 1 BcC3H33 Cloning of the gene 1、 BcC3H33 Obtaining of the CDS sequence of the gene Protein sequences, CDS sequences, GFF3 annotation files, and genome sequences of *Brassica rapa* were downloaded from the NCBI database. Subsequently, the Hidden Markov Model (HMM) configuration file for the conserved C3H family domains (PFAM ID: PF00642) was downloaded from the PFAM database. Using the HMM software HMMER, the obtained sequences were compared with the downloaded HMM configuration file to preliminarily screen candidate genes of the *Brassica rapa* BcC3H family. The protein sequences of these candidate genes were submitted to the CDD and SMART websites for domain verification. After verification, only proteins containing the zf-CCCH domain were retained. Using the GFF3 annotation file, the chromosomal location information of the *Brassica rapa* BcC3H family members was obtained, and the BcC3H family members were named BcC3H1-77 based on chromosomal localization. Figure 1 As shown. Subsequently, the Tbtools software was used to obtain data from the Chinese cabbage genome data. BcC3H33 The gene CDS sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0046] 2. Primer design Amplification was analyzed and designed using Primer 6.0 software. BcC3H33 The primers for the gene are shown in Table 1, with specific primer sequences as shown in SEQ ID NO. 3~4.

[0047] 3. RNA extraction and cDNA first-strand synthesis Total RNA was extracted from the leaf tip tissue of Chinese cabbage using a plant RNA extraction kit. The entire process was carried out in accordance with the instructions of the RNA extraction kit. Then, using the extracted total RNA as a template, cDNA was obtained using a reverse transcription kit.

[0048] 4. Gene cloning Using the obtained Chinese cabbage cDNA as a template, primers were used... BcC3H33 -F and BcC3H33 -R was used for PCR amplification. The PCR reaction system was as follows: 2 μL of cDNA template, 20 μL of 2×Primer STAR Max Mix, 2 μL each of forward and reverse primers, and ddH2O to a final volume of 40 μL. The PCR reaction program was as follows: PCR reaction conditions were 94°C pre-denaturation for 3 min, 98°C denaturation for 10 s, 60°C annealing for 10 s, 72°C extension for 30 s, 30 cycles, 72°C final extension for 5 min, and incubation at 16°C.

[0049] The PCR product was subjected to agarose gel electrophoresis, and the target fragment of 2100 bp was finally obtained.

[0050] 5. Gel recovery and sequencing The PCR amplification products were recovered from the gel and sequenced. Sequencing comparisons showed that the amplification products were consistent with... BcC3H33 The gene's coding region sequence (SEQ ID NO.1) is identical, and its encoded protein amino acid sequence (SEQ ID NO.2) is also identical, indicating successful cloning. BcC3H33 Gene.

[0051] Example 2 BcC3H33 Analysis of gene expression patterns 1. Material Collection Using 'Youlv 501' Chinese cabbage as experimental material, seedlings were raised in perlite. During the seedling stage, 1 / 4 Hoagland nutrient solution was applied as fertilizer. When the plants reached the three-leaf stage, they were transplanted into a substrate (peat:coconut coir:perlite = 3:1:1) and subjected to cadmium stress treatment, with a 100 μM / L Cd solution. 2+ The solution was used for stress treatment. Nutrient solution was applied every 2 days during the cultivation period. At 0, 1, 3, 6, 12 and 24 h after treatment, healthy Chinese cabbage plants with the same growth were selected, and their leaves and roots were treated with liquid nitrogen and stored in a -80 ℃ refrigerator for later use.

[0052] 2. RNA extraction and cDNA first-strand synthesis Total RNA was extracted from the leaves and roots of the above-mentioned experimental materials using a plant RNA extraction kit. The entire operation was carried out in accordance with the instructions of the RNA extraction kit. Then, using the extracted total RNA as a template, cDNA was obtained using a reverse transcription kit.

[0053] 3. Primer design Choy sum obtained by cloning BcC3H33 Using genes as a reference, fluorescent quantitative primers were designed, and Chinese cabbage was used as a reference. Action Gene-based crop internal references, with specific primer sequences shown in Table 1 as SEQ ID NO.5~8.

