Application of tomato histone variant H2A. Z in improvement of low-temperature resistance of tomatoes

Abstract

The invention discloses an application of a tomato histone variant H2A. Z in improving low-temperature resistance of tomatoes, and is characterized in that the histone variant H2A. Z is coded by three genes of S1HTA8, S1HTA9 and S1HTA11, the nucleotide sequence of the S1HTA9 is as shown in SEQ ID NO: 1, and the nucleotide sequence of the S1HTA11 is as shown in SEQ ID NO: 2. According to the application, the genes of the S1HTA9 and the S1HTA11 for coding the histone H2A. Z are knocked out through a gene knockout technology, and the tomato histone variant H2A. Z is obtained. The low-temperature resistance of the tomatoes is obviously improved. The invention discovers that the histone variant H2A. Z participates in regulating the low-temperature resistance of plants for the first time, provides a new gene resource for cultivating a new variety of low-temperature-resistant tomatoes, and has important potential application value.

Description

Technical Field

[0001] The present application relates to the fields of genetic engineering, molecular biology and physiology, and specifically to the application of a tomato histone variant H2A.Z in improving the low temperature resistance of tomatoes. Background Art

[0002] tomato( Solanum lycopersicum ) is native to the tropical regions of South America. It is one of the most widely cultivated vegetable crops in the world and an important thermophilic crop in my country's greenhouse vegetable cultivation. However, with the frequent occurrence of extreme climate phenomena around the world, and the common problems in tomato facility production such as low facility equipment level and poor controllability of temperature and light environment, tomatoes often suffer from low temperature damage in winter and spring, resulting in poor growth and development, decreased fruit yield and quality. Therefore, studying the key regulatory factors of tomato's low temperature response will not only help to reveal its temperature-responsive molecular network, but also provide important theoretical support for improving tomato yield and quality and reducing economic losses caused by low temperatures.

[0003] Epigenetic regulation plays an important role in plant response to stress, among which histone variant replacement is one of the important contents in the field of epigenetic research. In addition to the four conventional histones H2A / H2B / H3 / H4 in the plant genome, there is also a class of histone variants whose amino acid sequences are different from conventional histones and whose biological functions are also different. The histone variant H2A.Z is one of the variants of H2A, which is replaced with H2A through ATP-dependent chromatin remodeling factors (such as the SWR1 complex), thereby changing the structure of the nucleosome and the accessibility of chromatin. The enrichment of H2A.Z in different gene regions affects gene transcription, thereby regulating a variety of important physiological pathways (such as plant development, flowering and environmental response, etc.) and plays an important regulatory role. For example, in Arabidopsis, the enrichment of H2A.Z at nucleosome sites is reduced under high temperature stress, which promotes HSP70 and HSFA1 The expression of genes in response to high temperature stress [SV Kumar et al., "H2A.Z - Containing Nucleosomes Mediate theThermosensory Response in Arabidopsis." Cell, 2010, 140(1): 136-147]; Under salt stress, Arabidopsis activates the expression of genes by reducing the enrichment of H2A.Z. AtMYB44transcription, and further regulates salt stress response [NH Nguyen et al., "H2A.Z-containing nucleosomes are evicted to activate AtMYB44 transcription in response to salt stress." Biochemical and Biophysical Research Communications, 2018, 499 (4): 1039-1043]; in addition, under drought stress, the enrichment of H2A.Z in the coding region of genes significantly inhibits the transcription level of drought stress response genes [W Sura et al., "Dual Role of the Histone Variant H2A.Z in Transcriptional Regulation of Stress-Response Genes." The Plant Cell, 2017, 29 (4): 791-807].

[0004] Although the important role of epigenetic factors in plant stress response has been widely studied, the research on its role in tomato low temperature resistance pathway is still limited, especially the role and regulation mechanism of histone variant H2A.Z in tomato low temperature stress have not been reported. SUMMARY

[0005] Therefore, the embodiment of the present application provides a method for knocking out tomato histone variant H2A.Z to improve the low temperature tolerance of tomato.

