Application of CaCA2 gene in pepper resistance to low temperature and weak light stress

Abstract

The application provides a gene and an application thereof CaCA2 The application belongs to the technical field of gene breeding and relates to application of a gene in pepper resistance to low-temperature and weak-light stress. CaCA2 The application discloses a gene and an application thereof CaCA2 The transient overexpression of the gene can slow down wilting of pepper plants, improve net photosynthetic rate of pepper leaves, expression amount of antioxidant enzyme genes, soluble sugar, free proline and mineral element content, and simultaneously reduce intercellular CO2 concentration and malondialdehyde content. CaCA2 After the VIGS technology is used to transiently silence the gene, the plant height, stem diameter, biomass and root morphological parameters of the pepper are reduced in varying degrees, and the gene causes stoma to close, and the overall fluorescence intensity, photosynthetic performance index and trace element content are reduced. CaCA2 The transient overexpression of the gene in the pepper can relieve wilting of the pepper plants and improve the resistance to low-temperature and weak-light stress, and the resistance is weakened after the gene is silenced.

Description

Technical Field

[0001] This invention belongs to the field of gene breeding technology, and particularly relates to a... CaCA2 Application of genes in chili peppers' resistance to low temperature and low light stress. Background Technology

[0002] Chili peppers, a widely cultivated cash crop in both northern and southern my country, are closely linked to climatic conditions in their industrial development. However, the impact of low temperatures and weak light on the chili pepper industry is gradually becoming apparent, and its influence is expanding. In open-field cultivation, abnormal weather such as early spring frosts and early autumn frosts can cause seedling damage, wilting of mature plants, necrotic spots on leaves, and in severe cases, death of the entire plant. In greenhouse cultivation, prolonged periods of low light caused by continuous rain and snow can damage the photosynthetic system of chili pepper plants, accelerate chlorophyll degradation in leaves, resulting in "chlorosis and yellowing," and consequently, a sharp decline in the plant's nutrient synthesis capacity. According to survey data from production areas, a single period of low temperatures and weak light lasting more than 7 days can reduce chili pepper yield by more than 40% per crop, and significantly decrease the marketable yield of the fruit.

[0003] In recent years, with the rapid development of molecular biology techniques, research on genes involved in low-temperature and low-light tolerance in chili peppers has made some progress. Studies have shown that the expression regulation of multiple genes is involved in the chili pepper's response to low-temperature and low-light stress. These genes synergistically regulate the chili pepper's tolerance to low-temperature and low-light stress by participating in physiological processes such as photosynthesis, antioxidant defense systems, hormone signal transduction, and osmotic regulation. For example, some genes can regulate the synthesis and degradation of photosynthetic pigments in chili pepper leaves, maintaining normal photosynthesis; others participate in the synthesis of antioxidant enzymes, enhancing the plant's ability to scavenge reactive oxygen species and reducing oxidative damage. In addition, some genes influence plant growth, development, and stress response by regulating plant hormone levels.

[0004] Previously, through transcriptome sequencing, we discovered that low temperature and low light stress (LL) induced the gene mutation of the β-carotene hydroxylase gene in pepper (Capsicum annuum L.) seedlings. CaCA2 The downregulation of this gene suggests it may be involved in regulating the chili pepper's response to low temperature and low light stress, but the specific mechanism of action remains unknown. In-depth exploration and application of low temperature and low light genes will build a multi-dimensional stress resistance system for the chili pepper industry. In the long run, the development of these gene resources will also break down geographical planting limitations. By cultivating stress-resistant varieties adapted to high latitude and high altitude regions, areas previously unsuitable for chili pepper cultivation will form new industrial belts, effectively alleviating supply pressure in major producing areas. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a CaCA2 Application of genes in chili peppers' resistance to low temperature and low light stress.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides CaCA2 Application of gene in chili pepper's resistance to low temperature and low light stress, transient overexpression CaCA2 Genes that enhance chili peppers' resistance to low temperature and low light stress, and silencing genes... CaCA2 Genes reduce the resistance of chili peppers to low temperature and low light stress.

[0007] The present invention also provides CaCA2 Application of genes in regulating photosynthesis in peppers, transient overexpression CaCA2 Genes enhance photosynthesis in chili peppers, silencing CaCA2 Genes reduce photosynthesis in chili peppers.

