Application of CsCEPD1.1 and CsCEPD1.2 genes in detecting nitrogen nutrient abundance and deficiency in tea plants

Abstract

The present invention relates to the application of CsCEPD1.1 and CsCEPD1.2 genes in the detection of nitrogen nutrient abundance and deficiency in tea trees. CsCEPD1.1 and CsCEPD1.2 are polypeptide genes expressed in the young and tender parts of the aboveground part of tea trees. When the roots sense the low nitrogen stress signal, the transcriptional expression level of the gene in the aboveground part is upregulated, and is transported to the roots through the phloem as a downstream signal, thereby regulating the absorption of nitrogen by the roots. The transcriptional expression level of the CsCEPD1.1 and CsCEPD1.2 genes provided by the present invention can be used as a signal of nitrogen deficiency in tea trees, can conveniently and quickly detect the nitrogen nutritional status of tea leaves, and has important potential application value in the management of tea tree cultivation elements. In addition, the CsCEPD1.1 and CsCEPD1.2 genes are involved in regulating the expression of nitrogen absorption-related genes in the roots of tea trees, which can provide ideas and theoretical basis for improving tea tree germplasm resources.

Description

Technical Field

[0001] The invention relates to the field of tea tree nitrogen deficiency detection methods, and in particular to the application of CsCEPD1.1 and CsCEPD1.2 genes in tea tree nitrogen nutrient abundance and deficiency detection. Background Art

[0002] Nitrogen is an important nutrient element in the growth and development of tea trees and the formation of tea quality. It not only participates in the composition of important life substances such as polysaccharides, proteins, and nucleic acids, but also affects the metabolism of free amino acids, caffeine, polyphenols, and purine alkaloids in tea trees. However, the growth of tea trees is affected by external factors such as multiple harvests per year, time, climate, and soil type. At the same time, the growth of tea trees themselves is also rhythmic, which leads to uneven distribution of nitrogen in the soil and cannot meet the needs of tea trees. In order to meet the needs of tea trees for nitrogen, tea trees have evolved corresponding mechanisms to meet their growth and development needs by regulating nitrogen absorption, transport, and distribution. When the plant roots feel low nitrogen stress, they will produce a mobile small peptide hormone (C-Terminally encoded Peptide, CEP) that responds to low nitrogen stress, and are loaded through the xylem and transmitted to the aboveground part in the form of root-derived ascending signals. They are sensed by the leucine-rich repeat receptor kinase CEP receptor (CEPR 1), and induce CEP Downstream 1 (CEPD1) and CEPD2 to upregulate expression in the vascular tissue of the leaves. CEP Downstream-Like2 (CEPDL2) is a homologous gene of CEPD1, and its expression level is affected by the nitrogen level of plant leaves. Therefore, the transcription level expression of CEPD in plant leaves can be used as an important detection indicator of whether the plant is under low nitrogen stress. In the present invention, genes CsCEPD1.1 and CsCEPD1.2 were cloned from the tea plant genome, and their transcription levels were used as indicator signals of nitrogen deficiency in tea plants, which can reflect the nitrogen nutrition status of tea leaves in real time. The operation is simple and convenient, and has important application prospects in actual agricultural production. Summary of the invention

[0003] The technical problem to be solved by the present invention is the application of CsCEPD1.1 and CsCEPD1.2 genes in the detection of nitrogen nutrient abundance and deficiency in tea plants. The present invention adopts the following technical solutions:

[0004] Application of CsCEPD1.1 and CsCEPD1.2 genes in detecting nitrogen nutrient abundance and deficiency in tea plants, CsCEPD1.1 gene, the nucleotide sequence of which is as follows A) or B) or C):

[0005] A. having the nucleotide sequence shown in SEQ ID NO.1;

[0006] B. The coding sequence includes SEQ ID NO.1;

[0007] C. A nucleotide sequence having a similarity of more than 70% with the nucleotide sequence defined in A) or B), and having a homology with a nucleotide sequence that is up-regulated in young leaves of tea plants after sensing nitrogen stress;

