Application of StPIF4 gene in regulation and control of heat resistance of potatoes

Abstract

The invention provides application of an StPIF4 gene in regulation and control of heat resistance of potatoes, the CDS sequence length of the StPIF4 gene is 1554 bp, the sequence is as shown in SEQ ID No.1, 517 amino acids are encoded, and the sequence is as shown in SEQ ID No.2, the gene is located in a cell nucleus, the heat resistance of the potatoes is positively regulated and controlled, and oxidative damage induced by high temperature is effectively relieved by positively regulating and controlling an antioxidant enzyme system, so that the heat resistance of the potatoes is enhanced. A new target gene and a theoretical basis are provided for analyzing a potato heat-resistant molecular mechanism and improving heat-resistant breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic engineering technology and potato breeding technology, and particularly relates to application of StPIF4 gene in regulating potato heat tolerance. BACKGROUND

[0002] The growth and development of plants are limited by various environmental stresses in nature, and high temperature is a major environmental stress, which poses a significant threat to crop production and seriously inhibits the yield of potato (Solanum tuberosum L.). Potato is an important major food crop in the world and originates from the relatively cool areas of the Andes in South America, which is highly sensitive to high temperature. With the continuous rise in air temperature, the growth and yield of potato plants are significantly affected. Therefore, exploring the molecular mechanism of plant resistance to high temperature and cultivating heat-resistant high-yield potato varieties are of great importance to alleviate the adverse effects of high temperature on potato production.

[0003] One of the effects of heat stress on plants is to increase the production of reactive oxygen species (ROS), such as O 2– and H2O2. These molecules are rapidly produced after the onset of heat stress, thus serving as early messengers to activate heat stress responses and further regulate the expression of transcription factors and key heat-resistant genes. High temperature stress affects the expression of genes in plants, and a large number of heat stress-responsive genes have been reported. For example, the removal of H2O2 enhances the heat tolerance of plants, and mutations in the genes encoding ROS-producing oxidase homologs, such as AtRBOHB and AtRBOHD, result in defective heat tolerance of plants; high temperature stress leads to increased cytoplasmic membrane fluidity, activates the calcium signaling transduction pathway, and Ca 2+ can activate the SYTA transmembrane protein, which can repair damaged cytoplasmic membrane sites with the help of Ca 2+ and activate the expression of HSFs and HSPs.

[0004] The plant phytochrome interacting factor (PIF) family belongs to the 15th subfamily of basic helix-loop-helix (bHLH) transcription factor family, and is a key factor connecting light, temperature and other environmental signals. Some researchers have proved that the structural adaptation induced by high temperature is mediated by the bHLH transcriptional regulator phytochrome interacting factor 4 (PIF4). Under high environmental temperature, plants show significant stem elongation in the adaptive response to heat. This response is mediated by the increased level of plant hormone auxin, and requires the biosynthesis, signaling and transport pathways of auxin. It is reported that PIF4 binds to the CACATG variant of the E-box motif, which exists in the promoters of LONGIFOLIA 1 (LNG 1) and LNG 2 genes, and increases their expression at warm temperatures. The increased LNG proteins further activate the expression of YUC 8 and IAA29 through unknown mechanisms, and the resulting increase in auxin biosynthesis and signaling induces heat morphogenic growth.

[0005] Although PIF plays an important role in integrating various environmental signals, the mechanism of PIF4 in regulating growth at high temperature is still unclear. There is no report on PIF4 in potato, especially on the function of PIF4 in regulating the high temperature tolerance of potato. Therefore, exploring the function and mechanism of PIF4 in potato in response to high temperature stress is of great significance for revealing the molecular mechanism of heat tolerance of potato. SUMMARY

[0006] The inventors screened the potato PIFs family member StPIF4 that has a significant response to high temperature from the PGSC database environmental stress transcriptome data of potato. The gene is induced by high temperature stress, and the promoter activity of the gene increases after high temperature treatment. The inventors further constructed StPIF4 transgenic lines overexpressing and interfering StPIF4 gene, and subjected the StPIF4 transgenic lines and wild type to high temperature treatment. The results show that the heat tolerance of the overexpression lines is better than that of WT, and the interference lines are the opposite, indicating that overexpression of StPIF4 gene can enhance the heat tolerance of potato. The inventors further performed active oxygen staining and related index determination, and the results show that the active oxygen scavenging capacity of the overexpression lines is higher than that of WT after high temperature, and the interference lines are the opposite, indicating that StPIF4 enhances the heat tolerance of potato by regulating the active oxygen scavenging capacity under high temperature.

