Application of BrCNGC16 gene in regulating and controlling dry heartburn disease resistance of plants

Abstract

The invention discloses application of a BrCNGC16 gene in regulating and controlling dry heartburn disease resistance of plants, and belongs to the technical field of gene engineering. According to the invention, a Chinese cabbage variety susceptible to dry heartburn is used as a test material, transcriptome data of a core leaf of a Chinese cabbage in a seedling stage under low-calcium and contrast treatment is analyzed, a dry heartburn key candidate gene BrCNGC16 is obtained, and a nucleotide sequence of a CDS region of the BrCNGC16 gene is as shown in SEQ ID NO.1. Through functional verification, the regulation and control effect of the BrCNGC16 in the dry burning resistance of the Chinese cabbage is disclosed. Gene resources are provided for creation and breeding of new dry-heartburn-resistant crop varieties, and the high dry-heartburn-resistant gene is transferred into a dry-heartburn-resistant sensitive plant by using a transgenic technology to obtain a new dry-heartburn-resistant germplasm. Cultivation of the Chinese cabbage dry-core-burning-resistant new germplasm can improve the yield and quality of Chinese cabbages and reduce the use of fertilizers and pesticides, and has important significance on agricultural sustainable development.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to application of BrCNGC16 gene in regulating plant resistance to dry heartburn disease. Background Art

[0002] Chinese cabbage (Brassica rapa ssp.pekinensis) is a member of the genus Brassica in the family Cruciferae and is an important economic vegetable worldwide. Dry heart disease is one of the main diseases of Chinese cabbage, with a disease rate of 10% to 20%, and can be as high as 80% or more in severe cases. The disease usually begins in the rosette stage, when the leaf edges dry up and curl inward, and growth is inhibited; as the disease worsens, the edges of the young leaves become water-soaked and translucent, and wilt after dehydration to become white ribbons; in the late stage of the disease, the appearance of the diseased plants is normal, but the edges of the inner leaves become dry and yellow, the mesophyll becomes dry paper-like, with irregular lesions, and the boundary between the diseased and healthy plants is clear. The disease during storage is prone to bacterial infection, and the dry heart turns into rot, seriously affecting its commercial quality.

[0003] Scholars have always believed that the dry heart of Chinese cabbage is a physiological disease caused by a lack of calcium (Kuo et al., 1981). Calcium, as one of the essential nutrients for plant growth and development, participates in the entire process from plant seed germination, growth differentiation, morphological construction to flowering and fruiting. The occurrence of physiological diseases includes direct and indirect factors. The so-called indirect factors are attributed to the lack of calcium in the external environment, which has an adverse effect on plant growth (Saure, 1998), while the direct factors are manifested in the inability of the plant itself to fully absorb calcium, and the lack of calcium content directly leads to the stunted growth of its own. This calcium-related physiological disorder occurs frequently and has become one of the main diseases that seriously threaten the yield and quality of Chinese cabbage.

[0004] In recent years, there has been a certain understanding of the absorption and transport of calcium by plants. Studies have shown that Ca 2+ The interaction between ion channels, exchangers and pumps plays a key role in calcium signal transduction (Tian et al., 2019). At present, the molecular mechanism of dry heart is not well studied, and the key gene that causes dry heart in Chinese cabbage due to calcium deficiency is the primary goal of clarifying the molecular mechanism of dry heart in Chinese cabbage.

[0005] In Chinese cabbage, there are reports on genes related to dry heartburn defense induced by calcium deficiency. 2+ Metabolism-related gene ACA (P2B-type Ca 2+ -ATPase pump), CAX (Ca 2+ / H+antiporter) and ECA (P2A-typeCa 2+-ATPase pump) transporter is involved in the defense process of dry heartburn induced by calcium deficiency (Lee et al., 2016). In addition, circRNA-seq analysis found that some circRNAs may participate in the response of Chinese cabbage to calcium deficiency-induced heartburn through circRNA regulation pathways (Wang et al., 2019b). Sequence difference alignment, molecular marker verification and transgenic analysis showed that calreticulin BrCRT2 (Calreticulin2) is a Ca2+ transporter in Chinese cabbage. 2+ Important node genes in the interaction between the pathway and dry heartburn resistance (Su et al., 2019).

[0006] It has been reported that cyclic nucleotide-gated channels (CNGCs) are important regulators of Ca 2+ In Arabidopsis, CNGC2 and CNGC4 regulate calcium concentration by inducing calcium signals through pathogen-associated molecular patterns (PAMPs) (Tian et al. 2019). So far, there have been no reports on the functional involvement of CNGCs genes in Chinese cabbage in calcium absorption and transport. Summary of the invention

[0007] The purpose of the present invention is to explore the key gene causing dry heartburn of Chinese cabbage due to calcium deficiency, and to provide a theoretical basis and relevant gene resources for the breeding and production of Chinese cabbage resistant to dry heartburn.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention uses the dry heartburn-sensitive Chinese cabbage variety 'HK-8' obtained by the research team through multi-generation self-pollination of the commercial cabbage variety 'Fire Phoenix' as the experimental material, and treats the Chinese cabbage seedlings at the four-leaf stage with medium calcium (control) and low calcium. The differentially expressed genes are analyzed by transcriptome sequencing technology to obtain key candidate genes for dry heartburn, one of which is annotated as an ion channel protein controlled by cyclic nucleotides (Gene ID: Bra018089), and is named BrCNGC16.