[0054] 4. Quantitative Real-Time PCR Detection Gene expression levels were detected using the LightCycler 480 Real-Time PCR system from Musen Biotechnology (Shanghai). Experimental data were analyzed using 2... -ΔΔCt The method is used for calculation and analysis.

[0055] The measurement results are as follows Figure 2 As shown, BcC3H33 Gene expression trends differed across different tissues under cadmium stress. BcC3H33 The gene is highly expressed in the leaves, but its expression level gradually decreases over time. BcC3H33The expression level of the gene in the root gradually increases over time, reaching its highest value at 12 hours, and then significantly decreases after 24 hours.

[0056] Example 3 BcC3H33 Yeast resistance testing of genes 1. Construction of the carrier PCR amplification was performed according to the method in Example 1. BcC3H33 The CDS of the gene was ligated into the pSR-416 vector. Primers used for cloning are shown in SEQ ID NO. 9-10 in Table 1. BcC3H33 Yeast strains with overexpressed genes.

[0057] 2. Tolerance to cadmium stress in yeast Based on the overexpression constructed above BcC3H33 The pSR-416 vector of the gene served as the experimental group, while the empty vector (pRS-416) served as the control. Overexpression was performed in yeast cells. BcC3H33 And subject it to cadmium stress (SD / URA medium supplemented with 25µM Cd) 2+ In the study, normal culture conditions (SD / URA medium) were used as a control to observe and statistically analyze the growth of yeast cells in each group and the cadmium content in yeast cells in each group.

[0058] The results are as follows Figure 3 As shown, under cadmium stress, overexpression BcC3H33 The yeast cells overexpressed the vector grew faster than wild-type (EV-WT) cells transfected with the empty vector. Under normal culture conditions (SD / URA medium), overexpression... BcC3H33 Yeast cells and EV-WT cells showed the same growth trend. Furthermore, compared to EV-WT cells, overexpression of... BcC3H33 The intracellular cadmium content in yeast cells was significantly reduced. These results indicate that heterologous expression... BcC3H33 It can reduce the absorption and accumulation of cadmium in yeast cells, enhance the yeast's tolerance to cadmium stress, and play an important role in the yeast's tolerance to cadmium stress; therefore BcC3H33 It may become a candidate gene for resistance to cadmium stress.

[0059] Example 4 BcC3H33 Obtaining Chinese cabbage heart 1. Construction of overexpression vectors PCR amplification was performed according to the method in Example 1. BcC3H33 The CDS of the gene was then digested using XbaⅠ and SmaⅠ rapid digestion enzymes to digest the pCAMBIA3300-ter-35S-GFP-ter vector. After digestion, the vector was coupled with a primer-interfaced gene. BcC3H33The gene fragments were recombined on a PCR instrument, and the primers used for cloning are shown in Table 1 as SEQ ID NO. 11~12. The ligated BcC3H33 -3300 The overexpression vector was added to DH5a competent cells for expansion culture, and the plasmid was extracted for sequencing. After amplification detection and sequencing verification, the pCAMBIA-35S- BcC3H33 overexpression vector was successfully constructed. Figure 4 The detection results of the overexpression vector are shown in

[0060] 2. Agrobacterium transformation The constructed pCAMBIA-35S- BcC3H33 overexpression vector was transformed into GV3101 Agrobacterium by freeze-thaw method, and the Agrobacterium monoclonal containing the target vector was picked into 5 mL of LB liquid medium containing antibiotics (50 mg / L kanamycin and 20 mg / L rifampicin) and cultured at 28°C, 200 rpm overnight. 0.5 mL of the overnight cultured Agrobacterium was taken to 50 mL of LB liquid medium containing antibiotics (50 mg / L kanamycin and 20 mg / L rifampicin) for expansion culture, and the culture was continued until the Agrobacterium cell OD 600 =0.8. The cells were collected by centrifugation at 5000 rpm for 10 min at room temperature, and the Agrobacterium was resuspended with an equal volume of infiltration solution (5% sucrose, 0.03% silwit L-77).