[0007] Therefore, the purpose of the present application is to provide the application of StPIF4 gene in regulating the heat tolerance of potato.

[0008] In order to achieve the above purpose, the present application provides the following technical scheme:

[0009] The application provides application of a StPIF4 gene or a coded protein thereof in regulating heat resistance of potatoes, wherein a CDS sequence of the StPIF4 gene is shown as SEQ ID No. 1, and an amino acid sequence of the coded protein is shown as SEQ ID No. 2.

[0010] Specifically, the application is to enhance the heat resistance of the potatoes by means of genetic engineering technology to make the StPIF4 gene overexpress.

[0011] The application also provides a method for enhancing the heat resistance of the potatoes, which comprises making the StPIF4 gene overexpress by means of genetic engineering technology to enhance the heat resistance of the potatoes.

[0012] The application also provides a preparation for enhancing the heat resistance of the potatoes, which comprises a reagent capable of promoting overexpression of the StPIF4 gene.

[0013] The application also provides application of the StPIF4 gene, the StPIF4 gene coded protein and a recombinant expression vector of the StPIF4 gene in breeding of the potatoes.

[0014] The application also provides a method for creating the heat-resistant potatoes, which comprises making the StPIF4 gene overexpress by means of genetic engineering technology to obtain the potato mutant with enhanced heat resistance.

[0015] Further, the method comprises cloning the CDS sequence of the StPIF4 gene into a pCAMBIA1300 vector to obtain a 35S::StPIF4-GFP recombinant vector, and then using an agrobacterium-mediated potato slice genetic transformation to obtain the potato mutant with overexpression of the StPIF4 gene and enhanced heat resistance.

[0016] The application has the beneficial effects that the application provides application of the StPIF4 gene in regulating the heat resistance of the potatoes, the CDS sequence of the StPIF4 gene is 1554 bp long, the sequence is shown as SEQ ID No. 1, the gene encodes 517 amino acids, the sequence is shown as SEQ ID No. 2, the gene is located in the nucleus, positively regulates the heat resistance of the potatoes, positively regulates the antioxidant enzyme system, effectively alleviates the oxidative damage induced by high temperature, and thus enhances the heat resistance of the potatoes. This provides a new target gene and theoretical basis for analyzing the heat resistance molecular mechanism of the potatoes and heat resistance breeding improvement. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a sequence feature analysis of StPIF4; wherein A is a phylogenetic tree analysis; and B is an amino acid sequence alignment analysis.

[0018] Figure 2Figure 1 is StPIF4 expression pattern analysis; wherein A is qRT-PCR identification of StPIF4 tissue expression; B is proStPIF4: :GUS transgenic line identification; C is GUS staining of proStPIF4: :GUS transgenic line after 42°C high temperature treatment for 24 h; D is subcellular localization of StPIF4.

[0019] Figure 3 Figure 2 is StPIF4 transgenic line identification; wherein A is agarose gel electrophoresis identification of interference transgenic plants using vector primers; B is interference efficiency detection of StPIF4 interference transgenic lines; C is agarose gel electrophoresis identification of overexpression transgenic plants using vector primers; D is overexpression efficiency detection of StPIF4 overexpression transgenic lines.

[0020] Figure 4 Figure 3 is phenotype of wild type and StPIF4 transgenic lines after normal temperature treatment and 42°C high temperature treatment for 48 h; wherein A is phenotype of wild type and StPIF4 overexpression transgenic lines after normal temperature treatment and 42°C high temperature treatment for 48 h; B is relative conductivity analysis of StPIF4 overexpression lines; C is Fv / Fm analysis of StPIF4 overexpression lines; D is phenotype of wild type and StPIF4 interference transgenic lines after normal temperature treatment and 42°C high temperature treatment for 48 h; E is relative conductivity analysis of StPIF4 interference lines; F is Fv / Fm analysis of StPIF4 interference lines.