[0010] BrCNGC16 gene cloning and analysis: The BrCNGC16 gene was cloned from Chinese cabbage 'HK8', and the nucleotide sequence of its CDS region is shown in SEQ ID NO.1.

[0011] The CDS region of the BrCNGC16 gene is 2121 bp in length, and the protein sequence it encodes contains 706 amino acid residues. The amino acid sequence is shown in SEQ ID NO.2.

[0012] Protein structure analysis predicted that BrCNGC16 protein has two conserved functional domains: a conserved Ion_trans domain at the 53-387aa position of the N-terminus and a cNMP domain (Cyclic nucleotide-monophosphate binding domain) at the N-terminus.

[0013] By constructing a phylogenetic tree to analyze the evolutionary relationship between BrCNGC16 and other CNGC proteins, it was found that BrCNGC16 and AtCNGC16 were on the same branch.

[0014] In order to clarify the subcellular localization of BrCNGC16, BrCNGC16 was transiently transformed into tobacco, and the results showed that BrCNGC16 protein was localized on the cell membrane and nucleus.

[0015] In order to explore the effect of BrCNGC16 on dry heartburn resistance, BrCNGC16 was heterologously expressed in the model plant Arabidopsis. By analyzing the phenotype, relative expression of BrCNGC16 gene, dry heartburn incidence, calcium concentration and the ratio of calcium ion concentration in roots and leaves, it was found that under calcium deficiency treatment, BrCNGC16 promoted the transfer of calcium content in Arabidopsis plants from roots to leaves, thereby enhancing the dry heartburn resistance of plants. Furthermore, BrCNGC16 silenced Chinese cabbage plants were obtained by gene silencing technology. After the BrCNGC16 silenced and control plants were treated with calcium deficiency and normal calcium content conditions, the phenotypic differences, calcium content and chlorophyll content of the plants were analyzed. The results showed that BrCNGC16 silencing enhanced the dry heartburn of Chinese cabbage, inhibited the growth and development of plants, and reduced the chlorophyll content of plants by inhibiting the absorption of calcium ions.

[0016] The above studies show that the BrCNGC16 gene plays a positive regulatory role in the plant's resistance to dry heartburn physiological diseases.

[0017] Therefore, the present invention provides the use of the BrCNGC16 gene in regulating plant resistance to dry heartburn disease.

[0018] Furthermore, the application includes: utilizing biological techniques to up-regulate the expression of the BrCNGC16 gene in the plant, thereby enhancing the plant's resistance to dry heartburn disease.

[0019] Furthermore, the application includes: cloning the BrCNGC16 gene into an overexpression vector to construct a recombinant vector, using Agrobacterium-mediated technology to introduce the target fragment in the recombinant vector into a recipient plant, and cultivating transgenic plants with enhanced dry heartburn resistance.

[0020] In the present invention, suitable overexpression vectors and Agrobacterium are selected for transgenic operation according to the characteristics of the recipient plant. The method for upregulating the expression of the BrCNGC16 gene in the present invention is not limited to the use of transgenic technology, and the BrCNGC16 gene fragment can be obtained by artificial synthesis.

[0021] Furthermore, the mechanism of action of enhancing the plant's resistance to dry heartburn includes: under low calcium stress, the up-regulated expression of the BrCNGC16 gene in the plant promotes the transfer of calcium ions from the underground to the aboveground part.

[0022] Furthermore, the plant may be, but is not limited to, Chinese cabbage or Arabidopsis thaliana.

[0023] The present invention obtains BrCNGC16 overexpressing Chinese cabbage plants by genetic transformation method. Through comprehensive analysis of the phenotypes of transgenic and control plants and the relative expression of related genes, the results show that under calcium deficiency treatment, BrCNGC16 overexpression enhances the resistance of Chinese cabbage to dry heartburn, providing a theoretical basis for analyzing the mechanism of resistance to dry heartburn in Chinese cabbage.

[0024] Further mechanistic studies showed that under low calcium stress, the BrCNGC16 gene in Chinese cabbage plants was upregulated to increase the calcium content in the leaves of the plants.

[0025] The present invention also provides a method for creating Chinese cabbage germplasm with enhanced dry heartburn resistance, comprising the following steps:

[0026] (1) cloning the BrCNGC16 gene fragment with the nucleotide sequence shown in SEQ ID NO.1 into a plant overexpression vector to construct a recombinant plasmid;

[0027] (2) Using the cotyledons of Chinese cabbage as explants, the Agrobacterium-mediated genetic transformation method was used to obtain BrCNGC16 overexpressing Chinese cabbage plants.