[0006] In order to achieve the purpose of the present application, the technical solution adopted by the present application is as follows: The application of tomato histone variant H2A.Z in improving the low temperature resistance of tomato, wherein the histone variant H2A.Z is encoded by SlHTA8, SlHTA9, SlHTA11 three genes, wherein, SlHTA9 the nucleotide sequence is shown as SEQ ID NO: 1, SlHTA11 the nucleotide sequence is shown as SEQ ID NO: 2, and the application is to knock out the gene encoding histone H2A.Z by gene knockout technology SlHTA9 and SlHTA11 so as to improve the low temperature resistance of tomato.

[0007] Further, the SlHTA9, SlHTA11 encoded H2A.Z protein amino acid sequence is shown as SEQ ID NO: 3 and SEQ ID NO: 4.

[0008] Further, the gene knockout technology is specifically as follows: knocking out the gene encoding histone H2A.Z in tomatoSlHTA9 、 SlHTA11 The target fragment containing the protospacer sequence adjacent to the PAM structure was selected from the protein coding region, the corresponding primers were designed, and the CRISPR / Cas9 recombinant expression vector was constructed; The CRISPR / Cas9 recombinant expression vector was introduced into Agrobacterium, which was then used to infect tomato cotyledons. Positive transgenic plants were screened, and single mutant purified lines were obtained by self-pollination, and double mutants were obtained by hybridization. sl_h2a.z , sl_h2a.z By improving the cold response gene of tomato SlCBFs expression, thereby improving the low temperature resistance of tomatoes.

[0009] Furthermore, the recombinant expression vector is pCAMBIA1301-U6-26-sgRNA-SlHTA9-35S-cas9 or pCAMBIA1301-U6-26-sgRNA-SlHTA11-35S-cas9.

[0010] Furthermore, the SlHTA9, SlHTA11 The nucleotide sequences of the first 20 bases containing the protospacer sequence adjacent to the PAM structure motif are shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0011] Furthermore, the sl_h2a.z By increasing the cold response genes SlCBF1, SlCBF2, SlCBF3 The transcription level of the gene is expressed in 1777kJ / mL and 1777kJ / mL, thereby improving

[0012] The technical solutions provided by the embodiments of the present application may have the following beneficial effects: As can be seen from the above examples, this application first reveals the regulatory role of histone variant H2A.Z in tomato low temperature stress. sl_h2a.z The double mutant was selected and its low temperature resistance function was studied in depth. sl_h2a.z The knockout plants exhibited a significant cold-tolerance phenotype. This discovery, the first to identify the histone variant H2A.Z as involved in regulating cold resistance, provides a new genetic resource for breeding new cold-tolerant tomato varieties and has significant potential applications. Furthermore, this discovery lays a theoretical foundation for understanding the molecular mechanisms of tomato responses to stress signals and tolerance to adverse environments.

[0013] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0015] Figure 1 The tomato in Example 1 of the present invention sl_h2a.z Sequencing results of sgRNA sequences of gene knockout lines.

[0016] Figure 2 The wild-type tomato and sl_h2a.z The double mutant plants were grown at room temperature (25 o C) and low temperature (4 o C) Phenotype after 7 days of treatment.

[0017] Figure 3 The wild type tomato and sl_h2a.z The double mutant plants were grown at room temperature (25 o C) and low temperature (4 o C) Relative electrolyte permeability after 7 days of treatment.

[0018] Figure 4 The wild type tomato and sl_h2a.z The double mutant plants were grown at room temperature (25 o C) and low temperature (4 o C) Maximum photochemical efficiency of photosystem II (Fv / Fm) after 7 days of treatment.

[0019] Figure 5 For the wild type and sl_h2a.z Crispr / Cas9 gene knockout plants were grown at room temperature (25 o C) and low temperature (4 o C) Expression levels of cold-resistant genes after 6 h of treatment, where A represents tomato SlCBF1 Gene expression level, B is tomato SlCBF2 Gene expression, C is tomato SlCBF3 Gene expression level. DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to specific embodiments. It should be understood that the following are only specific embodiments of the present invention, and the scope of protection of the present invention is not limited thereto.