[0008] The nucleotide sequence of the tea plant CsCEPD1.2 gene is as follows A) or B) or C):

[0009] A. having the nucleotide sequence shown in SEQ ID NO.2;

[0010] B. The coding sequence includes SEQ ID NO.2;

[0011] C. A nucleotide sequence having a similarity of more than 70% with the nucleotide sequence defined in A) or B), and having homology with the nucleotide sequence that is up-regulated in young leaves of tea plants after sensing nitrogen stress.

[0012] Furthermore, the specific application method includes the following steps:

[0013] Step 1, detecting and recording the expression level of CsCEPD1.1 or CsCEPD1.2 gene in tea plants under the condition of sufficient nitrogen supply;

[0014] Step 2: Detect the expression level of CsCEPD1.1 or CsCEPD1.2 gene during the normal growth of tea trees, and compare it with the expression level of the corresponding gene under the condition of sufficient nitrogen supply in step 1. If the gene expression level is judged to be significantly upregulated by the one-way variance method, it is judged that the tea trees are in a nitrogen deficiency state.

[0015] Furthermore, the nitrogen supplied is in the form of ammonium nitrogen (NH4 + -N) and nitrate nitrogen (NO3 - -N) mixed nitrogen.

[0016] Furthermore, the sequences of the primer pair for detecting the CsCEPD1.1 gene of tea plant are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0017] Furthermore, the sequences of the primer pairs for detecting the CsCEPD1.2 gene of tea plant are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0018] After adopting the above technical solution, the present invention has the following advantages compared with the prior art:

[0019] The present invention provides genes that respond to low nitrogen in tea leaves, which can detect the nitrogen nutritional status of tea leaves in real time. The method is simple and fast, and has important potential application value in agricultural production. The present invention uses the transcription levels of CsCEPD1.1 and CsCEPD1.2 genes in young leaves of tea trees as indicator signals of nitrogen nutritional abundance or deficiency of tea trees. By directly detecting the transcription level expression of CsCEPD1.1 and CsCEPD1.2 genes in tea leaves, the nitrogen nutritional abundance or deficiency of tea trees can be detected in real time. Compared with the existing methods, it is convenient and fast to operate, and the diagnostic cost can be greatly reduced. The present invention directly detects the transcription levels of CsCEPD1.1 and CsCEPD1.2 genes regulated by nitrogen signals in tea leaves, avoids the interference of other factors such as tea tree varieties, seasons and tea garden cultivation measures, ensures the accuracy of the results, and has the universality of promotion and application.

[0020] The present invention is described in detail below with reference to the accompanying drawings and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the result of real-time quantitative fluorescence PCR detection of CsCEPD1.1 gene expression;

[0022] Figure 2 This is the result of real-time quantitative fluorescence PCR detection of CsCEPD1.2 gene expression;

[0023] Figure 3 The figure is a PBI121-35S-GUS vector for promoter fusion GUS expression of the present invention;

[0024] Figure 4 Analysis of expression pattern after promoter fusion with GUS gene Figure 4 A is the positive control Arabidopsis seedling GUS staining; Figure 4 B is the negative control Arabidopsis seedling GUS staining; Figure 4 C is the expression pattern analysis of proCsCEPD1.1 after fusion with GUS gene; Figure 4 D is the expression pattern analysis of proCsCEPD1.2 fused with GUS gene. Scale bar = 1 mm. The above plant materials are all 10-day-old Arabidopsis thaliana. DETAILED DESCRIPTION

[0025] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0026] The experimental methods in the following examples without specific experimental conditions are usually carried out under conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials and reagents used are commercially available unless otherwise specified.

[0027] In the following examples, Escherichia coli DH5α and Agrobacterium tumefaciens GV3101 are commonly used strains, which are stored in most molecular biology laboratories and can be purchased commercially; the tea tree variety is Fuding Dabaicha.