[0021] Figure 5 Figure 4 is determination of reactive oxygen species content of WT and StPIF4 overexpression transgenic plants before and after high temperature treatment in seedling stage; wherein A, B, C are DAB, NBT and Trpanblue staining of wild type and StPIF4 overexpression lines after normal temperature treatment and 42°C high temperature treatment for 48 h; D is determination of POD content of wild type and StPIF4 overexpression lines after normal temperature treatment and 42°C high temperature treatment for 48 h; E is determination of SOD content of wild type and StPIF4 overexpression lines after normal temperature treatment and 42°C high temperature treatment for 48 h; F is determination of CAT content of wild type and StPIF4 overexpression lines after normal temperature treatment and 42°C high temperature treatment for 48 h; G is determination of MDA content of wild type and StPIF4 overexpression lines after normal temperature treatment and 42°C high temperature treatment for 48 h.

[0022] Figure 6Determination of active oxygen content of WT and StPIF4 interference transgenic plant seedlings before and after high temperature treatment. A, B and C are DAB, NBT and Trpanblue staining of wild type and StPIF4 interference lines after 42°C high temperature treatment for 48 h; D is the determination of POD content of wild type and StPIF4 interference lines after 42°C high temperature treatment for 48 h; E is the determination of SOD content of wild type and StPIF4 interference lines after 42°C high temperature treatment for 48 h; F is the determination of CAT content of wild type and StPIF4 interference lines after 42°C high temperature treatment for 48 h; G is the determination of MDA content of wild type and StPIF4 interference lines after 42°C high temperature treatment for 48 h. DETAILED DESCRIPTION

[0023] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively described and explained the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized on the basis of the above description of the present application is covered within the scope intended to be protected by the present application. It should be noted that the experimental materials not mentioned in the examples of the present application are commercially available, and the experimental methods not mentioned in the specific conditions in the examples of the present application are usually carried out according to the conventional experimental methods or according to the recommended methods of the experimental material manufacturers. It should be noted that the potato E3 in the present application is the No. 3 potato variety in Hubei, and AT is the Atlantic potato variety, and WT represents the wild type.

[0024] Example 1 Cloning and sequence characteristic analysis of StPIF4

[0025] The inventors screened StPIF4, a member of PIFs family in potato, which has obvious response to high temperature from the PGSC database of environmental stress transcriptome data.

[0026] The sequence cloning analysis of StPIF4 was carried out with potato E3 leaf cDNA as template. The sequence cloning analysis results showed that the CDS sequence of StPIF4 gene was 1554 bp long, the nucleotide sequence was shown as SEQ ID No. 1, and it encoded 517 amino acids, and the amino acid sequence was shown as SEQ ID No. 2. The phylogenetic tree analysis of StPIF4 showed that the genetic distance of StPIF4 protein and the homologous protein of Solanum lycopersicum was closer (0.000) than that of Arabidopsis thaliana (0.005), Capsicum annuum (0.005) and Solanum melongena (0.005). Figure 1A). Sequence alignment with homologous proteins of Arabidopsis and congeneric plants showed that StPIF4 protein sequence has a conserved bHLH domain (Figure 1) Figure 1 B).

[0027] Example 2 StPIF4 expression pattern analysis

[0028] qRT-PCR expression analysis was performed on different tissues of potato E3, and the results of different tissue expression analysis showed that StPIF4 had the highest expression in leaves and lateral stems (Figure 2) Figure 2 A).