[0021] Unless otherwise indicated, the present invention will be practiced using conventional botanical techniques, tissue culture, molecular biology, physiology and biochemistry, and bioinformatics techniques familiar to those skilled in the art. The relevant techniques are fully explained in the literature, and all experimental reagents and materials used are commercially available.

[0022] Example 1: Tomatosl_h2a.z Construction of CRISPR / Cas9 gene knockout vector and acquisition of double mutant homozygous plants.

[0023] Find the gene encoding the histone variant H2A.Z on the SGN website (http: / / solgenomics.net / ) SlHTA9, SlHTA11 The full-length DNA sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0024] Site Design Using CRISPR-P SlHTA9, SlHTA11 The sgRNA sequence was synthesized, and the sgRNA forward and reverse primers were synthesized. The primers were annealed into double-stranded sgRNA containing sticky end adapters using a PCR instrument, and ligated with the vector AtU6-sgRNA-AtUBQ-Cas9 that had been digested with BbsI under the action of T4 ligase. The DH5α competent cells were heat-shocked and transformed. The positive single clone colonies were selected for testing. After the sequencing results were correct, the newly obtained AtU6-sgRNA-AtUBQ-Cas9 fragment was ligated to the pCAMBIA1301 vector that had been double-digested with Hind III / KpnI using T4 ligase. The competent cells were transformed into Escherichia coli DH5α, and single colonies were picked and placed in liquid LB medium containing 50 mg / L kanamycin. The cells were shaken and cultured at 37°C and 200 rpm overnight. The positive clones were verified by PCR and sequenced ( Figure 1 ).

[0025] The correctly sequenced gene editing vector was transformed into GV3101 Agrobacterium competent cells by electroporation, and tomato cotyledons were infected by the "leaf disc method" to induce callus. Hygromycin was used for resistance induction differentiation and rooting culture to initially obtain candidate transgenic plants. SlHTA9, SlHTA11 Specific primers were designed around the sequence position of the sgRNA of the gene, and 500bp were selected to detect the changes in the target gene sequence. Positive strains were obtained and homozygous strains were obtained by self-pollination. slhta9, slhta11 After the single mutant lines were hybridized, double mutants were obtained sl_h2a.z.

[0026] Example 2: sl_h2a.z Testing of low temperature resistance of genetically modified materials The tomato varieties used in the experiment were the wild type '1479' (WT) and the tomato obtained in Example 1. sl_h2a.z For the double mutant, seeds were sown in plug trays containing a 3:1 mixture of peat and vermiculite. Hoagland's nutrient solution was then watered to ensure the substrate was moist. When the tomatoes had five leaves and one heart, they were treated with a low-temperature treatment at 4°C. The control temperature was 25°C, and the photoperiod was 12 h.

[0027] The experiment set up four groups, WT normal temperature group, WT low temperature group,sl_h2a.z Normal temperature group, sl_h2a.z Low temperature group; the fourth leaf of tomato was taken at 0 h, 3 h, 6 h, 9 h and 24 h after the start of low temperature treatment for RNA extraction and RT-qPCR analysis of cold-resistant genes. After low temperature treatment, phenotype shooting, relative conductivity determination and maximum photochemical efficiency of photosystem II determination were performed.

[0028] The method for determining the maximum photochemical efficiency of photosystem II (Fv / Fm) of tomato was as follows: after dark adaptation for 30 min, the tomato plants were placed in the detection tray, and the chlorophyll fluorescence imager (IMAG-PAM) was used to determine the maximum quantum yield of PSII of tomato after 7 d of low temperature treatment.