[0028] Furthermore, the plant overexpression vector uses pTCK303 vector.

[0029] Furthermore, the genetic transformation method comprises: firstly, cutting off 1 / 3 of the cotyledon edge with the petiole retained, culturing in the dark on a preculture medium for 1-2 days, then soaking the explant in an Agrobacterium infection solution for 10-15 minutes, taking out and drying in the shade, and culturing in the dark on a coculture medium for 2 days, washing the explant after the coculture, and then transferring the explant to a screening medium and culturing until a resistant bud is formed, cutting a resistant bud of 2-3 cm in length, transferring it to a rooting induction medium for culturing, and screening to obtain transgenic plants;

[0030] The composition of the preculture medium is: MS basic medium + 20 g / L sucrose + 7 g / L agar powder + 0.5 mg / L 1-naphthylacetic acid (NAA) + 4 mg / L 6-benzylaminopurine (6-BA) + 4 mg / L AgNO3, pH = 5.8;

[0031] The composition of the co-culture medium is: MS basal medium + 20 g / L sucrose + 7 g / L agar powder + 0.5 mg / L 1-naphthylacetic acid + 4 mg / L 6-benzylaminopurine + 4 mg / L AgNO3 + 100 μmol / L acetosyringone (AS), pH = 5.8;

[0032] The screening medium is composed of: MS basal medium + 20 g / L sucrose + 7 g / L agar powder + 0.5 mg / L 1-naphthaleneacetic acid + 4 mg / L 6-benzylaminopurine + 4 mg / L AgNO3 + 200 mg / L cephalexin (Cef) + 100 mg / L carbenicillin (Car) + 20 mg / L hygromycin (Hyg), pH = 5.8;

[0033] The root induction medium is composed of: MS basic medium + 20 g / L sucrose + 7 g / L agar powder + 1 mg / L 1-naphthylacetic acid + 200 mg / L cephalexin + 100 mg / L carbenicillin, pH = 5.8.

[0034] The present invention improves the genetic transformation method based on the characteristics of Chinese cabbage and establishes an efficient and stable method for Agrobacterium-mediated genetic transformation of Chinese cabbage. Specifically, the sucrose content in the culture medium used in each stage is increased from 30 g.L -1 Change to 20g.L -1 This concentration is more suitable for the growth of Chinese cabbage tissue. The antibiotics in the screening medium and the root induction medium were selected at 200 mg.L -1 Cephalexin and 100mg.L -1 Compared with the addition of timentin, the culture medium provided by the present invention is more conducive to the regeneration of Chinese cabbage explants. The amount of hygromycin in the screening culture medium is 25g.L -1 Change to 20g.L -1 , the germination rate of antibiotic buds is improved.

[0035] Further, the seed germination method includes: selecting seeds of uniform size, sterilizing them, and placing them in a 20g·L -1 Sucrose and 7g·L -1 The seeds were germinated in 1 / 2MS basic medium containing agarose (pH 5.8) at 25±2°C and a photoperiod of 16h / 8h (light / dark).

[0036] Furthermore, before the explants were transferred to the screening medium and before the resistant buds were transferred to the rooting induction medium, they were washed 3-5 times with sterile water, and the antibacterial antibiotics cephalexin (final concentration 400-600 mg / L) and carbenicillin (final concentration 200-300 mg / L) were added for the last time. The entire cleaning time was controlled within half an hour.

[0037] The present invention has the following beneficial effects:

[0038] The present invention uses Chinese cabbage varieties susceptible to dry heartburn as test materials, analyzes the transcriptome data of the heart leaves of Chinese cabbage in the seedling stage under low calcium and medium calcium (control) treatments, mines the key candidate gene BrCNGC16 related to calcium ions based on GO and KEGG, and reveals the regulatory role of BrCNGC16 in Chinese cabbage resistance to dry heartburn through functional verification. Studies have shown that heterologous expression of BrCNGC16 in plants can enhance the resistance of plants to low calcium-induced dry heartburn and promote the transfer of calcium content from underground to aboveground parts; Silencing of the BrCNGC16 gene in Chinese cabbage plants inhibits the absorption of calcium ions; under low calcium stress, overexpression of the BrCNGC16 gene in Chinese cabbage can significantly increase the calcium content in plant leaves.

[0039] The present invention provides gene resources for the creation and breeding of new varieties of crops resistant to dryness and heartburn, and uses transgenic technology to transfer high dryness and heartburn resistance genes into dryness and heartburn sensitive plants to obtain new dryness and heartburn resistance germplasm. It can not only enrich my country's germplasm resources, but also save labor, reduce breeding links, shorten breeding years and improve breeding efficiency. In addition, cultivating new dryness and heartburn resistance germplasm of cabbage can increase cabbage yield and quality, reduce the use of fertilizers and pesticides, and is of great significance to the sustainable development of agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The effect of low calcium on the phenotype of Chinese cabbage 'HK8' seedlings.