[0028] The primers used in the following examples were synthesized by Wuhan Qingke Biotechnology Co., Ltd., and the sequencing was performed by Wuhan Qingke Biotechnology Co., Ltd.

[0029] Example 1

[0030] Cloning and Expression Detection of CsCEPD1.1 and CsCEPD1.2 Genes in Tea Plant

[0031] (1) Treatment of plant materials: The branches of Fuding Dabaicha with the same growth potential were placed in a tissue culture room (26°C, 12 hours during the day and 12 hours at night) for nutrient solution cutting culture to induce white new roots. The tea seedlings were randomly divided into three groups, and the control treatment (normal nitrogen concentration), 250μM NH4NO3 nitrogen deficiency treatment (1 / 20 of the normal nitrogen concentration) and nitrogen deficiency followed by restoration of normal nitrogen supply were carried out. The nutrient solution was changed every 7 days for each treatment. After 14 days of nitrogen deficiency treatment, the normal nitrogen nutrient solution was restored, and the young leaves of the three groups of tea seedlings were sampled at 0, 6, and 24 hours after the normal nitrogen nutrient solution was restored. The total RNA was extracted from the collected samples using the Rapid Universal Plant RNA Extraction Kit (purchased from Beijing Huayueyang Biotechnology Co., Ltd.), and cDNA was synthesized using the TRUEscript RT Kit with gDNA Eraser Reverse Transcription Kit (purchased from Hangzhou Xinjing Biological Reagent Development Co., Ltd.). The specific operation steps refer to the instruction manual of the response kit. The cDNA obtained by reverse transcription was used as a template for PCR amplification.

[0032] (2) Cloning of tea plant genes CsCEPD1.1 and CsCEPD1.2: Design specific primers for CsCEPD1.1 and CsCEPD1.2. The forward primer of CsCEPD1.1 gene cloning primer is

[0033] 5'-ATGGATAAGGTGACAAGGGTG-3'; reverse primer is

[0034] 5'-CTATATCAATGCTTGATATGGC-3';

[0035] The forward primer of CsCEPD1.2 gene cloning primers is 5'-ATGGAAAAAGTGACAAGATTG-3'; the reverse primer is 5'-TTAAGATCGAGCTTGATATGG-3'.

[0036] The amplified product was subjected to agarose gel electrophoresis, and the amplified fragment was recovered according to the length of the target gene fragment. The recovered product was connected to the pTOPO-Blunt Simple vector and transformed into Escherichia coli DH5α competent cells. The sequence of CsCEPD1.1 obtained by sequencing is shown in SEQ ID NO.1 in the sequence list; the sequence of CsCEPD1.2 obtained by sequencing is shown in SEQ ID NO.2 in the sequence list.

[0037] (3) Gene expression detection of tea tree genes CsCEPD1.1 and CsCEPD1.2:

[0038] Using the tea leaves cDNA after the above different nitrogen treatments as templates, real-time quantitative fluorescence PCR (qRT-PCR) was performed with the following primers to detect the expression of CsCEPD1.1 and CsCEPD1.2 genes at the transcriptional level in each material. The experiment was repeated 3 times and the results were averaged. Tea leaves with a continuous supply of normal nitrogen concentration nutrient solution were used as controls.

[0039] The qRT-PCR experiment was performed using Adlay fluorescent quantitative enzyme 2×Sybr Green qPCR Mix with High ROX. The reaction system (10 μL) was carried out according to the product instructions. The specific reaction was as follows: 5 μL 2×SybrGreen qPCRMix, 1 μL forward and reverse primer mixture (the forward and reverse primer concentrations were 10 μM), 3 μL ddH2O, 1 μL cDNA template (200 ng / μL). The specific reaction procedure was as follows: enzyme heat activation at 94°C, 30 min, 1 cycle; denaturation at 94°C, 10 s, extension at 60°C, 34 s, a total of 40 cycles.

