Tea plant CsARF9 gene and its application in promoting plant root growth

Abstract

The invention discloses the application of the tea tree CsARF9 gene in regulating plant root growth and cultivating varieties with high nitrogen utilization rate, and belongs to the field of genetic engineering technology. The CsARF9 gene encodes an amino acid sequence as shown in SEQ ID NO.2. The invention can increase the taproot length, the number of lateral roots and the utilization rate of nitrogen by increasing the expression of CsARF9, indicating that the gene can be used in genetic breeding for increasing the nitrogen utilization rate and growth rate of plants.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a tea plant CsARF9 gene sequence and its application in regulating plant root growth and cultivating varieties with high nitrogen utilization rate. Background Art

[0002] Currently, global agriculture is facing the challenge of balancing the growing population demand with limited resource supply. As one of the key elements for plant growth, the effective use of nitrogen has always been an important issue in agricultural production. Improving the efficiency of nitrogen use by plants and reducing nitrogen waste have become one of the important goals for improving agricultural sustainability and environmental protection.

[0003] In response to the above problems, we have discovered a new gene, the expression of which in plants can significantly improve the efficiency of nitrogen utilization by plants. Our research shows that the expression of this gene not only promotes the absorption and transport of nitrogen from the soil by plants, but also increases the length of the main root and the number of lateral roots, thereby increasing the amount of nitrogen accumulated by plants. In addition, the expression of this gene also leads to an increase in plant growth rate and dry matter content.

[0004] Therefore, based on this discovery, we proposed a method and application of plant genetic breeding using this gene. By introducing this gene into crops, the efficiency of nitrogen utilization by crops can be effectively improved, the amount of nitrogen applied can be reduced, the cost of agricultural production can be reduced, and the negative impact on the environment can be reduced. In addition, due to the increase in plant growth rate and dry matter content, we believe that this technology will also provide a new way to increase crop yields and improve the quality of agricultural products.

[0005] In summary, this invention has important agricultural and environmental significance, and is expected to provide new solutions to the current problems of inefficient nitrogen utilization and limited resources. Summary of the invention

[0006] Without solving the above technical problems, the present invention provides a tea plant CsARF9 gene and its application in regulating plant root growth and cultivating varieties with high nitrogen utilization rate.

[0007] The present invention clones the CsARF9 gene of tea tree, constructs and compares the root length, lateral root number, dry matter weight, nitrogen accumulation and nitrogen utilization rate of the CsARF9 overexpression strain and the wild-type strain. The results show that the overexpression strain has advantages in all the above indicators. The CsARF9 gene encodes a nucleotide sequence of the amino acid sequence shown in SEQ ID NO.2. The gene is expected to be applied to genetic engineering breeding of plants, providing a theoretical basis for the creation or improvement of new germplasm with high nitrogen utilization rate and high growth rate of plants. Taking into account the degeneracy of codons, the bases of the above nucleotide sequence are modified without changing the amino acid sequence, which also falls within the scope of protection of the present invention.

[0008] In one aspect, the present invention provides a protein for regulating plant root growth, characterized in that the sequence of the protein is shown in SEQ ID NO.2.

[0009] Furthermore, a gene for regulating plant root growth is provided, characterized in that the gene encodes an amino acid sequence as shown in SEQ ID NO.2.

[0010] Preferably, the gene sequence comprises a nucleotide sequence as shown in SEQ ID NO.1, or comprises a nucleotide sequence that is completely reverse complementary to the sequence shown in SEQ ID NO.1.

[0011] In another aspect, the present invention provides a use of the aforementioned protein or the aforementioned gene in regulating root growth.

[0012] Furthermore, the regulation of plant root growth is to increase the plant root length and / or the number of lateral roots by overexpressing the protein described in claim 1 or the gene described in claim 2, or to reduce the plant root length and / or the number of lateral roots by reducing the expression of the protein described in claim 1 or the gene described in claim 2.

[0013] In a preferred embodiment, the plant is a dicotyledonous plant.

[0014] Furthermore, the plant is a plant of the Cruciferae family or the Theaceae family.

[0015] Furthermore, the plant is a plant of the genus Arabidopsis or the genus Camellia.

