A SiTPS27 gene regulating millet rust resistance and its application

Abstract

The present invention discloses a SiTPS27 gene for regulating millet rust resistance and its application. In this study, the gene SiTPS27 with a nucleotide sequence as shown in SEQ ID NO.1 was isolated and cloned from millet. After the gene was overexpressed in millet, a transgenic plant with improved rust resistance was obtained, which was specifically manifested as follows: compared with the wild-type strain, the strain overexpressing the SiTPS27 gene had a reduced number of rust fungus summer spores on the leaves of the SiTPS27 transgenic offspring plants on the 14th day after inoculation with millet rust fungi, a smaller proportion of the leaf area, a lower severity, and improved disease resistance, indicating that SiTPS27 was involved in the rust resistance process of millet. Therefore, the SiTPS27 gene has important theoretical and practical significance for improving millet rust resistance, will play an important role in millet disease resistance breeding and improvement, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a SiTPS27 gene for regulating millet rust resistance and an application thereof. Technical Background

[0002] Millet (Setaria italica (L.) Beauv.) is drought-resistant and barren-tolerant, can be used as both food and grass, and is rich in nutrition. It is distributed in Asia and Europe. China's planting area and output of millet rank first in the world, and it is an important coarse grain crop in northern my country.

[0003] Millet rust is caused by Uromyces setariae-italicae Yoshino and is one of the main airborne diseases in millet production. It generally reduces yield by 10% to 30%. In epidemic years, plants fall over and grains are not harvested, which seriously affects the stable and high yield of millet. With the substantial improvement of the level of millet industrialization, the damage caused by rust is more serious in epidemic years and has become one of the important factors restricting the development of the millet industry. The most economical and effective measure to prevent and control millet rust is to cultivate and use millet rust-resistant varieties. To this end, it is a very necessary prerequisite to widely explore and clone millet rust resistance and related genes.

[0004] Phytoalexin, also known as phytoalexin and plant defensin, is an important secondary metabolite produced by plants. It mainly plays a toxic role on pathogens and participates in the chemical defense response of plants to pathogens. Among them, terpenoids are the most diverse and most abundant secondary metabolites in plants. The application of terpenoid Momilactone A to rice can improve the resistance of rice to rice blast. At the same time, this type of compound can also inhibit the spore germination of rice blast fungus, indicating that terpenoids have an important positive regulatory effect on the immune response of plants. The terpene synthase (TPS) family catalyzes the cyclization and rearrangement reactions of GPP (C10_monoterpene), FPP (C15_sesquiterpene), GGPP (C20_diterpene) and FGPP (C25_diterpene) precursors to form various terpene compounds. Therefore, TPS family genes play an important and unique role in the disease resistance of plants. However, there are no reports of TPS family members in millet. Summary of the invention

[0005] The present invention provides a SiTPS27 gene for regulating millet rust resistance and its application. The present invention isolates and clones the SiTPS27 gene from millet, connects its full-length CDS to an expression vector driven by a ubiqutin (abbreviated as Ubi) promoter, and transforms millet by infection with Agrobacterium. Experiments confirm that on the 14th day after inoculation with millet rust fungi, the number of rust fungus summer spores on the leaves of SiTPS27 transgenic offspring plants decreases, the proportion of leaf area decreases, and the severity decreases, thereby improving millet rust resistance.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] The present invention provides a SiTPS27 gene for regulating millet resistance to rust, and the nucleotide sequence of the SiTPS27 gene is shown in SEQ ID NO.1.

[0008] Furthermore, the CDS nucleotide sequence of the SiTPS27 gene is shown as SEQ ID NO.2; and the amino acid sequence of the SiTPS27 protein encoded thereby is shown as SEQ ID NO.3.

[0009] The invention provides application of the SiTPS27 gene in improving millet resistance to millet rust.

[0010] Furthermore, an overexpression vector containing the SiTPS27 gene was transformed into millet to obtain a millet strain in which the number of rust summer spores on millet leaves after rust infection was reduced, the proportion of leaf area was reduced, and the severity was reduced.

[0011] Furthermore, the overexpression vector containing the SiTPS27 gene is obtained by extracting millet RNA, reverse transcribing the RNA into cDNA, cloning the gene, and then connecting it with the Ubi-activated pTCK303 expression vector.

[0012] Furthermore, the primer sequences for gene cloning are:

[0013] TPS27-F: 5'-ATGGCCGCCGCCGCCTCCAGCT-3';

[0014] TPS27-R: 5'-TCACACGAAATTCACGTACAGTG-3'.

[0015] Furthermore, compared with the wild-type strain, the strain overexpressing the SiTPS27 gene had a reduced number of rust summer spores on millet leaves 14 days after millet rust infection, a smaller proportion of leaf area, and a lower severity, thereby improving millet's rust resistance.

[0016] Furthermore, under non-inoculated conditions, the strain overexpressing the SiTPS27 gene showed no significant changes in plant height and 1000-grain weight phenotypes compared with the wild-type strain.