[0041] Figure 2 Screening of differentially expressed gene BrCNGC16. A, principal component analysis of all genes in low calcium heart leaf samples and medium calcium heart leaf samples; B, Volcano Plot of differentially expressed genes; C, statistical analysis of the number of genes related to calcium absorption and transport in differentially expressed genes; D, expression level of differentially expressed gene BrCNGC12 under low calcium and medium calcium treatments; E, expression level of differentially expressed gene BrCNGC16 under low calcium and medium calcium treatments; F, expression level of differentially expressed gene BrCNGC20 under low calcium and medium calcium treatments; G, expression level of differentially expressed gene BrCNGC11 under low calcium and medium calcium treatments.

[0042] Figure 3The structure and evolutionary relationship of BrCNGC16. A, conservative structure and analysis of BrCNGC16, sequences with different background colors represent different domains, and sequences with the same background color represent the positions corresponding to the domains; B, phylogenetic relationship analysis of BrCNGC16 and CNGC proteins of other species, using MEGA5.0 software, neighbor-joining method to construct phylogenetic tree, showing bootstrap values ​​higher than 97% on nodes.

[0043] Figure 4 Subcellular localization analysis of BrCNGC16 in Nicotiana benthamiana leaf cells. Membrane labels: mCherry, pm-rbCD3-1008; GFP, dark stripe images of tobacco leaf cells; bright stripe images of tobacco leaf cells; merge, overlay of red fluorescence images, green fluorescence images, and bright fluorescence images; scale: 20 μm.

[0044] Figure 5 The response analysis of BrCNGC16 transgenic Arabidopsis plants to low calcium stress. A, phenotypes of BrCNGC16-transgenic Arabidopsis plants and wild-type plants under medium and low calcium conditions; B, expression analysis of BrCNGC16 in transgenic plants and wild-type plants under calcium treatment; C, statistics of total incidence of dry heartburn in BrCNGC16-transgenic Arabidopsis plants and wild-type plants under low calcium conditions; D, calcium content in roots and leaves of BrCNGC16-transgenic plants and wild-type plants; E, ratio of calcium in leaves to roots of BrCNGC16-transgenic plants and wild-type plants under low and medium calcium conditions.

[0045] Figure 6 The response analysis of BrCNGC16-silenced plants and control plants to low calcium stress. A, local phenotypes of BrCNGC16-silenced cabbage and control plants 20 days after virus inoculation; B, phenotypes of BrCNGC16-silenced cabbage and control plants under medium and low calcium treatments; C, relative expression levels of leaves of BrCNGC16-silenced cabbage and control plants under medium and low calcium treatments; D, calcium content of inner leaves of BrCNGC16-silenced cabbage and control plants under medium and low calcium treatments.

[0046] Figure 7 The genetic transformation of BrCNGC16 in Chinese cabbage plants. A, sowing; B, pre-culture; C, co-culture; D, bud induction; E, root induction; F, seedling.

[0047] Figure 8This is the positive verification of BrCNGC16 in Chinese cabbage plants and its involvement in calcium absorption. A, hygromycin resistance gene detection, band size is 481bp; B, GUS histochemical staining; C, target gene detection, amplified band size is 463bp, including 383bp of the 3' end of the target gene and 80bp of the vector sequence; Marker (M) is 1000bp. From bottom to top, they are 100, 250, 500, 750, 1000, and 2000 bp respectively; P is plasmid (positive control), WT is gene detection using DNA of wild-type Chinese cabbage (negative control) as template, H is water (negative control), and 1-4 is gene detection using DNA of tissue culture seedlings as template; D, relative expression level of BrCNGC16 gene in BrCNGC16-overexpressing Chinese cabbage plants and control plants (qRT-PCR); E, calcium content in outer leaves of BrCNGC16-overexpressing Chinese cabbage plants and control plants; F, calcium content in inner leaves of BrCNGC16-overexpressing Chinese cabbage plants and control plants. DETAILED DESCRIPTION

[0048] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0049] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0050] The Arabidopsis thaliana variety used in the following examples is the Columbia type Arabidopsis thaliana variety, and the cabbage varieties 'HK-8' and 'HK-13' are homozygous strains bred by the cabbage team of Henan Institute of Science and Technology for multiple generations of self-pollination of the commercial cabbage varieties 'Fire Phoenix' and 'Tokyo No. 9'.

[0051] Example 1: Screening of candidate gene BrCNGC16 for heartburn resistance

[0052] 1. The material used in this study is the Chinese cabbage variety 'HK-8', which is susceptible to dry heartburn. First, 100 Chinese cabbage seeds were sown on a petri dish containing filter paper for germination for 2-3 days. Then the germinated seeds were sown in a 50-hole plug tray and grown in a suitable environment. When the plants had 2 leaves and 1 heart (28 days), they were transplanted into a turnover box containing improved Hoagland nutrient solution for calcium treatment. This study set up two treatments: medium calcium (5mmol·L -1 ) and low calcium (0.01mmol·L -1) treatments, each treatment was allocated to 5 turnover boxes, each turnover box contained 4 seedlings. During the culture period, the nutrient solution completely covered the roots and was replaced every 3 days. The culture conditions were: 25°C, 16h light / 20°C, 8h dark, and relative humidity of 65%.