[0061] 3、 BcC3H33 Obtaining and identifying of gene overexpression Brassica parachinensis (1) BcC3H33 Obtaining of gene overexpression Brassica parachinensis: Wild type Brassica parachinensis seeds were sterilized with 75% alcohol for 2 min, washed twice with sterile water, then sterilized with 7.5% sodium hypochlorite (NaClO) for 10 min, and washed with sterile water. Then evenly sow on 1 / 2 MS medium. After 3 days, the petiole cotyledons of the sterile seedlings were cut from the growth point and inserted into the pre-culture medium for 2 days of callus differentiation. The positive single colony of Agrobacterium carrying the gene editing vector was picked into 5 mL of YEP liquid medium containing antibiotics for initial shaking overnight, and then transferred to 100 mL of fresh YEP liquid medium containing antibiotics for expansion culture until the bacterial liquid OD 600The concentration was 0.6. Then, centrifuged for 10 min, discarded the supernatant, and resuspended the bacterial culture in the infection solution. Activated at 28℃ and 200 rpm, this was used to infect the cotyledons of Chinese cabbage. The pre-cultured cotyledons were infected with Agrobacterium infection solution for 10 min, then transferred to a co-culture medium. A sterile filter paper moistened with sterile infection solution was placed on the co-culture medium, and co-cultured for 3 days. The culture was then transferred to an antibacterial medium. After new adventitious buds differentiated from the callus, they were transferred to a selection medium, with the medium changed periodically according to the selection results. Resistant and well-grown adventitious buds were cut from the top of the callus and inserted into a rooting medium for rooting. Two weeks later, the successfully rooted resistant plants were opened and bagged for hardening off. Four days later, they were transplanted into nutrient soil (peat:vermiculite = 3:1), bagged to retain moisture. After the plants survived transplanting, the plastic bag was removed, and normal water and fertilizer were applied. The Chinese cabbage tissue culture and selection process is as follows. Figure 5 As shown.

[0062] (2) BcC3H33 Identification of overexpressing Chinese cabbage: DNA was taken from the leaves of tissue-cultured Chinese cabbage plants. Using vector-specific primers, the specific primer sequences are shown in SEQ ID NO.13~14 in Table 1. The gene was detected by PCR. The resistant plants that amplified the correct target band indicated that the gene editing vector had been successfully transferred into the Chinese cabbage plants, and the overexpressing seedlings were finally obtained.

[0063] 4. BcC3H33 Gene expression level analysis Subsequently, referring to the real-time quantitative PCR detection method in Example 2, the overexpression lines obtained above were detected in seedlings. BcC3H33 Gene expression levels were compared with normal "Youlv 501" Chinese cabbage as a control.

[0064] Test results as follows Figure 6 As shown, this is evident in the overexpression of the overexpression line seedlings. BcC3H33 Gene expression levels were significantly increased.

[0065] Example 5 Overexpression BcC3H33 The Influence of Genes on the Cotyledon Stage of Chinese Pepper Overexpression obtained in Example 4 BcC3H33 Seeds of Chinese cabbage were used as experimental materials and were sown in 1 / 2 MS medium containing 5 μmol / L, 10 μmol / L, and 20 μmol / L cadmium, respectively, along with 1% sucrose and 0.6% agar. Wild-type Chinese cabbage (WT) was sown in medium without cadmium as a control. On the fifth day after sowing, the aboveground and underground growth of the overexpression line seedlings and control seedlings was measured using a ruler.

[0066] Statistical results of growth-related indicators of Chinese cabbage cotyledons under cadmium treatment are as follows: Figure 7As shown, under cadmium treatments at concentrations of 5 μmol / L and 10 μmol / L, the aboveground parts of the WT lines were significantly shortened, while the underground parts showed shortened taproots and a reduced number of fibrous roots. Under cadmium treatment at a concentration of 20 μmol / L, the growth of the WT lines was further inhibited, with the epicotyl bending and unable to extend, preventing the roots from emerging smoothly. In contrast, the overexpression lines, under cadmium treatments at concentrations of 5 μmol / L, 10 μmol / L, and 20 μmol / L, showed significantly lower growth inhibition due to cadmium stress than the WT lines, and epicotyl development was superior in all three cases. Only under cadmium treatment at a concentration of 20 μmol / L did the epicotyl shorten and begin to bend, indicating enhanced cadmium tolerance in the overexpression lines.