[0016] Furthermore, the plant is Arabidopsis thaliana or tea plant.

[0017] In another aspect, the present invention provides a use of the aforementioned protein or the aforementioned gene in regulating plant nitrogen utilization rate and / or nitrogen accumulation.

[0018] Furthermore, the regulation of plant nitrogen utilization rate and / or nitrogen accumulation is to increase the plant nitrogen utilization rate and / or nitrogen accumulation by overexpressing the protein according to claim 1 or the gene according to claim 2, or to reduce the plant nitrogen utilization rate and / or nitrogen accumulation by reducing the expression of the protein according to claim 1 or the gene according to claim 2.

[0019] In a preferred embodiment, the plant is a dicotyledonous plant.

[0020] Furthermore, the plant is a plant of the Cruciferae family or the Theaceae family.

[0021] Furthermore, the plant is a plant of the genus Arabidopsis or the genus Camellia.

[0022] Furthermore, the plant is Arabidopsis thaliana or tea plant.

[0023] In another aspect, the present invention provides a use of the aforementioned protein or the aforementioned gene in regulating plant growth.

[0024] Furthermore, the regulating plant growth is to increase the dry matter weight of the plant by overexpressing the protein of claim 1 or the gene of claim 2, or to reduce the dry matter weight of the plant by reducing the expression of the protein of claim 1 or the gene of claim 2.

[0025] In a preferred embodiment, the plant is a dicotyledonous plant.

[0026] Furthermore, the plant is a plant of the Cruciferae family or the Theaceae family.

[0027] Furthermore, the plant is a plant of the genus Arabidopsis or the genus Camellia.

[0028] Furthermore, the plant is Arabidopsis thaliana or tea plant.

[0029] In another aspect, the present invention provides a use of the aforementioned protein or the aforementioned gene in breeding plant varieties with high nitrogen utilization efficiency and / or nitrogen accumulation.

[0030] Furthermore, the plant variety cultivated with high nitrogen utilization rate and / or nitrogen accumulation amount is a plant variety with high nitrogen utilization rate and / or nitrogen accumulation amount obtained by overexpressing the aforementioned protein or the aforementioned gene.

[0031] In a preferred embodiment, the plant is a dicotyledonous plant.

[0032] Furthermore, the plant is a plant of the Cruciferae family or the Theaceae family.

[0033] Furthermore, the plant is a plant of the genus Arabidopsis or the genus Camellia.

[0034] Furthermore, the plant is Arabidopsis thaliana or tea plant.

[0035] In another aspect, the present invention provides a use of the aforementioned protein or the aforementioned gene in cultivating plant varieties with high growth rates.

[0036] Furthermore, the plant variety with high growth rate is cultivated by overexpressing the aforementioned protein or the aforementioned gene to obtain a plant variety with high growth rate.

[0037] Furthermore, the indicator of the high growth rate is high dry matter mass, and / or increased plant root length and / or lateral root number.

[0038] Compared with the prior art, the present invention has the following advantages:

[0039] 1) The present invention provides a new protein and its encoding gene, which has the function of regulating plant root length, lateral root number, dry matter weight, nitrogen accumulation and / or nitrogen utilization rate.

[0040] 2) The present invention provides a new method for regulating plant growth and nitrogen utilization efficiency, namely, increasing the growth rate and nitrogen utilization efficiency of plants by increasing the expression of the CsARF9 gene or the protein encoded by it.

[0041] 3) The present invention provides a new method for cultivating plant varieties with fast growth and high nitrogen utilization efficiency, namely, cultivating plant varieties with fast growth and high nitrogen utilization efficiency by increasing the expression of CsARF9 gene or the protein encoded by it.

[0042] 4) Effectively improve the nitrogen utilization efficiency of crops, reduce the amount of nitrogen applied, reduce the cost of agricultural production, and reduce the negative impact on the environment. In addition, due to the increase in plant growth rate and dry matter content, this technology will also provide a new way to increase crop yields and improve the quality of agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The beneficial effects of the present invention are described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0044] Figure 1 Shown are the expression levels of CsARF9 in wild-type and transgenic Arabidopsis.