[0008] Preferably, the indicators of photosynthesis include photosynthetic pigment content, net photosynthetic rate, transpiration rate, stomatal conductance, and photosynthetic performance index.

[0009] The present invention also provides CaCA2 Application of the gene in regulating the activity of antioxidant enzymes in peppers, transient overexpression CaCA2 Genes enhance the activity of antioxidant enzymes in chili peppers.

[0010] Preferably, the antioxidant enzyme genes include SOD, POD, CAT, and APX enzyme genes.

[0011] The present invention also provides CaCA2 Application of genes in regulating the content of osmotic regulatory substances in chili peppers, overexpression CaCA2 Genes increase the content of osmotic regulatory substances in chili peppers.

[0012] Preferably, the osmotic regulating substance includes soluble sugars, soluble proteins, and free proline.

[0013] The present invention also provides CaCA2 Application of genes in regulating the mineral element content of chili peppers, overexpression CaCA2 Genes increase the mineral content of chili peppers, silencing CaCA2 Genes reduce the mineral content of chili peppers.

[0014] Preferably, the mineral elements include P, Cu, Fe, Mn, and Zn.

[0015] Preferably, the overexpression CaCA2 The genetic approach includes the following steps: Build CaCA2 Gene overexpression vectors were used to genetically transform peppers using Agrobacterium infection, and overexpression vectors were screened. CaCA2 Genetically modified strains of chili peppers CaCA2 Gene overexpression; The CaCA2 The gene overexpression vector used P-Super-1300 as the initial vector, and cloned gene expression vectors between the Xba1 and Kpn1 restriction enzyme sites of P-Super-1300. CaCA2 Gene fragments.

[0016] The silence CaCA2 The genetic approach includes the following steps: Build CaCA2 The VIGS gene silencing vector was used to infect peppers with Agrobacterium tumefaciens, and the silencing vector was screened. CaCA2 Genetically modified strains of chili peppers CaCA2 Gene silencing; The CaCA2 The VIGS gene silencing vector uses TRV2 as the initial vector and clones a gene between the EcoRI and BamHI enzymes at the TRV2 restriction site. CaCA2 Silent segments of genes.

[0017] Compared with the prior art, the present invention has the following beneficial effects: In this invention, chili peppers CaCA2 The gene contains cis-regulatory elements involved in light response, and its expression level is highest in green fruits. After 168 hours of low temperature and low light stress, CaCA2 The expression level was downregulated. Under low temperature and low light stress, CaCA2 Transient overexpression of [a specific substance] increased net photosynthetic rate (Pn), light energy absorbed per unit reaction center (ABS / RC), expression levels of SOD, POD, CAT, and APX enzyme genes, soluble sugar content, free proline content, and mineral element (P, Na, Cu, and Fe) content, while decreasing intercellular CO2 concentration (Ci) and malondialdehyde (MDA) content. Transient silencing using VIGS technology [further details needed]. CaCA2 Following gene modification, pepper plants became smaller, with varying degrees of reduction in plant height, stem diameter, biomass, and root morphology parameters. Stomatal closure was also induced, and overall fluorescence intensity, photosynthetic performance index (PIabs), and trace element (Cu, Fe, Mn, and Zn) content all decreased. In summary, CaCA2 Transient overexpression of the gene in pepper can enhance resistance to low temperature and low light stress by increasing photosynthesis, antioxidant enzyme activity, accumulation of osmotic regulators, and maintenance of mineral balance, while silencing the gene... CaCA2 Genetic modification can weaken its resistance. Attached Figure Description

