A SiTAF10 gene for improving drought resistance and salt tolerance in millet and its application

Abstract

The present invention provides a SiTAF10 gene for improving the drought resistance and salt tolerance of millet and its application. The present invention isolates and clones the gene SiTAF10 whose nucleotide sequence is shown in SEQ ID NO.1 from millet, and fluorescence quantitative expression analysis shows that it is involved in a variety of abiotic stresses. After the gene is overexpressed in millet, in drought and salt stress experiments, the survival rate and chlorophyll content of the strain overexpressing the SiTAF10 gene are increased by more than 2 times compared with the wild-type strain, and the accumulation of reactive oxygen species (ROS) is significantly reduced. It is explained that the SiTAF10 gene provided by the present invention and the protein encoded by it can significantly improve the drought resistance and salt tolerance of plants. Therefore, the SiTAF10 gene is a new gene resource for millet drought resistance and salt tolerance, which will play an active role in the improvement of millet drought resistance and salt tolerance varieties and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a SiTAF10 gene capable of improving the drought resistance and salt tolerance of millet and an application thereof. Background Art

[0002] Millet is an important crop in the arid, semi-arid and saline-alkali barren regions of northern my country. In recent years, the consumption demand for millet, farmers' income and willingness to grow millet have all shown a clear growth trend. However, the unclear molecular mechanism of millet drought and salt tolerance and the small amount of available germplasm are the main factors limiting its production. Therefore, identifying millet drought and salt tolerance genes and clarifying their mechanism of action can provide new gene resources and germplasm for millet stress molecular breeding, which is of great production significance.

[0003] TAFs are an independent protein family with transcription initiation recognition and regulation, and each family member has its own unique function. They all contain the HFD histone fold domain. There are 18 genes encoding TAFs in Arabidopsis. TAFs whose functions have been identified so far are mostly involved in plant light and hormone signal transduction, morphological construction and reproductive growth. TAF1 is involved in light signal transduction; TAF1, TAF5 and TAF6 can all regulate pollen tube germination; TAF4b controls chromosome exchange during meiosis and gene transcription in the germ cell line; TAF12b is involved in ethylene signal transduction and cytokinin pathway; TAF13 participates in regulating plant seed development by regulating the development of plant embryos and endosperms; TAF15b is involved in plant autoimmunity. Therefore, TAFs are essential for plant growth and development and stress resistance. However, no members of the TAFs family have been reported in millet. Summary of the invention

[0004] The present invention provides a SiTAF10 gene for improving the drought resistance and salt tolerance of millet and its application. The present invention isolates and clones the SiTAF10 gene from millet, connects its full-length cDNA to an expression vector driven by a 35S promoter, and transforms millet by Agrobacterium infection. Experiments confirm that the SiTAF10 gene can significantly improve the drought resistance and salt tolerance of millet.

[0005] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0006] The present invention provides a SiTAF10 gene for improving drought resistance and salt tolerance of millet. The nucleotide sequence of the SiTAF10 gene is shown as SEQ ID NO.1.

[0007] Furthermore, the CDS nucleotide sequence of the SiTAF10 gene is shown as SEQ ID NO.2; and the amino acid sequence of the SiTAF10 protein encoded thereby is shown as SEQ ID NO.3.

[0008] The present invention provides the application of the SiTAF10 gene in breeding for improving the drought resistance and salt tolerance of millet.

[0009] Furthermore, the overexpression vector containing the SiTAF10 gene is obtained by extracting millet RNA, reverse transcribing the RNA into cDNA, cloning the gene, and then connecting it with the pCAMBIA 1305.1 expression vector driven by 35S.

[0010] Furthermore, the primer sequences for gene cloning are:

[0011] P1: 5'-ATGATGGGCAGCAACAGCA-3';

[0012] P2: 5'-CTCCTCCTTGTCGAAGGAGC-3'.

[0013] Furthermore, the expression of SiTAF10 gene was upregulated by drought and salt stress.

[0014] Furthermore, under drought stress conditions, the photosynthetic rate, fresh weight and survival rate of millet strains overexpressing the SiTAF10 gene were significantly higher than those of wild plants, and the water loss rate was significantly lower than that of wild plants, thereby improving the drought stress tolerance of millet.