[0040] Among them, the forward primer for detecting the expression of CsCEPD1.1 gene is 5'-AAGGCTCTCTTGAGGTTGGG-3' (SEQ ID NO.5); the reverse primer is 5'-CCATTGGAAGGAGAGAGCC-3' (SEQ ID NO.6); the forward primer for detecting the expression of CsCEPD1.2 gene is 5'-CAAGACCCTGAAGGAAGGGA-3' (SEQ ID NO.7); the reverse primer is 5'-AGGGACATGACTTCGTTGGT-3' (SEQ ID NO.8). Tea tree CsGAPDH is used as the internal reference gene, the internal reference gene forward primer is 5'-TTGGCATCGTTGAGGGTCT-3', and the reverse primer is 5'-CAGTGGGAACACGGAAAGC-3'.

[0041] The data were processed using the Comparative Ct method, that is, the Ct value was the number of cycles experienced when the fluorescence signal in the PCR tube reached the set threshold value, ΔCt = Ct (CsCEPD1.1 or CsCEPD1.2) - Ct (CsNAPDH), with 2 －△△Ct The value of was used to measure the gene transcription level, and the expression of CsCEPD1.1 and CsCEPD1.2 genes in each material was compared and analyzed.

[0042] In each experimental material, the qRT-PCR detection results of CsCEPD1.1 gene are as follows Figure 1 As shown: Compared with tea leaves that were continuously supplied with normal nitrogen nutrient solution, the expression of the CsCEPD1.1 gene in tea leaves under low nitrogen conditions was significantly upregulated, and the gene expression was significantly downregulated 6 hours and 24 hours after nitrogen supply was restored compared with before nitrogen supply was restored. This indicates that when tea leaves are under low nitrogen conditions, the expression of the CsCEPD1.1 gene will increase, and when normal nitrogen supply is restored, the nitrogen content of the leaves will increase, and the expression of the CsCEPD1.1 gene will decrease.

[0043] The results of qRT-PCR detection of CsCEPD1.2 gene expression were as follows Figure 2 As shown: The expression pattern of CsCEPD1.2 gene is similar to that of CsCEPD1.1.

[0044] Example 2

[0045] Tissue localization of CsCEPD1.1 and CsCEPD1.2 genes in tea tree (GUS staining)

[0046] (1) Cloning of tea leaf-specific promoter

[0047] Using the extracted DNA as a template, restriction endonuclease sites and protective bases were added to the 5' and 3' ends of the amplification primers, respectively. The restriction sites added to the CsCEPD1.1 promoter were ScaⅠ (5' end) and XbaⅠ (3' end), and its forward primer was 5'-AAA AGTACT AAGGTGGGACTTTAGGCG-3' (the underline indicates the ScaⅠ restriction site); the reverse primer is 5'-TGC TCTAGA ACTTCGAATGCAACAGCT-3' (the underline indicates the XbaⅠ restriction site), the cloned promoter was named proCsCEPD1.1, and its nucleotide sequence is shown in SEQ ID NO.3 in the sequence listing.

[0048] The restriction sites added to the CsCEPD1.2 promoter are SbfⅠ (5' end) and XbaⅠ (3' end). The forward primer is 5'-C CTGCAGGGCACGAGTCACAGGTTTC-3' (the underline indicates the SbfⅠ restriction site); the reverse primer is 5'-TGC TCTAGA ACTCTTGAGACTTTCAAACTC-3' (the underline indicates the XbaⅠ restriction site), the cloned promoter was named proCsCEPD1.2, and its nucleotide sequence is shown in SEQ ID NO.4 in the sequence listing.

[0049] (2) Construction of recombinant expression vector and acquisition of transgenic Arabidopsis

[0050] The above fragments were constructed into pBI121-35S-GUS vector by double enzyme digestion and ligation, and pBI121-proCsCEPD1.1-GUS vector and pBI121-proCsCEPD1.2-GUS vector were constructed respectively, and they were transferred into Arabidopsis thaliana by Agrobacterium-mediated genetic transformation. At the same time, pBI121-35S-GUS was transferred into Arabidopsis thaliana as a positive control. Kan resistance was used to screen transgenic Arabidopsis thaliana positive plants.