[0018] Figure 1 yes CaCA2 Gene clone electrophoresis diagram and plasmid map (A is the clone electrophoresis diagram, B is the plasmid map, the red part is the insertion). CaCA2 (gene fragments) Figure 2 yes CaCA2 Electrophoresis diagram and plasmid map of gene silencing fragment clones (A is the clone electrophoresis diagram, B is the plasmid map, the red part is the insertion). CaCA2 (gene silencing fragments); Figure 3 yes CaCA2 Tissue-specific expression of genes in chili peppers; Figure 4 yes CaCA2 Changes in gene expression at different time points under LL stress; Figure 5 It is an instantaneous overexpression CaCA2 At that time, its expression level in chili peppers; Figure 6 It is transient overexpression under LL stress. CaCA2 Images showing changes in the growth morphology of chili peppers after genetic modification; Figure 7 It is transient overexpression under LL stress. CaCA2 Changes in photosynthetic pigment content and gas exchange parameters (where A is photosynthetic pigment content, B is net photosynthetic rate (Pn), C is transpiration rate (Tr), D is stomatal conductance (Gs), and E is intercellular CO2 concentration (Ci)). Figure 8 The changes in chlorophyll fluorescence parameters during transient overexpression of CaCA2 under LL stress are shown in the figure (where A is the chlorophyll fluorescence image, B is the initial fluorescence (Fo), C is the maximum fluorescence (Fm), D is the maximum photochemical efficiency of PSII (Fv / Fm), E is the actual photochemical efficiency of PSII [Y(II)], F is the photochemical quenching coefficient (qP), and G is the non-photochemical quenching (NPQ)). Figure 9 Transient overexpression under LL stress CaCA2 The changes in OJIP curves, JIP test parameters, unit interfacial energy distribution, and leaf activity ratio parameters are as follows (where A and B are chlorophyll fluorescence kinetics (OJIP) curves, C and D are JIP test parameters, E and F are electron absorption, capture, transport, and dissipation fluxes for each cross section, and G and H are electron absorption, capture, transport, and dissipation fluxes for each RC). Figure 10 It is transient overexpression under LL stress. CaCA2 Changes in ROS content and antioxidant enzyme activity over time (where A is O2) - Content, B is H2O2 content, C is SOD activity, D is POD activity, E is CAT activity, F is APX activity, G is relative SOD gene expression level, H is relative POD gene expression level, I is relative CAT gene expression level, J is relative APX gene expression level. Figure 11It is transient overexpression under LL stress. CaCA2 Changes in the content of osmotic regulators over time (where A is relative conductivity (REC), B is malondialdehyde (MDA) content, C is soluble sugar content, D is soluble protein content, E is free proline content, F is total phenol content, and G is flavonoid content). Figure 12 It is transient overexpression under LL stress. CaCA2 Changes in mineral ions over time (where A is P content, B is K content, C is Ca content, D is Na content, E is Mg content, F is Cu content, G is Fe content, H is Mn content, and I is Zn content). Figure 13 It was a momentary silence under LL's coercion. CaCA2 Images showing changes in the growth morphology of chili peppers and albino growth after genetic modification; Figure 14 It was silence under LL's coercion CaCA2 Changes in stomatal morphology and parameters over time (where A is a stomatal image, B is stomatal length, C is stomatal width, D is pore length, and E is pore width). Figure 15 Silence under LL's coercion CaCA2 The changes in OJIP curves, JIP test parameters, unit interfacial energy distribution, and leaf activity ratio parameters are as follows (where A is the chlorophyll fluorescence kinetics (OJIP) curve, B is the JIP test parameter, C, D, E, and F are the electron absorption, capture, transport, and dissipation fluxes for each cross section, and G, H, I, and J are the electron absorption, capture, transport, and dissipation fluxes for each RC). Figure 16 It was silence under LL's coercion CaCA2 Changes in mineral ions over time (where A is P content, B is K content, C is Ca content, D is Na content, E is Mg content, F is Cu content, G is Fe content, H is Mn content, and I is Zn content). Detailed Implementation

[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] 1. Obtaining the gene sequence data of capsicum β-carotene hydroxylase

[0021] A β-carotene hydroxylase protein sequence was downloaded from the Arabidopsis Genome Database (TAIR) (https: / / www.arabidopsis.org / ) as the target sequence. The amino acid sequence of "Zunla-1" pepper was obtained from the Solanaceae Genome Database (http: / / peppergenome.snu.ac.kr / -download.php). Blastop alignment was performed using Blast Compare Two Seq in TBtools software, with an E value set to 10. -5 A single chili β-carotene hydroxylase gene was obtained, and its domains were validated using the online software SMART and PFAM. The full-length gene, CDS sequence, amino acid sequence, and cDNA sequence were then downloaded. The gene ID for CaCA2 is Capana06g002492.