[0015] Furthermore, under salt stress conditions, the survival rate and chlorophyll content of millet lines overexpressing the SiTAF10 gene were significantly higher than those of wild plants, thereby improving the salt stress tolerance of millet.

[0016] Furthermore, under salt stress conditions, the reactive oxygen species (ROS) accumulated in millet lines overexpressing the SiTAF10 gene were significantly lower than those in the wild type.

[0017] Furthermore, under drought stress, the accumulated ROS in millet lines overexpressing the SiTAF10 gene was significantly lower than that in the wild type.

[0018] Compared with the prior art, the advantages and positive effects of the present invention are: the present invention connects the full-length cDNA of the SiTAF10 gene to the 1305.1 expression vector initiated by 35S, and uses Agrobacterium to infect and transform millet. It has been experimentally verified that overexpression of the SiTAF10 gene can enhance the tolerance of C4 model plant millet transgenic strains to drought and salt stress. In view of the conservatism of the SiTAF10 gene in plants and the phenotype presented in transgenic millet, it can be considered that the gene has application value. The SiTAF10 gene provides a new gene resource for the molecular breeding of millet drought and salt resistance, and will play an important role in the improvement of crop drought and salt resistance varieties, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A is the fluorescence quantitative RT-qPCR analysis of the expression level of SiTAF10 in different tissues of millet, including root, stem, leaf, leaf sheath, flag leaf, young panicle, immature panicle and mature seed. Figure 1 B is the expression characteristics of SiTAF10 gene analyzed by fluorescence quantitative RT-qPCR under treatment with 200 mM NaCl, 100 μM ABA (abscisic acid), 100 μM IAA (auxin), 20% PEG6000 and 100 μM MeJA (methyl jasmonate) for different time periods.

[0020] Figure 2 A is the SiTAF10 gene connected to the 1305.1 expression vector; Figure 2 B is the fluorescence quantitative RT-qPCR analysis of the expression level of the target gene in the overexpressing SiTAF10 strain.

[0021] Figure 3 A shows the phenotypes of overexpressing SiTAF10 and wild-type Ci846 millet under normal conditions, drought stress treatment, and rewatering after drought; Figure 3 B, C, D, and E are the analysis and comparison of photosynthetic rate, water loss rate, fresh weight, and survival rate of overexpressing SiTAF10 and wild-type Ci846 millet after drought stress treatment.

[0022] Figure 4 A shows the phenotypes of overexpressed SiTAF10 and wild-type Ci846 millet under normal conditions and after high salt stress treatment; Figure 4 B and 4C are the analysis and comparison of the survival rate and chlorophyll content of overexpressing SiTAF10 and wild-type Ci846 millet after high salt stress treatment.

[0023] Figure 5 A and 5B are the results of nitro blue tetrazolium chloride (NBT) and diaminobenzidine (DAB) staining to detect hydrogen peroxide (H2O2) and superoxide anion (O2 。- )content; Figure 5 C and 5D show the H2O2 and O2 in overexpressing SiTAF10 and wild-type Ci846 millet under normal conditions and salt stress conditions. 。- Content determination.

[0024] Figure 6A and 6B show the H2O2 and O2 in overexpressing SiTAF10 and wild-type Ci846 millet under normal conditions and drought stress. 。- Content determination; Figure 6 C shows the results of ROS fluorescent dye H2DCFDA staining of overexpressing SiTAF10 and wild-type Ci846 millet under normal conditions and drought stress conditions. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0026] Example 1: Sequence analysis, cloning and vector construction of foxtail millet SiTAF10 gene:

[0027] The present invention uses the foxtail millet genome database website phytozome to find the gene SiTAF10, compares the amino acid sequence encoded by the gene on the ClustalW website, and finds that the SiTAF10 protein is highly homologous in different species. The full length of the SiTAF10 genome sequence is 3862bp (the sequence is shown in SEQ ID NO.1), the CDS sequence is 474bp (the sequence is shown in SEQ ID NO.2), and the SiTAF10 protein sequence encoded by it is shown in SEQ ID NO.3.