[0045] Figure 2 Shown is the phenotypic analysis of CsARF9 overexpressing Arabidopsis and wild-type Arabidopsis

[0046] A: Phenotypic comparison of wild type and CsARF9 overexpressing Arabidopsis; B: Main root length of wild type and CsARF9 overexpressing Arabidopsis; C: Number of lateral roots of wild type and CsARF9 overexpressing Arabidopsis.

[0047] Figure 3 Shown are the dry matter weights of CsARF9-overexpressing Arabidopsis and wild-type Arabidopsis.

[0048] Figure 4 Shown are the total nitrogen contents of CsARF9-overexpressing Arabidopsis and wild-type Arabidopsis.

[0049] Figure 5 The results show that CsARF9 overexpressing Arabidopsis and wild-type Arabidopsis 15 N-NO3 - 10 min in roots at different concentrations of markers 15 N-NO3 - . DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0052] The nucleotide sequence of the tea plant CsARF9 gene in the embodiment is shown in SEQ ID NO.1:

[0053]

[0054] The amino acid sequence of the tea plant CsARF9 protein is shown in SEQ ID NO.2:

[0055] MLSPLAHGLSCTYSLFRHYVVSITVYLRLKRSNHERERVEAVFRAGKAALRRFLAVFGVE

[0056] IEETLGGYGGDDLYTELWKACAGPLVDVPRSGERVFYFPQGHMEQLEASTNQELNEKIPH

[0057] FNLPSKILCRVINVHLRAEQETDEVYAQITLVPEQDQTEPESPDSCSPEPPKPTVYSFCK

[0058] VLTASDTSTHGGFSVLRKHANECLPSLDMSQQTPTQELISKDLHGLEWRFKHIFRDILTF

[0059] KNLFPRAENGELRVGVRRVARQQSSMPSSVISSQSMHLGVLATASHAVSTQTLFVVYYKP

[0060] RTSQFIISLNKYLEAVNNEFAVGMRFRMRFEGDESPERRFTGTIVGVEDLSRQWEKSKWR

[0061] SLKVQWDEPTSIARPERVSPWEIEPFVPSVPSGLVQQVIKKRPRPPIEIPGHETASAVWN

[0062] Example 1 Construction of tea plant CsARF9 gene overexpression vector

[0063] Based on the unigene sequence obtained by previous sequencing in our laboratory, a pair of specific primers CsARF9-F (ATGAAGGCTGCATCAAATGGGTATC) and CsARF9-R (TCAGTTTTCTACTGTGTCCAAGCT) were designed. RNA was extracted from the Shuchazao variety of tea trees and reverse transcribed into cDNA. Tea trees were planted in the greenhouse of Nanjing Agricultural University. The PCR amplification system was 20 μL: 10 μL Prime STAR Max Premix enzyme (TaKaRa, Dalian, China), 7 μL ddH2O, 1 μL template cDNA, and 1 μL each of upstream and downstream primers. The RT-PCR reaction procedure was: 98℃ pre-denaturation for 3 min; 35 cycles: 98℃ denaturation for 10 s, 54℃ annealing for 30 s, 72℃ extension for 60 s; 72℃ extension for 5 min. The PCR product was recovered using a DNA recovery kit, ligated to the pMD19-T vector, and transformed into Escherichia coli DH5α competent cells. Single colonies were picked for PCR detection, and finally the plasmids were screened and extracted for sequencing (GenScript, Nanjing, China). Specific recombination primers CsARF9-F (ATGAAGGCTGCATCAAATGGGTATC) and CsARF9-R (TCAGTTTTCTACTGTGTCCAAGCT) were designed according to the vector sequence and CsARF9 gene sequence to amplify the CsARF9 gene, and then recombined into the pCAMBIA1301 vector to obtain the recombinant vector pCAMBIA1301-CsARF9.