[0022] 2. Functional verification of the chili β-carotene hydroxylase gene

[0023] 2.1 Total RNA extraction and RT-PCR analysis

[0024] Total RNA was extracted using an RNA extraction kit (Accurate Biotechnology Co., Ltd., Hunan, China). The extracted RNA samples were reverse transcribed using a cDNA synthesis kit (Accurate Biotechnology Co., Ltd., Hunan, China) to obtain cDNA. Gene sequences were obtained from the Sol Genomics Network database (https: / / solgenomics.net / ). Primers were synthesized by Beijing Qingke Biotechnology Co., Ltd., and their sequences are shown in Table S1. Real-time PCR reactions were performed using a Light Cycler® 96 Real-Time PCR system (Roche, Switzerland) according to the instructions of the SYBR Green Priemix Pro Taq HS qPCR kit (Yaque Biotechnology Co., Ltd.). Relative quantification of mRNA levels was calculated using the 2-ΔΔCt method. Each treatment consisted of three biological replicates.

[0025] Table 1 qRT-PCR primer sequences

[0026] 2.2 Cloning, transient overexpression, and VIGS silencing vector construction of the β-carotene hydroxylase gene

[0027] The full-length coding sequence (CDS) and silent fragment sequence of CaCA2 were cloned from the cDNA of “L1211” pepper leaves. The PCR amplification products were recovered, purified, and transformed into E. coli. Positive clones were screened and sequenced. Results ( Figure 1 and 2 The results showed that CaCA2 exhibited the target band at 927 bp, and the silenced fragment exhibited the target band at 300 bp, both matching the expected band lengths. The full-length CDS and silenced fragment sequences of CaCA2 were then ligated into the overexpression vector P-Super-1300 and the silencer vector TRV2, respectively. The plasmid map of the full-length CaCA2 is shown below. Figure 1 As shown, the plasmid map of the CaCA2 silencing fragment is as follows: Figure 2 As shown.

[0028] 2.3 Transient overexpression and silencing of CaCA2 in chili peppers

[0029] Transient overexpression of chili pepper was determined using Ma Xiao's method (Ma Xiao. Study on the response and regulatory mechanism of chili pepper CaCIPKs to drought and low temperature stress [D]. Northwest A&F University, 2022.), and VIGS silencing of chili pepper was determined using Gou Bingdiao's method (Gou Bingdiao. Expression analysis of chili pepper CaTPS family members and functional analysis of CaTPS1 response to low temperature and salt stress [D]. Gansu Agricultural University, 2022.).

[0030] Agrobacterium suspensions containing ps1300:GFP and ps1300:CaCA2:GFP were treated with MES resuspension (10 mM MES, 10 mM CaCl2, 200 μM acetylsuccinone, pH 5.7), respectively. OD 600 Adjusting the pH to around 0.8, the suspension was gently injected into the five-leaf-one-heart chili pepper leaves using a syringe with the needle removed, avoiding any additional damage to the leaves. After culturing in the dark at 22℃ for 2 days, the GFP signal in the leaves was detected using a fluorescent protein observation flashlight (LUYOR-3280RB), and RNA was extracted for real-time quantitative PCR (qRT-PCR) to detect the transient expression level of the gene in the chili pepper. Positive plants were selected for subsequent experiments.

[0031] The contents of TRV2:00 and TRV2: respectively CaCA2 The Agrobacterium tumefaciens solutions of TRV2:PDS and TRV1 were mixed with the IM bacterial solution at a ratio of 1:25 and incubated at 200 rpm for 27-36 h until the OD reached. 600The concentration was 0.5-0.8. The bacterial culture was then transferred to 50 mL centrifuge tubes and centrifuged at 3000g for 10 min at 4°C. The supernatant was discarded, and the culture was resuspended in an equal volume of MES. The culture was then centrifuged again at 3000g for 10 min at 4°C, and the bacterial cells were collected and resuspended in half a volume of MES. Acetyleugenol (AS) was then added to TRV1 to a final concentration of 400 mM. TRV1 was then mixed with TRV2:00, TRV2:CaCA2, and TRV2:PDS at a 1:1 ratio to achieve a final AS concentration of 200 mM. OD 600 The concentration was 0.6. After standing at room temperature for 5 hours, the cotyledons of the pepper plant were inoculated using a 1 mL syringe needle at the two-leaf-one-heart stage. The infected plants were then placed in the dark at 18°C ​​for two days, followed by normal culture. Once the white leaves of the pepper seedlings appeared, the silencing efficiency was tested, and silenced plants were selected for subsequent experiments.