[0067] Example 6 Overexpression BcC3H33 The Influence of Genes on the Growth of Chinese Spinach Seedlings The overexpression obtained in Example 4 BcC3H33 The seeds of Chinese cabbage were used as experimental materials and sown in a substrate (peat:coconut coir:perlite = 3:1:1). The plants were transplanted when they reached the three-leaf stage. The plants were irrigated with clean water for one week after sowing, and then watered every 2 days with 1 / 2 Hoagland nutrient solution containing 25 μmol / L and 50 μmol / L cadmium, respectively. The results were measured when the plants reached the five-leaf stage. Wild-type Chinese cabbage was watered every 2 days with nutrient solution without cadmium as a control.

[0068] Nine uniformly growing whole Chinese cabbage plants were randomly selected from each treatment. Their total fresh weight, aboveground fresh weight, and root fresh weight (g) were measured using a 0.01 g electronic balance. The root length and plant height (from the lower end of the hypocotyl to the highest point of the leaf extension) were measured using a ruler. The stem diameter (approximately 0.5 cm above the cotyledons, unit: cm) was measured using a 0.1 g digital vernier caliper. After that, the plants were placed in an oven at 105℃ for 30 min to blanch, and then dried at 75℃ to constant weight. The leaf dry weight and aboveground dry weight were determined using a 0.01 g electronic balance.

[0069] Statistical results of growth-related indicators of Chinese cabbage seedlings under cadmium treatment are as follows: Figure 8 and Figure 9 As shown, in the control (CK) group without stress, there was no significant difference between the overexpressing lines and the WT lines. Under 25 μmol / L Cd treatment, the WT lines were slightly affected by stress, and compared with the overexpressing lines, there were significant differences in total fresh weight, total dry weight, plant height, aboveground fresh weight, root fresh weight, and aboveground dry weight. The overexpressing lines were significantly better than the WT lines in all these indicators. Under 50 μmol / L Cd treatment, the WT lines were further affected by stress, and compared with the overexpressing lines, there were significant differences in total fresh weight, plant height, aboveground fresh weight, root fresh weight, and root length. The overexpressing lines were significantly better than the WT lines in all these indicators.

[0070] Example 7 Overexpression BcC3H33 Effects of genes on the absorption activity of Chinese cabbage roots The overexpression obtained in Example 4 BcC3H33 Using Chinese cabbage seeds as experimental material, root activity was measured when the plants reached the five-leaf stage. Fresh roots of the Chinese cabbage were cut, rinsed with deionized water, and dried. The roots were cut into uniform small segments (approximately 0.5-1 cm). The root segments were placed in test tubes containing TTC solution and phosphate buffer and incubated at 37°C in the dark for 40 min. Dilute sulfuric acid was added. The root segments were removed, dried, and placed in a mortar with ethyl acetate and a small amount of quartz sand, and ground thoroughly. The extract was filtered or centrifuged, transferred to a volumetric flask, and diluted to volume with solvent. The absorbance of the extract was measured at a wavelength of 485-490 nm using a spectrophotometer.

[0071] Results of cadmium treatment on the root absorption activity of Chinese cabbage: Figure 10 As shown in the figure, the root absorption activity of the overexpression lines was significantly higher than that of the WT lines in all treatment groups. Compared with the CK group, the average root absorption activity of both the overexpression and WT lines was enhanced to some extent after treatment with 25 μmol / L and 50 μmol / L Cd. The results indicate that overexpression... BcC3H33 Genes can enhance the absorption activity of Chinese cabbage roots.