[0053] like Figure 1 As shown, phenotypic analysis found that with the increase of culture time, the growth of plants and the number of leaves under low-calcium conditions were significantly inhibited compared with medium-calcium culture. At the same time, low-calcium culture caused the inner leaves of Chinese cabbage to show yellowing, scorch and necrosis.

[0054] 2. This study used the heart leaves of Chinese cabbage treated with calcium (low calcium and medium calcium) for 14 days as samples, and each treatment was repeated 3 times, for a total of 6 samples. First, RNA was extracted according to the RNAsimple total RNA extraction kit, and the purity, concentration and integrity of RNA were analyzed using NanoDrop (Desjardinsand Conklin, 2010), a Qubit 2.0 (Subramanian and Gomez, 2014) and an Agilent 2100 (Panaro et al., 2000). Then, about 10 μg of total RNA was taken, and the RNA fragments were reverse transcribed to create the final cDNA library. After the library was qualified, Illumina Novaseq TM 6000 (Modi et al., 2021) for sequencing, with a paired-end 2×150 bp (PE150) read length. Transcriptome data have been submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) with the registration number PRJNA1130097.

[0055] The cabbage heart leaves treated with normal calcium (medium calcium) were used as controls, and transcriptome sequencing was performed on the cabbage heart leaves treated with low calcium. Principal component analysis found that principal component 1 and principal component 2 accounted for 92.0% and 4.9% of the variance, respectively, and the repeated samples under each calcium treatment were significantly clustered together ( Figure 2 A), thus indicating that the transcriptome sequencing results are reliable.

[0056] A total of 5041 differentially expressed genes were found in this study. Volcano Plot showed that 2574 genes were upregulated and 2467 genes were downregulated ( Figure 2 B).

[0057] By performing Gene Ontology (GO) analysis and KEGG annotation analysis on the differentially expressed genes, we found that there were 61 genes related to calcium signal transduction, absorption and transport, including calcium binding protein (CML) genes (20 genes), cyclic nucleotide-gated ion channel (CNGC) genes (4 genes), Calmodulin (CaM) genes (2 genes), Calmodulin-binding protein (CBP) genes (7 genes), calmodulin-interacting protein (CIP) genes (2 genes), Calcium-dependent channel (CSC) genes (6 genes), calcium-transporting ATPase (CT ATPase) genes (6 genes), Cation / calcium exchanger (CCX) genes (4 genes) ( Figure 2 C).

[0058] Considering the importance of CNGC in calcium signal transduction, we found that 4 CNGC genes among 35 CNGC genes in Chinese cabbage responded to calcium deficiency stress, among which the transcript of BrCNGC16 under medium calcium treatment was 12.79 times that under low calcium treatment ( Figure 2 D-2G). Therefore, it is speculated that BrCNGC16 may play an important role in calcium absorption and transport.

[0059] Example 2: Cloning and sequence analysis of BrCNGC16 gene

[0060] 1. In order to clone the nucleotide sequence of BrCNGC16, we used the cDNA of Chinese cabbage 'HK8' as a template and first designed specific primers for PCR amplification. After the amplified fragment was connected to the intermediate vector pEASY-Blunt Simple Vector (Quanshijin Company), the positive single clone was obtained by transformation of Escherichia coli DH5α and screening. The monoclonal bacterial solution was sequenced and analyzed to obtain the reading frame sequence of BrCNGC16. Specifically, the nucleotide sequence of the CDS region of the BrCNGC16 gene is shown in SEQ ID NO.1.

[0061] The specific primer is named BrCNGC16clone, and the sequence is as follows:

[0062] BrCNGC16clone forward primer: 5′-ATGAGCAACCTCCACCTCCA-3′;

[0063] BrCNGC16 cloning primer: 5′-TCAGAAGAATCCTGGATCCT-3′.

[0064] 2. Use SMART (http: / / smart.embl-heidelberg.de / ) (Letunic et al., 2012) to analyze the conserved domains of BrCNGC16 protein.

[0065] like Figure 3 As shown in A, it contains a conserved Ion_trans domain at the 53rd to 387th aa position of the N-terminus, and also contains a cNMP domain (Cyclic nucleotide-monophosphate binding domain) at the N-terminus.

[0066] 3. The NJ method of MEGAR 5.0 program was used to construct a phylogenetic tree of BrCNGC16 and CNGC proteins of other species.

[0067] like Figure 3 As shown in B, BrCNGC16 and AtCNGC16 are on the same branch, indicating that their protein homology is close. In addition, BrCNGC16 is not on the same evolutionary branch as BrCNGC20, BrCNGC12, and BrCNGC11, indicating that their homology is distant.