[0032] 2.4 Plant materials and culture conditions

[0033] The chili pepper variety selected for this invention is "L1211". The seeds were first soaked in 55℃ warm water for 30 minutes, then soaked in 25℃ tap water for 6 hours. The soaked seeds were placed on a clean towel and germinated in an artificial climate chamber (RDN-400E-4, Ningbo, Zhejiang). Once the seeds turned white, seeds with uniform germination (1 mm) were selected and sown in plastic seedling pots (9×9 cm, volume ratio) containing substrate, one seed per pot. Seedlings were cultured at 28 / 18℃ and 300 μmol·m³. -2 ·s -1 Cultivated in an artificial climate chamber with illumination.

[0034] 2.5 Experimental Design

[0035] Select healthy, uniformly growing pepper seedlings and treat them at 10 / 5℃ with 100 μmol·m⁻²·m⁻²·℃. -2 ·s -1 Seedlings were subjected to low temperature and low light treatment under stress conditions. Seedlings under normal growth conditions served as a control, with conditions of 28 / 18℃ and 300 μmol·m⁻²·m⁻²·℃. -2 ·s -1 The experiment employed a randomized block design with two treatments.

[0036] CK: (28 / 18°C, day / night, 300 μmol·m -2 ·s -1 )

[0037] LL: (Low temperature and low light, 10 / 5°C, day / night, 100 μmol·m -2 ·s -1 )

[0038] (1) Chili peppers were treated continuously for 7 days under low temperature and low light conditions. Chili peppers from each treatment were randomly selected at 0, 6, 12, 24, 48 and 168 h to determine the expression level of related genes.

[0039] All data were analyzed using Excel 2020. Experiments were repeated three times, and results are expressed as mean ± standard error. Analysis of variance (ANOVA) was performed using SPSS 20.0 (SPSS Institute Inc., USA). Means of each treatment group were compared using Duncan's multiple range test (significance level of 0.05) to determine if significant differences existed. Figures and tables were created using OriginPro 2022.

[0040] ① CaCA2 tissue-specific expression

[0041] Experimental results: such as Figure 3 As shown. CaCA2 The expression level was highest in green pepper fruits, followed by red fruits and leaves, and lowest in roots. At 6 h of LL treatment, compared to the control group, LL treatment increased expression levels. CaCA2 Expression; at LL treatment times of 12, 24, 48, and 168 h, LL treatment significantly reduced expression compared to CK. CaCA2 The expression ( Figure 4 ).

[0042] ② Expression of β-carotene hydroxylase gene

[0043] Experimental results: such as Figure 5 and Figure 6 As shown. Compared with the control plants (ps1300:GFP), the transient expression plants showed significantly higher levels of... CaCA2 The expression level of was significantly increased.