[0072] Example 8: Effects of overexpression of the BcC3H33 gene on seedling stage When the plants in Example 6 reached the five-leaf stage, leaf surface area, photosynthetic characteristics, chlorophyll fluorescence characteristics, MDA content, antioxidant enzyme activity, and hormone content were measured. Specifically, plant leaf images with a scale bar were taken using a camera, and the leaf area (cm²) of the true leaves was measured using ImageJ software. 2 The leaves of Chinese cabbage plants in the east, west, south, and north directions were tracked and measured using a TARGAS-1 portable photosynthesis instrument (PPSystem brand). The measurements included net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular carbon dioxide concentration (Ci).

[0073] The SPAD values ​​of leaves in each group were measured using a SPAD-502PLUS chlorophyll meter. On the day of harvest, six plants with uniform growth were randomly selected from each treatment and subjected to 20 min of dark treatment. The chlorophyll fluorescence parameters Y(II), Y(NPQ), Y(NO), and ETR of the first true leaf were then measured using an IMAGINE PAM chlorophyll meter.

[0074] SOD activity was determined according to the method of Gianopolitis and Ries, with the amount of enzyme inhibiting 50% of the photochemical reduction of NBT per minute representing the unit enzyme activity (U). POD activity was determined according to Jiang's method, with the amount of enzyme with a change of absorbance of 0.01 per minute at a wavelength of 470 nm representing the amount of enzyme in U. CAT activity was determined according to Wang's method, with the amount of enzyme with a change of absorbance of 0.01 per minute at a wavelength of 240 nm representing the amount of enzyme in U.

[0075] Hormone extraction involved grinding plant samples into powder using liquid nitrogen. 0.2 g of the sample was weighed and added to 1.6 mL of PBS buffer (pH 7.4, 4℃), and incubated at 4℃ for 24 h. The mixture was then centrifuged at 4000 rpm for 20 min. The supernatant was aliquoted and stored at -20℃ for hormone assays. IAA and ABA contents in the plants were determined using an enzyme-linked immunosorbent assay (ELISA). The ELISA kit was purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.

[0076] Phenotypes of Chinese cabbage leaves under cadmium treatment Figure 11 As shown, the leaves of the overexpressing strain are rounder and greener than those of the WT strain.

[0077] Results of cadmium treatment on leaf surface area and photosynthesis in Chinese cabbage: Figure 12 As shown, the leaf surface area was not significantly different from that of the WT line. Under 25 μmol / L Cd treatment, the SPAD value of the overexpression line was significantly higher than that of the WT line, indicating a higher relative chlorophyll content. Under 50 μmol / L Cd treatment, both the overexpression and WT lines were affected by cadmium stress, with significant decreases in transpiration rate and stomatal conductance. However, the overexpression line was less affected by the stress than the WT line. This indicates that overexpression... BcC3H33 The gene can alleviate the impact of high concentrations of cadmium on the photosynthetic capacity of Chinese cabbage.

[0078] Results of chlorophyll fluorescence in Chinese cabbage leaves under cadmium treatment: Figure 13 As shown, there were no significant differences in any of the indicators between the overexpression line and the WT line in the CK group. Under 25 μmol / L Cd treatment, the Fv / Fm parameter value of the overexpression line was significantly higher than that of the WT line. Under 50 μmol / L Cd treatment, the Y(II), qP, and ETR values ​​of the overexpression line were significantly higher than those of the WT line, while the Y(NO) value of the WT line was higher than that of the overexpression line.

[0079] Results of MDA content and antioxidant enzyme activity in Chinese cabbage under cadmium treatment are as follows: Figure 14As shown, it is shown that in the CK group, the MDA content, POD enzyme activity and CAT enzyme activity of the overexpression strain have no significant difference with the WT strain, and the average value of SOD enzyme activity is higher than that of the WT strain. Under the treatment of 25 μmol / L Cd, the POD enzyme activity and SOD enzyme activity of the overexpression strain are significantly higher than those of the WT strain, and the POD enzyme activity and CAT enzyme activity of the overexpression strain and the WT strain are significantly increased compared with the CK group. Under the treatment of 50 μmol / L Cd, the MDA content of the overexpression strain and the WT strain is significantly increased, and the MDA content of the overexpression strain is significantly lower than that of the WT strain, while the CAT enzyme activity is significantly higher than that of the WT strain. The results show that the overexpression strain has stronger cadmium stress resistance than the WT strain, and after cadmium stress treatment, the MDA content of the WT strain rises faster, and the stress damage is deeper, while the antioxidant enzyme activity of the overexpression strain rises faster, and the stress response ability is stronger.