[0028] Primers were designed based on the CDS sequence of the SiTAF10 gene for cloning. The cloning method was as follows:

[0029] (1) RNA extraction: The total RNA of millet was extracted using the Biotek Universal Plant Total RNA Extraction Kit.

[0030] (2) Synthesis of the first-strand cDNA by reverse transcription: The extracted RNA was dissolved and the RNA concentration was determined. The RNA was then reverse transcribed using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Reverse Transcription Kit.

[0031] Take 5 μg of total RNA, add 10 μL of 2× reaction buffer, 1 μL of primer oligo dT (0.5 μg / μL), 1 μL of reverse transcriptase, 1 μL of de novo genomic enzyme, add water to 20 μL, incubate at 42°C for 30 min, and inactivate the enzyme at 85°C for 5 min.

[0032] (3) Cloning of SiTAF10 gene:

[0033] Upstream primer: 5′-ATGATGGGCAGCAACAGCA-3′;

[0034] Downstream primer: 5′-CTCCTCCCTTGTCGAAGGAGC-3′;

[0035] Vazyme high-fidelity enzyme (Vazyme#P505) was used for amplification, and the reaction system was: 12.5 μL of 2× reaction buffer, 0.5 μL of deoxyribonucleic acid (dNTP), 1 μL of upstream primer, 1 μL of downstream primer, 0.5 μL of high-fidelity enzyme, 1 μL of cDNA template, and water was added to 25 μL.

[0036] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 1 minute, for a total of 35 cycles; post-extension at 72°C for 5 minutes; and insulation at 16°C.

[0037] After the reaction, agarose gel electrophoresis was performed. After the target band was detected, the gel was cut and the gel was recovered. The gel recovery method was performed according to the quick agarose gel DNA recovery kit (Cat# DP1722) of BioTeke Company.

[0038] (4) Take the above 3.5 μL gel recovery product and connect it with the 35S-activated pCAMBIA 1305.1 expression vector. The operation steps are carried out according to the instructions of EasyFusion Assembly Master Mix of New Cyme. The ligation product is transformed into Escherichia coli DH5α strain using the heat shock method and grown overnight on an LB plate containing kanamycin to obtain positive clones. Pick a single white colony for colony PCR. The reaction system is the same as above. Select the positive colony and grow it in LB liquid culture medium overnight.

[0039] (5) Extraction of plasmid DNA: Plasmid DNA was extracted using the Kangwei Century High Purity Plasmid Mini Extraction Kit (CW0500A).

[0040] (6) Sequence determination: The extracted plasmid DNA was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequence determination.

[0041] Example 2: Tissue and induced expression characteristics of SiTAF10 gene

[0042] In order to confirm whether the SiTAF10 gene is involved in the regulation of plant stress tolerance, the present invention uses real-time fluorescence quantitative RT-PCR technology to analyze the expression pattern of the SiTAF10 gene in different tissues of millet and under different stress and hormone treatments.

[0043] 1. Analysis of SiTAF10 gene tissue expression under normal growth conditions

[0044] Millet roots, stems, leaves, leaf sheaths, flag leaves, young panicles, developing panicles and mature seeds were collected under normal growth conditions, frozen in liquid nitrogen, ground into powder in liquid nitrogen, and RNA was extracted using the Biotech Universal Plant RNA Extraction Kit. The RNA extracted from each part was used as a template for reverse transcription using the TransScript One-Step gDNA Removal and cDNA SynthesisSuperMix Reverse Transcription Kit. Real-time fluorescence quantitative RT-PCR was used to analyze the expression of the SiTAF10 gene. The millet SiACTIN gene was used as an internal reference gene, and the primers for SiACTIN gene amplification were Actin_F and Actin_R; the primers for SiTAF10 gene amplification were SiTAF10_F and SiTAF10_R.

[0045] The real-time fluorescence quantitative RT-PCR reaction system is:

[0046]

[0047] The amplification procedure is as follows:

[0048] (1) 95.0℃ for 60s;

[0049] (2) 95.0℃ for 10s;

[0050] (3) 60.0℃ for 10s;

[0051] (4) 72.0℃ for 15s;

[0052] (5) Plate Read;

[0053] (6)Incubate at 65℃for 20s;

[0054] (7) Melting curve from 65℃ to 95℃, read every 0.5℃, hold 1s;

[0055] (8)End.