[0044] (2) At 168 h, the functional leaves of the plant were rapidly frozen with liquid nitrogen and placed in a -80℃ freezer for physiological index determination. Plant height and stem diameter were measured using a ruler and vernier calipers, respectively. The fresh weight of the above-ground parts and roots of the plant was weighed using a 0.01% balance. Then, each part was heated to 105℃. C. Blanch in a forced-air drying oven for 30 minutes, then adjust the temperature to 80°C. C. The roots were dried to constant weight, and their dry weight was measured. After washing, the pepper roots were placed in a root analysis system (Win RHIZO ProLA2400, Canada) to obtain images, and root morphology parameters were analyzed using Win RHIZO 5.0 software. Chlorophyll content was determined using the acetone leaching method. Photosynthetic gas exchange parameters (Gs, Pn, Ci, and Tr) were measured using a portable photosynthesis system (CIRAS2, PPsystem, UK) between 9:00 AM and 11:00 AM on a sunny day. Chlorophyll fluorescence induction kinetics (OJIP curve) and JIP test parameters were collected using a plant efficiency analyzer (Handy PEA, Hansatech, UK). Relative conductivity (REC) was determined according to the method of Huang et al. (HUANG J, SUN R, CAO X, et al. Preservation effect of Lactobacillus plantarum O2 fermentation supernatanton postharvest pepper and its induced resistance to Phytophthoracapsici. Plant Physiology and Biochemistry, 2023, 204 (https: / / doi.org / ). MDA content was determined using the thiobarbituric acid method. Soluble protein content was determined according to the method of Li et al. (LI LEI LL, YINXIANHUI YX, LONG YOUHUA LY, et al. Effects of amino acid water soluble fertilizer on amino acid content and quality of pepper. 2019, https: / / doi.org / ). Soluble sugar content was determined using the anthrone colorimetric method described by Li et al. (LI J, YANG P, GAN Y, et al. Brassinosteroid alleviates chilling-induced oxidative stress in pepper by enhancing antioxidation systems and maintenance of...). photosystem II. ActaPhysiologiae Plantarum, 2015, 37(11):1-11. https: / / doi.org / ).Free proline was determined using the sulfosalicylic acid method, and total phenols and flavonoids were determined using the method of Wang et al. (WANG C, ZHANG J, XIE J, et al. Effects of Preharvest Methyl Jasmonate and Salicylic Acid Treatments on Growth, Quality, Volatile Components, and Antioxidant Systems of Chinese Chives. Frontiers in Plant Science, 2022, 12 (https: / / doi.org / ).) 2-H2O2 content was determined according to the method described by Bu et al. (BU R, XIE J, YU J, et al. Autotoxicity in cucumber (Cucumissativus L.) seedlings is alleviated by silicon through an increase in the activity of antioxidant enzymes and by mitigating lipid peroxidation. Journal of Plant Biology, 2016, 59(3):247-59. https: / / doi.org / ). SOD, POD, CAT and APX enzyme activities were determined according to the method of Li et al. (LI J, YANG P, GAN Y, et al. Brassinosteroid alleviates schilling-induced oxidative stress in pepper by enhancing antioxidation systems and maintenance of photosystem II. Acta Physiologiae Plantarum, 2015, 37(11):1-11. https: / / doi.org / ). Following the method of Yang et al., the content of phosphorus (K, Ca, Na, Mg, Cu, Fe, Mn and Zn) in leaves was determined using atomic absorption spectrometry (ZEEnit 700P, Analytik Jena, Germany) (YANG Y, XIE J, LIJ, et al. Trehalose alleviates salt tolerance by improving photosynthetic performance and maintaining mineral ion homeostasis in tomatoplants. Frontiers in Plant Science, 2022, 13 (https: / / doi.org / ).

[0045] All data were analyzed using Excel 2020. Experiments were repeated three times, and results are expressed as mean ± standard error. Analysis of variance (ANOVA) was performed using SPSS 20.0 (SPSS Institute Inc., USA). Means of each treatment group were compared using Duncan's multiple range test (significance level of 0.05) to determine if significant differences existed. Figures and tables were created using OriginPro 2022.

[0046] ① Photosynthetic pigment content and gas exchange parameters

[0047] Experimental results: such as Figure 7 As shown in the figure. Under CK conditions, compared with WT, the contents of Chla, Chlb, Chl-T, Ccar, and Gs in ps1300:CaCA2 treatment significantly increased by 18.43%, 10.76%, 16.18%, 22.95%, and 30.28%, respectively. Compared with ps1300:00, the contents of Chla, Chla+Chlb, Ccar, Pn, Gs, and Ci in ps1300:CaCA2 treatment significantly increased. Under LL conditions, compared with WT and ps1300:00, Pn in ps1300:CaCA2 treatment significantly increased, but Ci decreased.

[0048] ②Chlorophyll fluorescence parameters

[0049] Experimental results: such as Figure 8 As shown. Compared with ps1300:00, the Fv / Fm and Y(II) of ps1300:CaCA2 treatment were significantly increased ( Figure 8 (A, B, DG in the text).

[0050] ③ Linear electron transport in PSII

[0051] Experimental results: such as Figure 9 As shown. Under CK conditions, compared with WT, the overall fluorescence intensity of ps1300:CaCA2 treatment was enhanced. Compared with WT and ps1300:00, Sm / t(Fm), ΦPo, ΦEo, and PIabs were significantly increased in ps1300:CaCA2 treatment, while Vj, dV / dto, DIo / RC, and DIo / CSm were significantly decreased. Under LL conditions, compared with WT and ps1300:00, ABS / RC and DIo / RC were significantly increased in ps1300:CaCA2 treatment, while ETo / CSm was significantly decreased.

[0052] ④ Antioxidant defense system

[0053] Experimental results: such as Figure 10As shown. Under CK conditions, compared with WT and ps1300:00, the O2 of ps1300:CaCA2 is... - The contents were significantly reduced by 47.05% and 52.99%, respectively. Furthermore, the activities of SOD, CAT, and APX in ps1300:CaCA2 were all increased. Under LL conditions, compared with WT, the O2- content of ps1300:CaCA2 decreased, while the activities of SOD, POD, and CAT increased. Compared with WT and ps1300:00, the expression of SOD, POD, CAT, and APX enzyme genes in ps1300:CaCA2 increased.