[0068] Example 3: Construction of subcellular localization vector of BrCNGC16 and transient transformation in tobacco

[0069] In order to construct the subcellular localization vector of BrCNGC16, the cloned nucleotide sequence of BrCNGC16 was inserted into the Nde I and BamH I restriction sites of the pRI101 vector by homologous recombination, and the recombinant vector was named pRI101-BrCNGC16. The constructed pRI101-BrCNGC16 and the empty pRI101 were transformed into Agrobacterium GV3101 by freeze-thaw method (Dupadahalli, 2007), and BrCNGC16 was transiently expressed in tobacco according to the method of Wu et al. (2010). After 48 hours of dark culture, the cell fluorescence was observed by laser confocal microscope. The empty pRI101 was used as a control in the experiment.

[0070] like Figure 4 As shown, the GFP protein on the empty pRI101 emits green fluorescence throughout the cell. The GFP fusion protein with BrCNGC16 emits green fluorescence on the cell membrane, overlapping with the red fluorescent protein of the membrane tag. In addition, GFP also emits light in the cell nucleus and is embedded in the cell membrane. Therefore, it is speculated that the BrCNGC16 protein is localized on the cell membrane and the cell nucleus.

[0071] Example 4: Functional analysis of heterologous expression of BrCNGC16 in Arabidopsis thaliana in the context of dry heartburn resistance

[0072] 1. Overexpression vector construction

[0073] Based on the intermediate vector pEASY-Blunt Simple Vector (Quanshijin Company) containing the target gene fragment, the target fragment was cloned using specific primers, and the correct target fragment was constructed into the expression vector pTCK303. The correct recombinant vector was obtained by transforming Escherichia coli DH5α and sequencing the monoclonal bacterial solution, and was named pTCK303-BrCNGC16.

[0074] The specific primer is named BrCNGC16, and the sequence is as follows:

[0075] BrCNGC16 forward primer: 5′-GGTACCATGAGCAACCTCCACCTCCA-3′;

[0076] BrCNGC16 rear primer: 5'-ACTAGTTCAGAAGAATCCTGGATCCT-3'.

[0077] 2. Transgenic T1 generation Arabidopsis plants were obtained by using the Agrobacterium freeze-thaw method (Yu Yunzhou et al., 2003) and the flower dipping method (Xu Hongmei et al., 2010). After that, T3 generation homozygous strains were finally obtained through multiple generations of self-pollination and single plant harvesting.

[0078] 3. The transgenic positive seedlings and wild-type plants were treated with medium calcium and low calcium, using the same method as in Example 1.

[0079] like Figure 5 As shown in A, there was no significant difference in the phenotypes of transgenic plants and wild-type plants under medium calcium treatment, however, Arabidopsis plants heterologously expressing BrCNGC16 enhanced dry heartburn resistance under low calcium stress.

[0080] like Figure 5 As shown in B-5C, qRT-PCR analysis further found that low calcium reduced the expression level of BrCNGC16, and the total incidence of dry heartburn statistics showed that BrCNGC16 enhanced the resistance of Arabidopsis to dry heartburn.

[0081] By measuring the calcium content in the leaves and roots of the plants, Figure 5 E shows that under low calcium stress, BrCNGC16 has little effect on the calcium content in the roots, but increases the calcium content in the leaves of the plants, and the ratio of calcium in the leaves to the roots of the transgenic plants is significantly higher than that of the wild-type plants. Therefore, it is speculated that BrCNGC16 transfers the calcium content in Arabidopsis plants from the roots to the leaves under low calcium stress. Example 5: Functional analysis of partial silencing of BrCNGC16 in Chinese cabbage for dry heartburn resistance

[0082] Construction of silencing vector: The specific fragment of BrCNGC16 gene of 40 bp was identified through sequence analysis, and it was combined with the reverse complementary sequence to form a DNA fragment of 80 bp in length (5'-ATGATTTGGTTTGTGATCCCAAATGCGGGAGAATTCAGATATCTGAATTCTCCCGCATTTGGGATCACAAACCAAATCAT-3'), and the sequence was phosphorylated. The vector pTY was digested and dephosphorylated with SnaB I restriction enzyme. The phosphorylated DNA fragment and the dephosphorylated pTY vector were connected with T4 ligase in turn, transformed into DH5α, screened and sequenced, and finally a positive single clone was obtained.

[0083] After the positive clones were expanded and cultured, the plasmids were extracted for later use. The pTY-BrCNGC16 silenced Chinese cabbage plants were obtained using the gene gun method and the friction method (Yu et al., 2018).

[0084] Figure 6 As shown in A, pTY and pTY-BrCNGC16 Agrobacterium were used to infect Chinese cabbage leaves. 20 days after virus inoculation, the leaves of BrCNGC16-silenced and pTY Chinese cabbage plants showed significant chlorosis compared with the leaves of wild-type (WT) plants, and exhibited typical Turnip Yellow Mosaic Virus symptoms.

[0085] The pTY and BrCNGC16 silenced Chinese cabbage plants were treated with medium calcium and low calcium using the same method as in Example 1.