[0017] Compared with the prior art, the advantages and positive effects of the present invention are:

[0018] The present invention connects the full-length CDS of the SiTPS27 gene to the pTCK303 expression vector initiated by Ubi, and uses Agrobacterium to infect and transform millet. It is experimentally verified that the overexpression of the SiTPS27 gene can reduce the number of rust fungus summer spores on the leaves of the millet transgenic strain on the 14th day after inoculation with millet rust fungus, reduce the proportion of leaf area, and reduce the severity, thereby improving the rust resistance of millet. The SiTPS27 gene belongs to the TPS family gene unique to plants. In view of the phenotype presented by the SiTPS27 gene in transgenic millet, it can be considered that the gene has important theoretical significance and potential application value for improving the disease resistance of millet. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure is a schematic diagram of the constructed overexpression vector and the restriction sites. The expression vector is pTCK303, the promoter is Ubi, and the restriction sites at both ends of the target gene SiTPS27 are KpnⅠ and SpeⅠ respectively.

[0020] Figure 2 A is a schematic diagram of cloning the target gene SiTPS27, which is 1653 bp in size; Figure 2 B is the positive clone obtained by connecting the target gene to the pTCK303 expression vector.

[0021] Figure 3 To identify the expression level of positive seedlings of transgenic lines.

[0022] Figure 4 A is a schematic diagram of the leaves of the transgenic line Ubi::SiTPS27 and the wild type Ci846 on the 14th day after inoculation with rust fungi; Figure 4 B is the statistical data of leaf severity of transgenic lines Ubi::SiTPS27 and wild type Ci846 on the 14th day after inoculation with rust fungus.

[0023] Figure 5 A and 5B are schematic diagrams of transgenic line Ubi::SiTPS27 and wild-type Ci846 plants and grain sizes, respectively; Figure 5 C and 5D are the statistical data of plant height and 1000-grain weight of transgenic line Ubi::SiTPS27 and wild type Ci846, respectively. DETAILED DESCRIPTION

[0024] 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.

[0025] Example 1: Acquisition of SiTPS27 transgenic strain

[0026] 1. Sequence cloning and vector construction of foxtail millet SiTPS27 gene:

[0027] The present invention uses the foxtail millet reference genome database phytozome (https: / / phytozome-next.jgi.doe.gov / info / Sitalica_v2_2) to find the gene SiTPS27. The full length of the SiTPS27 gene sequence is 2621 bp (the sequence is shown in SEQ ID NO.1), the CDS sequence is 1635 bp (the sequence is shown in SEQ ID NO.2), and the sequence of the SiTPS27 protein encoded by it is shown in SEQ ID NO.3.

[0028] Primers were designed based on the CDS sequence of the SiTPS27 gene for cloning. The cloning method was as follows:

[0029] (1) RNA extraction: The total RNA of millet was extracted using a plant tissue RNA rapid extraction kit (Tian Gen #DP452).

[0030] (2) Synthesis of the first strand of reverse transcribed cDNA: The extracted RNA was dissolved and the RNA concentration was determined, and then reverse transcription was performed using the Fastking cDNA First Strand Synthesis Kit (Tiangen #KR116).

[0031] Take 3 μg of total RNA and 5×gDNA Buffer, incubate at 42°C for 3 min to remove genomic DNA; add 10×King RT Buffer 2 μL, FastKing RT Enzyme Mix 1 μL, and FQ-RT PrimerMix 2 μL to the gDNA removal tube, add water to 20 μL, incubate at 42°C for 15 min, and inactivate at 95°C for 3 min to obtain cDNA solution.

[0032] (3) Cloning of SiTPS27 gene:

[0033] Upstream primer 5'- GGTACC ATGGCCGCCGCCGCCTCCAGCT-3' (SEQ ID NO.4);

[0034] Downstream primer 5'- ACTAGTTCACACGAAATTCACGTACAGTG-3' (SEQ ID NO. 5);

[0035] The underlined parts are restriction sites. The restriction site of the upstream primer is KpnⅠ, and the restriction site of the downstream primer is SpeⅠ.

[0036] The cDNA of the leaves of Scutellaria baicalensis infected with grain rust was used as a template and amplified using Vazyme#P520. 50μL PCR reaction system: 2×Phanta Flash Master Mix (Dye Plus) 25μL, template DNA 2μL, 10μmol / L upstream and downstream primers 2μL each, sterilized ddH2O 19μL. PCR amplification program: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 56℃ annealing for 5s, 72℃ extension for 5s, 35 cycles; finally 72℃ extension for 1min.

[0037] After the reaction, the target band ( Figure 2 A) Cut and recycle the rubber.