[0056] Actin_F sequence is: 5′-TTGCTGACAGGATGAATGGC-3′;

[0057] Actin_R sequence is: 5′-CACATCTGCTGGAATGTGCT-3′;

[0058] The sequence of SiTAF10_F is: 5′-CCAGAAGTTCCTTTCAGA-3′;

[0059] The sequence of SiTAF10_R is: 5′-CATCCATAGTCAGTACAAGA-3′.

[0060] The results are as follows Figure 1 As shown in A, it can be seen that SiTAF10 is mainly expressed in roots during the seedling stage. Although it is also expressed in stems and leaves, the expression levels are relatively low. In the seedling stage, it is mainly expressed in ears and seeds, and secondly, it is also highly expressed in flag leaves.

[0061] 2. Expression analysis of SiTAF10 gene under stress and hormone treatment conditions

[0062] The wild-type Ci846 millet was used as the material, grown for 1 week, and 200mM NaCl, 100μM ABA, 100μMIAA, 20% PEG6000 and 100μM MeJA culture solutions were used for stress treatment. Fresh materials were collected at 0, 3, and 24h for each treatment time and quick-frozen in liquid nitrogen. The total RNA of the millet material was extracted using the Biotech Universal Plant RNA Extraction Kit, and the first-chain cDNA of the SiTAF10 gene was synthesized using a reverse transcription kit (the operating steps are the same as the title of this embodiment), and then the expression of the SiTAF10 gene was analyzed using fluorescent quantitative RT-PCR. Among them, the fluorescent quantitative RT-PCR experiment, the PCR system was configured according to the SYBR Premix Ex TaqⅡ kit, and the real-time quantitative fluorescent PCR detection was performed on the CFX96 Touch fluorescent quantitative PCR instrument.

[0063] The results are as follows Figure 1 As shown in B, salt stress treatment for 24 h and PEG6000 treatment for 3 h induced a significant upregulation of the expression of the SiTAF10 gene in foxtail millet; in addition, the expression of this gene was also significantly upregulated after MeJA induction for 24 h.

[0064] Example 2 shows that the SiTAF10 gene is expressed in millet roots, ears and seeds. This gene is a salt stress and drought stress response gene and is involved in the response of millet to high salt and drought stress.

[0065] Example 3: Transgenic SiTAF10 to cultivate drought-resistant and salt-tolerant millet

[0066] 1. Genetic transformation of foxtail millet SiTAF10 and screening of homozygous transgenic lines

[0067] Take 2 μL of the vector plasmid ( Figure 2A) Transformed into Agrogan EHA105 and infected millet embryonic callus cells. Millet embryonic callus cells were derived from the wild-type millet variety Ci846. Through hygromycin screening, 35S::SiTAF10 overexpression materials were obtained. Through RT-qPCR detection, five stable overexpression transgenic lines were obtained. The results are shown in Figure 2 As shown in B.

[0068] II. Drought resistance evaluation of transgenic SiTAF10 lines

[0069] Vermiculite and nutrient soil were mixed in a 1:1 ratio, and the same weight was weighed and placed in a small pot. Water was absorbed through the small holes at the bottom to completely moisten the soil. Transgenic and wild-type millet seeds that had been imbibed overnight were planted in the small pots, with 9 seeds in each small pot. After planting, a layer of soil was covered and lightly compacted. The seeds were grown normally in the greenhouse for 30 days. Millet seedlings with consistent growth were selected for drought stress treatment, and the position of the small pots needed to be changed frequently to reduce the impact of the position on the growth of millet seedlings under stress. The plants grew for 7 days under stress and watering was resumed ( Figure 3 A), photosynthetic rate and water loss rate were measured after 5 days of drought treatment; fresh weight was measured after 10 days of drought treatment, and survival rate was calculated after 3 days of rehydration.

[0070] The results showed that the photosynthetic rate of SiTAF10 transgenic millet was significantly higher than that of wild type ( Figure 3 B), the water loss rate is significantly lower than that of the wild type ( Figure 3 C), fresh weight and survival rate were significantly higher than those of wild plants ( Figure 3 D, E), indicating that the drought resistance of transgenic SiTAF10 millet is significantly higher than that of wild plants.