[0086] Figure 6 As shown in C, the qRT-PCR results of the BrCNGC16 gene further demonstrated that BrCNGC16 was partially silenced and low calcium significantly downregulated the expression level of BrCNGC16.

[0087] Figure 6 As shown in B, under medium calcium treatment, there was no significant difference in the aboveground part between BrCNGC16 silenced and pTY plants, but the underground root system was relatively weak. Under low calcium treatment, BrCNGC16 silenced plants showed obvious dry heart phenomenon compared with pTY plants, and the plant shape was smaller and the root system was underdeveloped.

[0088] Figure 6 As shown in D, low calcium reduced the calcium content in new leaves, while partial silencing of BrCNGC16 reduced the calcium content in new leaves.

[0089] Example 6: Functional verification of BrCNGC16 overexpressing Chinese cabbage plants in dry heartburn resistance

[0090] In order to obtain Chinese cabbage plants overexpressing BrCNGC16, the Agrobacterium-mediated genetic transformation method of Li et al. (Li et al., 2021) was improved, and the cotyledons and petioles of Chinese cabbage 'HK13' were used as explants to obtain Chinese cabbage plants overexpressing BrCNGC16. The specific method is as follows:

[0091] Soak seeds of roughly the same size in 75% ethanol and shake for 30 seconds for surface sterilization, soak in 10% sodium hypochlorite for 1 minute, soak in 2% sodium hypochlorite for 15 minutes, or soak in 10% sodium hypochlorite for 10 minutes (can be reduced appropriately), and rinse with sterile water 4-5 times. Place about 20 seeds in a 5.5cm×9cm glass culture bottle, each bottle containing 30mL containing 20g·L -1 Sucrose and 7g·L -1 The seeds were germinated in 1 / 2MS basic medium containing agarose (pH=5.8) at (25±2)°C and a photoperiod of 16h / 8h (light / dark).

[0092] The petiole of the cotyledon was retained, and 1 / 3 of the cotyledon edge was cut off and cultured on the pre-culture medium in the dark for 1-2 days. Then, the explant was immersed in Agrobacterium (OD 600 0.5) for 10-15min (shake gently every 5min). After infection, remove the bacterial suspension, dry the explants on sterile paper towels, and then culture them in the co-culture medium under dark conditions for 2 days. After the co-culture is completed, wash the bacteria with sterile water 3-5 times, and add appropriate antibacterial antibiotics (Cef cephalosporin + Car) for the last time, which is 2-3 times the amount used, and the washing time is controlled within half an hour.

[0093] Transfer the explants to the screening medium and change the screening medium every 18 days (if the medium is thick enough and not contaminated), until resistant buds are formed. Cut 2-3 cm long hygromycin-resistant buds and transfer them to the rooting induction medium (wash the bacteria before transfer, as above).

[0094] Figure 7 A-7F show the phenotypes at various stages of tissue culture.

[0095] The culture media used in each stage are as follows:

[0096] (1) Germination medium: 1 / 2MS + 20g.L -1 Sucrose +7g.L -1 Agar powder, pH = 5.8;

[0097] (2) Pre-culture: MS + 20g.L -1 Sucrose +7g.L -1 Agar powder + 0.5mg.L -1 NAA+4mg.L-1 6-BA+4mg.L - 1 AgNO3, pH = 5.8;

[0098] (3) Co-cultivation:

[0099] Resuspension (DM): MS + 100umol.L -1 AS+20g.L -1 Sucrose, pH = 5.2;

[0100] Symbiotic culture medium: MS+20g.L -1 Sucrose +7g.L -1 Agar powder + 0.5mg.L -1 NAA+4mg.L -1 6-BA+4mg.L - 1 AgNO3+100μmol.L -1 AS, pH = 5.8;

[0101] (4) The screening medium is:

[0102] MS+20g.L -1 Sucrose +7g.L -1 Agar powder + 0.5mg.L -1 NAA+4mg.L -1 6-BA+4mg.L -1 AgNO3+200mg.L -1 Cef+100mg.L -1 Car+20mg.L -1 Hyg, pH = 5.8;

[0103] (5) Root induction medium: MS+20g.L -1 Sucrose +7g.L -1 Agar powder + 1mg.L -1 NAA+200mg.L -1 Cef+100mg.L -1 Car, pH = 5.8;

[0104] In order to verify the transgenic Chinese cabbage plants, the leaves of T2 transgenic Chinese cabbage plants were used as materials, and the DNA of the samples was extracted and tested for hygromycin resistance (Hyg r )Gene. Figure 8 As shown in A, Hyg r Gene.

[0105] The primer sequences for the hygromycin resistance gene are as follows:

[0106] Hygr-F: 5'-CTGCCCGCTGTTCTACAACCGG-3';

[0107] Hygr-R: 5'-GGAGCATATACGCCCGGAGTC-3';

[0108] GUS histochemical staining test positive plants, such as Figure 8 As shown in B, the leaves of wild-type plants were not stained blue, while the leaves of positive plants appeared blue.