[0054] ⑤ Content of osmotic adjustment substances

[0055] Experimental results: such as Figure 11 As shown. Under CK conditions, compared with WT, the soluble protein and free proline content of ps1300:CaCA2 increased significantly by 11.47% and 202.59%, respectively, but the total phenol and flavonoid content decreased significantly by 38.40% and 40.48%, respectively. Figure 11 (B, D, EG in the text). Under LL conditions, compared with WT and ps1300:00, the soluble sugar and free proline content of ps1300:CaCA2 was significantly increased, but MDA was decreased. Compared with ps1300:00, the REC content of ps1300:CaCA2 was significantly decreased. Compared with WT, the total phenol and flavonoid content of ps1300:CaCA2 was significantly decreased. Figure 11 AC and EG in the middle.

[0056] ⑥ Mineral element content

[0057] Experimental results: such as Figure 12 As shown. Under CK conditions, compared with WT and ps1300:00, the P, Cu, Fe, and Zn contents of ps1300:CaCA2 increased, but the Na content decreased significantly by 17.64% and 16.93%, respectively. The Cu content of ps1300:CaCA2 increased significantly (…). Figure 12 (A, B, D, F, G, I). Under LL conditions, compared with WT and ps1300:00, the P, Na, Cu, and Fe contents of ps1300:CaCA2 increased, while the Ca content of ps1300:CaCA2 decreased significantly compared with WT. Figure 12 (A, CG in the text).

[0058] (3) Relevant indicators of chili peppers after silencing the CaCA2 gene under low temperature and low light conditions

[0059] ① Growth morphology indicators and root morphology parameters

[0060] Experimental results: as shown in Tables 2 and 3. Figure 13 As shown. Under CK and LL conditions, the peppers produced by TRV2:CaCA2 were smaller than those produced by TRV2:00.

[0061] Table 2. Growth morphological indicators of pepper after transient silencing of the CaCA2 gene under LL stress

[0062] As shown in Table 2, under CK and LL conditions, compared with TRV2:00, TRV2:CaCA2 showed a decrease in plant height, stem diameter, aboveground fresh weight, belowground fresh weight, aboveground dry weight, and belowground dry weight.

[0063] Table 3. Root morphological parameters of pepper after transient silencing of the CaCA2 gene under LL stress.

[0064] As shown in Table 3, under CK and LL conditions, compared with TRV2:00, the total root length, total root surface area, total root volume, and number of branches of TRV2:CaCA2 were all reduced.

[0065] ② Stomatal morphology and parameters

[0066] Experimental results: such as Figure 14 As shown. Under CK and LL conditions, compared to TRV2:00, TRV2: CaCA2 The stomata of the pepper leaves were closed to varying degrees. Under CK and LL conditions, compared with TRV2:00, TRV2: CaCA2 The pore length, pore width, pore length, and pore width all decreased. Figure 14 (BE in the middle).

[0067] ③ Linear electron transport in PSII

[0068] Experimental results: such as Figure 15 As shown. Under CK and LL conditions, the overall fluorescence intensity of TRV2:CaCA2 was reduced compared to TRV2:00. Compared to TRV2:00, TRV2: CaCA2 The treated values ​​of Vj, Vi, and dV / dto increased significantly, while PIabs decreased significantly. Figure 15 (A and B in the text). Under CK conditions, compared to TRV2:00, TRV2: CaCA2 The treated DIo / CSm increased significantly, while ETo / CSm and ETo / RC decreased. Figure 15 (D, F, J in the text); Under LL conditions, compared to TRV2:00, TRV2: CaCA2 The treated ABS / RC and DIo / RC increased significantly, while ETo / RC decreased. Figure 15 (G, H, J in the text).

[0069] ④ Mineral element content

[0070] Experimental results: such as Figure 16 As shown. Under CK conditions, compared with TRV2:00, the contents of P, Ca, Na, Cu, and Zn in TRV2:CaCA2 were all reduced ( Figure 16 (A, B, D, F, I in the text). Under LL conditions, compared to TRV2:00, the TRV2:CaCA2 treatment increased P, K, and Mg, while Na, Cu, Fe, Mn, and Zn decreased ( ). Figure 16 (A, B, D, E, F, G, H, I in the alphabet).