[0071] III. Identification of salt tolerance of transgenic millet lines carrying SiTAF10 gene

[0072] Vermiculite and nutrient soil were mixed in a 1:1 ratio, and the same weight was weighed and placed in a small pot. Water was absorbed through the small holes at the bottom to completely moisten the soil. Transgenic and wild-type millet seeds that had been swollen overnight were planted in small pots, with 9 seeds in each small pot. After planting, a layer of soil was covered and lightly compacted. The seeds were grown normally in a greenhouse for 14 days. Millet seedlings with consistent growth were selected for salt stress treatment, and the position of the small pots needed to be changed frequently to reduce the impact of the position on the growth of millet seedlings under stress. After the plants grew in 300mM NaCl for 7 days, the chlorophyll content of the plants was measured; the survival rate was measured after 21 days.

[0073] The results showed that the survival rate of transgenic SiTAF10 plants treated with 300 mM NaCl ( Figure 4 B) and chlorophyll content ( Figure 4C) is significantly higher than that of wild plants; the test results show that the salt tolerance of transgenic millet with SiTAF10 gene is significantly higher than that of wild plants ( Figure 4 A).

[0074] IV. Detection of Reactive Oxygen Species (ROS) Content in Transgenic Millet Lines with SiTAF10 Gene

[0075] Vermiculite and nutrient soil were mixed in a 1:1 ratio, and the same weight was weighed and placed in a small pot. Water was absorbed through the small holes at the bottom to completely moisten the soil. The transgenic and wild-type millet seeds that had been swollen overnight were planted in the small pots, with 9 seeds in each small pot. After planting, a layer of soil was covered and lightly compacted. The seeds were grown normally in the greenhouse for 14 days. Millet seedlings with consistent growth were selected for salt stress treatment, and the position of the small pots needed to be changed frequently to reduce the impact of the position on the growth of millet seedlings under stress. After the plants grew in 300mM NaCl for 3 days, the transgenic lines and wild types were stained with NBT and DAB, and O2 ·- The content of H2O2 was detected.

[0076] Vermiculite and nutrient soil were mixed in a 1:1 ratio, and the same weight was weighed and placed in a small pot. Water was absorbed through the small holes at the bottom to completely moisten the soil. The transgenic and wild-type millet seeds that had been swollen overnight were planted in the small pots, with 9 seeds in each small pot. After planting, a layer of soil was covered and lightly compacted. The seeds were grown normally in the greenhouse for 30 days. Millet seedlings with consistent growth were selected for drought stress treatment, and the position of the small pots needed to be changed frequently to reduce the impact of the position on the growth of millet seedlings under stress. When the plants grew for 5 days under stress, protoplasts were extracted from the transgenic lines and the wild type, and H2DCFDA was added. After that, the fluorescence was observed under a fluorescence electron microscope. The intensity of the fluorescence was proportional to the ROS content. At the same time, O2 ·- and H2O2 content.

[0077] The results showed that the ROS accumulation in SiTAF10-transfected millet was significantly lower than that in the wild type ( Figure 5 A, B, C, D), the ROS accumulation in SiTAF10-transformed millet after 5 days of drought treatment was significantly lower than that in the wild type ( Figure 6 A, B, C). The experimental results show that millet with SiTAF10 gene accumulates less reactive oxygen under salt and drought stress.

[0078] Example 3 shows that compared with wild plants, the tolerance of transgenic millet with SiTAF10 gene to drought and high salt stress is significantly improved, and it has a very good application prospect in drought-resistant and salt-tolerant breeding.

[0079] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A SiTAF10 The application of the gene in breeding for improving drought resistance and salt tolerance of millet is characterized in that: Said SiTAF10 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Containing the SiTAF10 The gene overexpression vector is obtained by extracting millet RNA, reverse transcribing the RNA into cDNA, cloning the gene, and then connecting it with the pCAMBIA 1305.1 expression vector promoted by 35S.

3. The use according to claim 2, characterized in that: The primer sequences for the gene cloning are: P1: 5'-ATGATGGGCAGCAACAGCA-3'; P2: 5'-CTCCCTCCCTTGTCGAAGGAGC-3'.

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