[0109] The RNA of the sample is further extracted and reverse transcribed into cDNA, and the expression profile of the target gene is detected using qRT-PCR technology. The target gene is detected by designing specific primers (the amplified fragment includes a partial fragment of the target gene and a partial fragment of the overexpression vector). Figure 8 As shown in C, the target gene can be detected in the positive seedlings.

[0110] The target gene-specific primer sequences are as follows:

[0111] NOS-F: 5'-TGGTTTTATACAAGCTGCAT-3';

[0112] NOS-R: 5'-CAAGACCGGCAACAGGATTCAATC-3';

[0113] after, Figure 8 As shown in D, the relative expression level of BrCNGC16 in transgenic plants was much higher than that in wild-type plants.

[0114] The quantitative primer sequence of BrCNGC16 is as follows:

[0115] BrCNGC16-front primer: 5′-AGAGATACAGAGGAATGGATG-3′;

[0116] BrCNGC16-later primer: 5′-TAGAGCGAGACAGAGATGA-3′;

[0117] The calcium content in Chinese cabbage leaves was further determined by low calcium treatment. Figure 8 E-8F showed that the calcium content in the outer and inner leaves of BrCNGC16-overexpressing Chinese cabbage plants was significantly increased compared with that in the control plants.

Claims

1. Application of BrCNGC16 gene in regulating plant resistance to dry heartburn disease, characterized in that: The nucleotide sequence of the CDS region of the BrCNGC16 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The amino acid sequence of the protein encoded by the BrCNGC16 gene is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that The application includes: using biological technology to up-regulate the expression of the BrCNGC16 gene in the plant, thereby enhancing the plant's resistance to dry heartburn disease.

4. The use according to claim 3, characterized in that The application includes: cloning the BrCNGC16 gene into an overexpression vector to construct a recombinant vector, using Agrobacterium-mediated technology to introduce the target fragment in the recombinant vector into a recipient plant, and cultivating a transgenic plant with enhanced dry heartburn resistance.

5. The use according to claim 3, characterized in that Under low calcium stress, the up-regulated expression of the BrCNGC16 gene in plants promotes the transfer of calcium ions from underground to aboveground parts.

6. The use according to any one of claims 1 to 5, characterized in that: The plants are Chinese cabbage and Arabidopsis thaliana.

7. The use according to claim 6, characterized in that Under low calcium stress, the BrCNGC16 gene in Chinese cabbage plants is upregulated to increase the calcium content in the leaves of the plants.

8. A method for creating Chinese cabbage germplasm with enhanced dry heartburn resistance, characterized in that: The following steps are involved: (1) cloning the BrCNGC16 gene fragment with the nucleotide sequence shown in SEQ ID NO.1 into a plant overexpression vector to construct a recombinant plasmid; (2) Using the cotyledons of Chinese cabbage as explants, the Agrobacterium-mediated genetic transformation method was used to obtain BrCNGC16 overexpressing Chinese cabbage plants.

9. The method for creating Chinese cabbage germplasm with enhanced dry heartburn resistance according to claim 8, characterized in that: The plant overexpression vector used was pTCK303 vector.

10. The method for creating Chinese cabbage germplasm with enhanced dry heartburn resistance according to claim 8, characterized in that: The genetic transformation method comprises: firstly, cutting off 1 / 3 of the cotyledon edge with the petiole retained, and then culturing in a pre-culture medium in the dark for 1-2 days, then soaking the explant in an Agrobacterium infection solution for 10-15 minutes, taking out and drying in the shade, and then culturing in a co-culture medium under dark conditions for 2 days, washing the explant after the co-culture, and then transferring the explant to a screening culture medium and culturing until a resistant bud is formed, cutting a resistant bud of 2-3 cm in length, transferring it to a rooting induction culture medium for culturing, and screening to obtain a transgenic plant; The composition of the preculture medium is: MS basic medium + 20 g / L sucrose + 7 g / L agar powder + 0.5 mg / L 1-naphthylacetic acid + 4 mg / L 6-benzylaminopurine + 4 mg / L AgNO3, pH = 5.8; The composition of the co-culture medium is: MS basal medium + 20 g / L sucrose + 7 g / L agar powder + 0.5 mg / L 1-naphthylacetic acid + 4 mg / L 6-benzylaminopurine + 4 mg / L AgNO3 + 100 μmol / L acetosyringone, pH = 5.8; The screening medium is composed of: MS basal medium + 20 g / L sucrose + 7 g / L agar powder + 0.5 mg / L 1-naphthylacetic acid + 4 mg / L 6-benzylaminopurine + 4 mg / L AgNO3 + 200 mg / L cephalexin + 100 mg / L carbenicillin + 20 mg / L hygromycin, pH = 5.8; The root induction medium is composed of: MS basic medium + 20 g / L sucrose + 7 g / L agar powder + 1 mg / L 1-naphthylacetic acid + 200 mg / L cephalexin + 100 mg / L carbenicillin, pH = 5.8.

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