[0038] (4) Take 3 μL of the gel recovery product and connect it with the pTCK303 expression vector initiated by Ubi after enzyme digestion ( Figure 1 ), and the operation steps were carried out according to the instructions of ClonExpress Ultra One Step Cloning Kit (Novozyme #C115). The ligation product was transformed into Escherichia coli DH5α competent cells using the heat shock method and grown overnight on LB plates containing kanamycin (Kan+). A single white colony was picked and cultured in LB liquid medium (Kan+) with shaking overnight, and colony PCR was performed using DNA polymerase (Kangwei Century #CW0690) ( Figure 2 B), PCR detection 20μL system: 2×Es Taq MasterMix (Dye) 10μL, template DNA 2μL, 10μmol / L upstream and downstream primers 0.5μL each, sterile ddH2O 7μL. PCR amplification program: 94℃ pre-denaturation 10min; 94℃ denaturation 30s, 56℃ annealing 30s, 72℃ extension 1min, 35 cycles; finally 72℃ extension for 5min.

[0039] (5) Sequence determination: The bacterial culture of the positive clones was sent to Shanghai Biotechnology Co., Ltd. for sequence determination.

[0040] 2. Genetic transformation of foxtail millet SiTPS27 and screening of homozygous transgenic lines

[0041] The positive clones with correct sequencing were expanded in LB liquid medium (Kan+), and plasmid DNA was extracted using a high-purity plasmid extraction kit (MeiGen Biotechnology #P1001).

[0042] Take 2 μL of plasmid and transform it into Agrobacterium EHA105 to infect millet embryonic callus cells. Millet embryonic callus cells come from the wild-type millet variety Ci846. Through hygromycin screening, the overexpression material of Ubi::SiTPS27 / Ci846 was obtained. Through Real-time PCR detection, three stable overexpression transgenic materials #8, #12, and #23 were obtained.

[0043] Example 2: SiTPS27 affects plant resistance to rust

[0044] I. Rust resistance phenotype of transgenic lines overexpressing SiTPS27

[0045] 1. Using wild-type foxtail millet Ci846 as a control, the expression of SiTPS27 gene was detected in the homozygous transgenic material Ubi::SiTPS27. The results are as follows Figure 3 As shown, compared with the wild type Ci846, the expression levels of SiTPS27 gene in transgenic materials #8, #12, and #23 were significantly increased, proving that the present invention has indeed obtained the millet transgenic strains overexpressing SiTPS27.

[0046] 2. The millet transgenic line Ubi::SiTPS27 and the wild-type millet Ci846 were cultivated under the same conditions and inoculated with millet rust at the 6-leaf stage. The summer spores on the diseased leaves of the transgenic line Ubi::SiTPS27 and the wild-type Ci846 were observed and counted on the 14th day after inoculation. The results are as follows: Figure 4 As shown, compared with the wild type, the number of rust uredia on the leaves of the millet transgenic lines decreased and the proportion of uredia in the leaf area became smaller, specifically, the proportion of uredia in the leaf area decreased by about 37%.

[0047] 2. Agronomic phenotypes of transgenic lines overexpressing SiTPS27

[0048] The plant height and grain size of the mature transgenic strains and the wild type Ci846 were observed and compared, and the plant height and thousand-grain weight were measured and counted. Figure 5 As shown, the plant height and 1000-grain weight of the transgenic lines overexpressing SiTPS27 were similar to those of the wild type, with no significant changes.

[0049] The above results indicate that the SiTPS27 gene can significantly reduce the number of rust fungus summer spores on millet leaves without affecting millet agronomic traits such as plant height and 1000-grain weight. Therefore, the SiTPS27 gene is of great significance in improving millet resistance to rust.

[0050] 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. An application of the SiTPS27 gene for regulating millet rust resistance, wherein the nucleotide sequence of the SiTPS27 gene is shown in SEQ ID NO.1; the CDS nucleotide sequence of the SiTPS27 gene is shown in SEQ ID NO.2; the amino acid sequence of the SiTPS27 protein encoded thereby is shown in SEQ ID NO.3; characterized in that: The SiTPS27 gene is used to improve millet's resistance to rust.

2. The use according to claim 1, characterized in that: The overexpression vector containing the SiTPS27 gene was transformed into millet to obtain millet lines with improved rust resistance.

3. The use according to claim 2, characterized in that: The overexpression vector containing the SiTPS27 gene is obtained by extracting millet RNA, reverse transcribing the RNA into cDNA, cloning the gene, and then connecting it with the pTCK303 expression vector started by Ubi.

4. The use according to claim 3, characterized in that: The primer sequences for gene cloning are: TPS27-F: 5′-ATGGCCGCCGCCGCCTCCAGCT-3′; TPS27-R: 5′-TCACACGAAATTCACGTACAGTG-3′.

5. The use according to claim 1, characterized in that: Compared with the wild-type strain, the strain overexpressing the SiTPS27 gene had fewer summer spores on the millet leaves 14 days after inoculation with millet rust, a smaller proportion of the leaf area, and lower severity, thereby improving the rust resistance of millet.

6. The use according to claim 1, characterized in that: Compared with the wild-type strain, the plant height and 1000-grain weight of millet in the strain overexpressing the SiTPS27 gene did not change significantly.

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