[0080] The results of the brassica chinensis hormone content under cadmium treatment are as shown in Figure 15 As shown, it is shown that in the CK group and the 25 μmol / L Cd treatment group, the IAA and ABA concentrations of the overexpression strain have no obvious difference with the WT strain. In the 50 μmol / L Cd treatment group, the ABA concentration of the overexpression strain is significantly higher than that of the WT strain, and the IAA concentration is significantly lower than that of the WT strain.

[0081] In summary, the results show that after overexpression of the BcC3H33 gene in brassica chinensis, the biological accumulation of brassica chinensis is increased, the morphological index is improved, the photosynthetic capacity is enhanced, the resistance to stress is enhanced, BcC3H33 the gene has the effect of promoting growth and tolerating cadmium stress under cadmium stress, and overexpression of the BcC3H33 gene in brassica chinensis can relieve the inhibition effect of cadmium stress, promote the growth of brassica chinensis, increase the fresh weight, dry weight and plant height of brassica chinensis, increase the plant biomass accumulation, promote the absorption of root system, enhance the photosynthetic capacity and resistance to stress, and can alleviate the influence of cadmium stress on the growth and development of brassica chinensis, provide basis and resources for brassica chinensis stress resistance breeding, and provide more gene resources for cadmium pollution environment and improvement of cadmium stress tolerance of plants.

[0082] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement modes, and all shall be included in the protection scope of the present application.

Claims

1. As shown in SEQ ID NO.1 BcC3H33 The application of genes or their encoded proteins in improving the tolerance of Chinese cabbage to cadmium stress is characterized by, Expressing through the heart of the vegetable BcC3H33 Gene.

2. As shown in SEQ ID NO.1 BcC3H33 The application of the gene or its encoded protein in promoting the growth of Chinese cabbage and / or promoting the growth of Chinese cabbage under cadmium stress, characterized in that, Expressing through the heart of the vegetable BcC3H33 Gene.

3. As shown in SEQ ID NO.1 BcC3H33 The application of the gene or its encoded protein in the cultivation of cadmium-tolerant Chinese cabbage plants is characterized by, Expressing through the heart of the vegetable BcC3H33 Gene.

4. Overexpression of the compound shown in SEQ ID NO.1 BcC3H33 Application of the gene or its encoded protein in improving the tolerance of Chinese cabbage to cadmium stress.

5. Overexpression of the compound shown in SEQ ID NO.1 BcC3H33 Application of the gene or its encoded protein in promoting the growth of Chinese cabbage and / or promoting the growth of Chinese cabbage under cadmium stress.

6. Overexpression of the compound shown in SEQ ID NO.1 BcC3H33 Application of genes or their encoded proteins in the cultivation of cadmium-tolerant Chinese cabbage plants.

7. Overexpression of the compound shown in SEQ ID NO.1 BcC3H33 Application of the gene or its encoded protein in the preparation of products that promote the growth of Chinese cabbage or tolerance to cadmium stress.

8. A method for improving the tolerance of Chinese cabbage to cadmium stress, characterized in that, By overexpressing the substance shown in SEQ ID NO.1 in Chinese broccoli. BcC3H33 Genes or their encoded proteins could be used to improve the tolerance of Chinese cabbage to cadmium stress.

9. A method for promoting the growth of Chinese cabbage and / or promoting the growth of Chinese cabbage under cadmium stress, characterized in that, overexpression BcC3H33 Genes or their encoded proteins are transferred into plants to promote the growth of Chinese cabbage.

10. The application according to any one of claims 4 to 7, characterized in that, The overexpression BcC3H33 The preparation of the gene or its encoded protein is an overexpression BcC3H33 Gene expression cassettes, recombinant expression vectors, or recombinant bacteria.

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