[0051] (3) GUS staining experiment

[0052] The 10-day-old pBI121-proCsCEPD1.1-GUS and pBI121-proCsCEPD1.2-GUS transgenic Arabidopsis seedlings were stained with GUS using the Biosharp BL622A kit. For detailed steps, refer to the instructions in the kit. Figure 4 As shown, Figure 4 Figure A in the middle is the expression pattern analysis after proCsCEPD1.1 was linked to the GUS gene; Figure 4 Figure B in the middle is the expression pattern analysis after proCsCEPD1.2 was linked to the GUS gene; Figure 4 C is the negative control Arabidopsis seedling GUS staining; Figure 4 Figure D in the middle shows the GUS staining of the positive control Arabidopsis seedlings. As can be seen from the figure, the negative control failed to stain, while the positive control expressed and stained the whole plant; while the Arabidopsis transformed with pBI121-proCsCEPD1.1-GUS and pBI121-proCsCEPD1.2-GUS vectors were only stained in the leaves, and could not be stained in the stems and roots, indicating that CsCEPD1.1 and CsCEPD1.2 are specifically expressed in tea leaves.

[0053] In summary, it can be seen that the tea tree genes CsCEPD1.1 and CsCEPD1.2 provided by the present invention are specifically expressed in the leaves of tea trees, and can be used as indicator signals of the nitrogen nutritional status of tea trees in actual production.

[0054] The above is an example of the best implementation of the present invention, and the parts not described in detail are common knowledge of ordinary technicians in the field. The protection scope of the present invention shall be based on the content of the claims, and any equivalent transformation based on the technical enlightenment of the present invention is also within the protection scope of the present invention.

Claims

1. CsCEPD1.1 and CsCEPD1.2 The application of genes in detecting nitrogen nutrient abundance and deficiency in tea trees is characterized in that: CsCEPD1.1 gene, the nucleotide sequence of which is shown in SEQ ID NO.1, tea tree CsCEPD1.2 The gene has a nucleotide sequence as shown in SEQ ID NO.

2.

2. According to claim 1 CsCEPD1.1 and CsCEPD1.2 The application of genes in detecting nitrogen nutrient abundance and deficiency in tea plants is characterized in that: The specific application method includes the following steps: Step 1: Check tea trees with adequate nitrogen supply CsCEPD1.1 or CsCEPD1.2 Gene expression levels are recorded; Step 2: Detection during the normal growth of tea trees CsCEPD1.1 or CsCEPD1.2 The gene expression level is compared with the expression level of the corresponding gene under the condition of sufficient nitrogen supply in step 1. If the gene expression level is judged to be significantly upregulated according to the one-way variance method, it is judged that the tea tree is in a nitrogen deficiency state.

3. According to claim 2 CsCEPD1.1 and CsCEPD1.2 The application of genes in detecting nitrogen nutrient abundance and deficiency in tea plants is characterized in that: The nitrogen supplied is in the form of a mixture of ammonium nitrogen and nitrate nitrogen.

4. According to claim 2 CsCEPD1.1 and CsCEPD1.2 The application of genes in detecting nitrogen nutrient abundance and deficiency in tea trees is characterized in that: tea tree CsCEPD1.1 The sequences of the detection primer pair of the gene are shown in SEQ ID NO.5 and SEQ ID NO.

6.

5. According to claim 2 CsCEPD1.1 and CsCEPD1.2 The application of genes in detecting nitrogen nutrient abundance and deficiency in tea plants is characterized in that: tea tree CsCEPD1.2 The sequences of the detection primer pair of the gene are shown in SEQ ID NO.7 and SEQ ID NO.8.

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