[0029] The method for determining the relative conductivity of tomato was as follows: the same functional part of tomato leaves was taken, avoiding the leaf veins, and cut into strips of appropriate length. Fresh samples were quickly weighed in triplicate, 0.1 g each, and placed in 20 mL deionized water. The samples were extracted in a 28℃, 200 rpm shaking bed for 2 h. The conductivity value R1 of the extraction solution was determined using a conductivity meter. The samples were heated in a 95℃ hot water bath for 15 min, and the conductivity value R2 was measured after cooling to room temperature. The relative conductivity = R1 / R2*100%.

[0030] The method for real-time fluorescence quantitative PCR (RT-qPCR) was as follows: the Roche Light Cycler® 480ⅡReal-Time PCR detection system (Roche, Swiss) was used, and the SYBR Green RT-PCR Kit (Takara, RR420A) was used. The total reaction system was 20 μL, which contained 10 ul of fluorescent dye SYBR Green PCR MasterMix, 1 μL of cDNA template, 0.4 μL of forward primer, 0.4 μL of reverse primer, and 8.2 μL of dd H2O. The PCR reaction conditions were as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 10 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 40 cycles, and 72℃ extension for 5 min. The relative expression of the gene was analyzed by the delta-delta Ct method.

[0031] The results showed that, under low temperature conditions, compared with the control group WT, sl_h2a.z a significant cold-resistant phenotype was exhibited, the degree of wilting of the plants was lower ( Figure 2 ), the relative conductivity was lower ( Figure 3 ), the Fv / Fm value was higher ( Figure 4 ), and the cold-induced SlCBF1,SlCBF2, SlCBF3 The transcriptional level of the genes was higher in the plants overexpressing H2A.Z (Fig. 6B) Figure 5 , indicating that the histone variant H2A.Z negatively regulates the cold resistance of tomato.

[0032] Although the present application has been described in detail with general description and specific embodiments above, the present application is not limited to the above embodiments, but can have many variations or improvements, which will be apparent to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present application are within the scope of the present application claimed.

Claims

1. A use of a tomato histone variant H2A.Z in improving the low temperature resistance of tomatoes, characterized in that: The histone variant H2A.Z is composed of SlHTA8, SlHTA9, SlHTA11 Three genes encode, among which, SlHTA9 The nucleotide sequence is shown in SEQ ID NO: 1, SlHTA11 The nucleotide sequence is shown in SEQ ID NO:

2. The application is to knock out the gene encoding histone H2A.Z by gene knockout technology. SlHTA9 and SlHTA11 , which improves the low temperature resistance of tomatoes.

2. The use according to claim 1, characterized in that described SlHTA9、SlHTA11 The amino acid sequences of the encoded H2A.Z protein are shown in SEQ ID NO: 3 and SEQ ID NO:

4.

3. The use according to claim 2, characterized in that The gene knockout technology is specifically as follows: In tomato SlHTA9 、 SlHTA11 The target fragment containing the protospacer sequence adjacent to the PAM structure was selected from the protein coding region, the corresponding primers were designed, and the CRISPR / Cas9 recombinant expression vector was constructed; The CRISPR / Cas9 recombinant expression vector was introduced into Agrobacterium, which was then used to infect tomato cotyledons. Positive transgenic plants were screened, and single mutant purified lines were obtained by self-pollination, and double mutants were obtained by hybridization. sl_h2a.z , sl_h2a.z By improving the cold response gene of tomato SqF expression, thereby improving the low temperature resistance of tomatoes.

4. The use according to claim 3, characterized in that The recombinant expression vectors are pCAMBIA1301-U6-26-sgRNA-SlHTA9-35S-cas9 and pCAMBIA1301-U6-26-sgRNA-SlHTA11-35S-cas9.

5. The use according to claim 3, characterized in that described SlHTA9、SlHTA11 The nucleotide sequences of the first 20 bases containing the protospacer sequence adjacent to the PAM structure motif are shown in SEQ ID NO: 5 and SEQ ID NO:

6.

6. The use according to claim 3, characterized in that described sl_h2a.z By increasing the cold response genes SlCBF1, SlCBF2, SlCBF3 The transcription level of the gene is expressed in 1777kJ / mL and 1777kJ / mL, thereby improving

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