[0064] Example 2 Acquisition and identification of CsARF9 transgenic Arabidopsis

[0065] The recombinant plasmid was sent to Nanjing GenScript Biotechnology Co., Ltd. for sequencing (Nanjing, China). The recombinant plasmid pCAMBIA1301-CsARF9, which was verified by sequencing, was introduced into Agrobacterium tumefaciens strain (GV3101) by electroporation, and genetic transformation of Arabidopsis thaliana was performed by floral dipping method (Zhang et al., 2006). Transgenic positive seedlings were screened using 1 / 2MS medium containing hygromycin (35 mg / L), and then PCR amplification was performed using specific primers (CsGADPH-qF:TTGGCATCGTTGAGGGTCT; CsGADPH-qR:CAGTGGGAACACGGAAAGC; CsARF9-qF:CTACTACAAGCCGAGGACGAG; CsARF9-qR:TGGGATGCCAGACAACACTA) for further verification. RT-qPCR was used to detect the expression level of CsARF9 in positive transgenic lines. RT-qPCR was performed on the Bio-Rad IQ5 fluorescent quantitative PCR platform using the SYBR PreMix Ex Taq kit (TaKaRa, Dalian, China). Amplification system: SYBR PreMix Ex Taq 10 μL, dd H2O 7.2 μL, cDNA 2 μL, forward and reverse primers 0.4 μL each. RT-qPCR reaction conditions were: 95°C 30 s, 95°C 10 s, 60°C 20 s, 40 cycles; melting curve analysis range was 65°C-95°C, and 0.5°C was increased every 5 s to verify amplification specificity. CsTBP was used as an internal reference gene (Wu et al., 2016). Each sample was subjected to 9 biological replicates, according to 2 -ΔΔCT Methods The relative expression level of genes was calculated (Pfaffl, 2001). Two different transgenic lines (OE-1 and OE-2) were selected for subsequent experiments.

[0066] The results are as follows Figure 1 As shown, the expression levels of the CsARF9 gene in the two strains were greatly increased, and these two overexpression strains were subsequently selected for experiments.

[0067] Example 3: Root phenotype analysis of CsARF9 overexpressing plants

[0068] Homozygous overexpression and wild-type Arabidopsis materials were placed in a closed container and sterilized with chlorine (Cl2) produced by the reaction of 100 mL sodium hypochlorite (NaClO) and 3 mL concentrated hydrochloric acid (HCl). After sterilization for at least 6 h, the residual Cl2 in the centrifuge tube was blown by a super-clean typhoon. After blowing for at least 2 h, the seeds were planted on 1 / 2MS solid agar medium (containing 1% sucrose, pH 5.7). After vernalization at 4 °C for 2 days, the seeds were transferred to a full-day incubator with a light intensity of 100 μmol m –2 s–1 , the temperature was 22℃. After 14 days, the root phenotype differences between CsARF9 overexpressing Arabidopsis and wild-type Arabidopsis were statistically analyzed.

[0069] Phenotypic analysis of Arabidopsis revealed that the taproot length and lateral root number of the CsARF9 overexpression strain were significantly higher than those of wild-type Arabidopsis ( Figure 2 ), which indicates that the expression of CsARF9 gene can promote the growth of plant roots.

[0070] Example 4: Measurement of dry matter in plants overexpressing CsARF9

[0071] After 2 days of vernalization at 4°C, wild-type and CsARF9 transgenic Arabidopsis seeds were sown in nutrient pots filled with peat soil to ensure that the amount of soil in the nutrient pots was basically the same (the nitrogen content in the nutrient pots was 2 g / kg). After 20 days of growth, the whole plant was washed, sterilized at 120°C for 30 minutes, and then dried at 80°C to constant weight. The dry matter mass of each part was weighed, and each sample was biologically replicated ten times.

[0072] The results show that ( Figure 3 ), the dry matter weight of the CsARF9 overexpression line was significantly higher than that of the wild type, indicating that overexpression of CsARF9 can promote the growth of transgenic Arabidopsis and increase the biomass of Arabidopsis.

[0073] Example 5 Measurement of nitrogen content in plants overexpressing CsARF9

[0074] After 2 days of vernalization at 4°C, wild-type and CsARF9 transgenic Arabidopsis seeds were sown in nutrient pots filled with peat soil to ensure that the amount of soil in the nutrient pots was basically the same (the nitrogen content in the nutrient pots was 2g / kg). After 20 days of growth, the whole plant was washed, dried at 105°C for 30 minutes, and then dried again at 65°C for 3 days, and digested with sulfuric acid until the mixture was clear. Then a small amount of H2O2 solution (30%) was added for further digestion until the brown mixture became colorless. The total nitrogen content in the digestion solution was determined by the phenol-hypochlorite method, with ten biological replicates for each sample.