[0071] As can be seen from the above embodiments, the chili pepper of the present invention... CaCA2 The gene expression level was highest in green fruit. After 168 hours of low temperature and low light stress, CaCA2 The expression level was downregulated. Under low temperature and low light stress, CaCA2 Transient overexpression of the gene slowed down wilting in pepper plants and increased net photosynthetic rate (Pn), light energy absorbed per unit reaction center (ABS / RC), expression levels of SOD, POD, CAT, and APX enzyme genes, soluble sugar content, free proline content, and mineral element (P, Na, Cu, and Fe) content in pepper leaves, while decreasing intercellular CO2 concentration (Ci) and malondialdehyde (MDA) content. However, transient silencing of the CaCA2 gene using VIGS technology resulted in smaller pepper plants, with varying degrees of reduction in plant height, stem diameter, biomass, and root morphology parameters. It also caused stomatal closure and decreased overall fluorescence intensity, photosynthetic performance index (PIabs), and trace element (Cu, Fe, Mn, and Zn) content. In conclusion, CaCA2 Transient overexpression of the CaCA2 gene in peppers can alleviate plant wilting and enhance resistance to low temperature and low light stress by increasing photosynthesis, antioxidant enzyme activity, accumulation of osmotic regulators, and maintenance of mineral element balance. Silencing the CaCA2 gene, on the other hand, weakens its resistance.

[0072] 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. CaCA2 The application of genes in the resistance of peppers to low temperature and low light stress is characterized by, Transient overexpression CaCA2 Genes that enhance chili peppers' resistance to low temperature and low light stress, and silencing genes... CaCA2 Genes reduce the resistance of chili peppers to low temperature and low light stress.

2. CaCA2 The application of genes in regulating photosynthesis in peppers is characterized by, Transient overexpression CaCA2 Genes enhance photosynthesis in chili peppers, silencing CaCA2 Genes reduce photosynthesis in chili peppers.

3. The application according to claim 2, characterized in that, The indicators of photosynthesis include photosynthetic pigment content, net photosynthetic rate, transpiration rate, stomatal conductance, and photosynthetic performance index.

4. CaCA2 The application of genes in regulating the activity of antioxidant enzymes in chili peppers is characterized by, Transient overexpression CaCA2 Genes enhance the activity of antioxidant enzymes in chili peppers, silencing them. CaCA2 Genes reduce the activity of antioxidant enzymes in chili peppers.

5. The application according to claim 4, characterized in that, The antioxidant enzyme genes include SOD, POD, CAT, and APX enzyme genes.

6. CaCA2 The application of genes in regulating the content of osmotic regulatory substances in chili peppers is characterized by, Transient overexpression CaCA2 Genes increase the content of osmotic regulatory substances in chili peppers, silencing... CaCA2 Genes reduce the content of osmotic regulatory substances in chili peppers.

7. The application according to claim 6, characterized in that, The osmotic regulating substances include soluble sugars, soluble proteins, and free proline.

8. CaCA2 The application of genes in regulating the mineral element content of chili peppers is characterized by, Transient overexpression CaCA2 Genes increase the mineral content of chili peppers, silencing CaCA2 Genes reduce the mineral content of chili peppers.

9. The application according to claim 8, characterized in that, The mineral elements include P, Cu, Fe, Mn, and Zn.

10. The application according to any one of claims 1 to 9, characterized in that, The overexpression CaCA2 The genetic approach includes the following steps: Build CaCA2 Gene overexpression vectors were used to infect peppers with Agrobacterium tumefaciens, and overexpressing vectors were screened. CaCA2 Genetically modified strains of chili peppers CaCA2 Transient overexpression of genes; The CaCA2 The gene overexpression vector used P-Super-1300 as the initial vector, and cloned gene expression vectors between the Xba1 and Kpn1 restriction enzyme sites of P-Super-1300. CaCA2 Gene fragments; The silence CaCA2 The genetic approach includes the following steps: Build CaCA2 The VIGS gene silencing vector was used to infect peppers with Agrobacterium tumefaciens, and the silencing vector was screened. CaCA2 Genetically modified strains of chili peppers CaCA2 Gene silencing; The CaCA2 The VIGS gene silencing vector uses TRV2 as the initial vector and clones a gene between the EcoRI and BamHI enzymes at the TRV2 restriction site. CaCA2 Silent segments of genes.

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