[0075] The results show that ( Figure 4 ), the nitrogen content of the CsARF9 overexpression line was significantly higher than that of the wild type, which indicates that overexpression of CsARF9 can promote the accumulation of nitrogen in plants.

[0076] Example 6 Plants overexpressing CsARF9 15 Measurement of N-NO3-absorption rate

[0077] Wild-type and CsARF9 transgenic Arabidopsis seeds were vernalized at 4°C for 2 days and then transferred to a full-day incubator with a light intensity of 100 μmol m –2 s –1, the temperature was 22℃. After 14 days, the plants were transferred to 1 / 4 Hoagland nutrient solution for hydroponics. After 10 days of culture, the conventional depletion method was used to eliminate the effect of residual nitrogen in the free space of the plant roots on the absorption test. Arabidopsis seedlings after starvation culture for 1 day were taken and then the rice roots were soaked in 0.1mM CaSO4 solution for about 1 minute. After that, the liquid on the surface of the roots was dried with absorbent paper and then transferred to the solution containing different concentrations (0.25, 0.5, 0.75, 1.0mmol·L -1 )15N labeled N-NO3 - In the culture solution, after the roots absorbed it for 10 minutes, 0.1mM CaSO4 solution was used to wash away the 15N-NO3 remaining on the root surface. - After the liquid on the root surface was absorbed by absorbent paper, the roots of Arabidopsis thaliana were removed, baked at 105℃ for 15min, and then placed in a 75℃ oven. After drying, the samples were weighed and finally ground with a sample grinder. 0.05mg of each sample was weighed and the 15N absorption rate was measured.

[0078] The results show that ( Figure 5 ), CsARF9 transgenic Arabidopsis thaliana to N-NO3 - The absorption rate of CsARF9 is higher than that of the wild type, which indicates that the expression of CsARF9 can increase the nitrogen absorption rate of the plant and improve nitrogen utilization efficiency.

[0079] Based on the above results, it is confirmed that the CsARF9 gene can improve the utilization of nitrogen by plants, promote the accumulation of nitrogen by plants, increase the length of the main root and the number of lateral roots, and increase the dry matter content of plants. Therefore, it can be used in genetic breeding to improve the nitrogen utilization efficiency and growth rate of plants.

[0080] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of CsARF9 gene in regulating root growth of Arabidopsis thaliana, characterized in that: The nucleotide sequence of the CsARF9 gene is shown in SEQ ID NO.

1. The regulating Arabidopsis root growth is to increase the Arabidopsis root length and / or the number of lateral roots by overexpressing the CsARF9 gene.

2. Use of the CsARF9 gene in regulating nitrogen utilization and / or nitrogen accumulation in Arabidopsis thaliana, characterized in that: The nucleotide sequence of the CsARF9 gene is shown in SEQ ID NO.

1. The regulation of nitrogen utilization rate and / or nitrogen accumulation of Arabidopsis thaliana is to increase the nitrogen utilization rate and / or nitrogen accumulation of Arabidopsis thaliana by overexpressing the CsARF9 gene.

3. Application of CsARF9 gene in regulating the growth of Arabidopsis thaliana, characterized in that: The nucleotide sequence of the CsARF9 gene is shown in SEQ ID NO.

1. The method of regulating the growth of Arabidopsis thaliana is to increase the dry matter weight of the plant by overexpressing the CsARF9 gene.

4. Use of the CsARF9 gene in cultivating plant varieties with high nitrogen utilization efficiency and / or nitrogen accumulation, characterized in that: The nucleotide sequence of the CsARF9 gene is shown in SEQ ID NO.

1. The plant variety with high nitrogen utilization rate and / or nitrogen accumulation is cultivated by overexpressing the CsARF9 gene. The plant is Arabidopsis thaliana.

5. Use of the CsARF9 gene in cultivating plant varieties with high growth rates, characterized in that: The nucleotide sequence of the CsARF9 gene is shown in SEQ ID NO.

1. The plant variety with high growth rate is cultivated by overexpressing the CsARF9 gene. The plant is Arabidopsis thaliana.

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