[0029] To explore the potential function of StPIF4 in high temperature stress response, a StPIF4::GUS reporter gene vector was constructed and transgenic potato plants were obtained by Agrobacterium-mediated genetic transformation (Figure 3) Figure 2 B). After PCR identification to determine positive lines, GUS chemical staining analysis was performed on leaves of transgenic potato after 24 h of high temperature stress at 42°C, and the results showed that GUS staining signal was enhanced after high temperature stress, indicating that the StPIF4 promoter activity was induced by high temperature stress, suggesting that the gene may be involved in the high temperature stress response regulation pathway of potato (Figure 4) Figure 2 C).

[0030] To explore the cellular localization of StPIF4 protein, pCAMBIA1300-StPIF4-eGFP vector with green fluorescent marker (eGFP) was used to infect tobacco for transient expression after being transformed into Agrobacterium, and the experimental results showed that StPIF4 protein was localized in the nucleus (Figure 5) Figure 2 D).

[0031] Example 3 StPIF4 transgenic line screening and identification

[0032] To further analyze the biological function of StPIF4 in potato high temperature stress response, StPIF4 overexpression vector was constructed, and the CDS of StPIF4 was cloned into pCAMBIA1300 vector to obtain 35S::StPIF4-GFP recombinant vector; the specific segment of StPIF4 CDS was cloned into pHELLSGATE-8 vector to obtain interference vector. Using AT and E3 as receptors, 3 overexpression lines (OE-6, OE-20, OE-24) and 3 RNAi lines (RNAi-2, RNAi-4, RNAi-12) were obtained by Agrobacterium-mediated potato slice genetic transformation, and positive identification and efficiency identification were performed by PCR and qRT-PCR, respectively (Figure 6)A-D). Figure 3

[0033] Example 4 Phenotype identification

[0034] (1) StPIF4 enhances the heat resistance of potato seedlings

[0035] To further elucidate the biological function of StPIF4 in the potato response to high-temperature stress, StPIF4 transgenic lines were subjected to high-temperature treatment. After 4 weeks of normal culture at 22℃, wild-type and StPIF4 transgenic lines were transferred to a 42℃ high-temperature stress environment for 48 h. Phenotypic analysis showed that under normal temperature conditions, there was no significant difference in growth status between wild-type and StPIF4 transgenic lines. After high-temperature stress, both wild-type (AT) and overexpression lines exhibited significant wilting, but compared with wild-type (AT) plants, the wilting degree of the overexpression lines was significantly reduced, demonstrating stronger heat tolerance. Figure 4 A). Conversely, RNAi lines exhibited a more severe heat-sensitive phenotype, with significantly increased leaf wilting compared to the wild type (E3). Figure 4 D).

[0036] Further measurements of the relative electrical conductivity of leaves before and after treatment showed that there was no significant difference in the relative electrical conductivity between wild-type AT and StPIF4 transgenic lines at room temperature. However, after 48 h of high-temperature stress treatment at 42℃, the relative electrical conductivity of leaves increased for both wild-type AT and overexpression transgenic lines, but the relative electrical conductivity of the overexpression lines was significantly lower than that of wild-type AT. Figure 4 B). The relative conductivity of both wild-type E3 and RNAi lines increased after the same high-temperature treatment, but unlike the over-treated lines, the relative conductivity of the interference lines was significantly higher than that of wild-type E3. Figure 4 E). Fv / Fm measurements showed that high-temperature treatment inhibited photosynthetic activity in plants, and the Fv / Fm values ​​of different potato genotypes decreased. However, the Fv / Fm value of the StPIF4 overexpression lines was significantly higher than that of the wild type. Figure 4 C), while RNAi strains compared to wild-type ( Figure 4 (F) The photosynthetic system is more damaged under high temperatures, and the Fv / Fm value decreases significantly. These results indicate that StPIF4 acts as a positive regulator in the heat stress response process of potatoes.

[0037] (2) StPIF4 enhances heat resistance by reducing the accumulation of reactive oxygen species.

[0038] To investigate the regulatory role of StPIF4 in ROS metabolism under high temperature stress, DAB, NBT, and trypan blue histochemical staining analyses were performed on leaves of wild-type and transgenic lines subjected to both normal temperature (22℃) and high temperature treatment (42℃ / 48h). The results showed no significant difference in staining intensity among genotypes under normal temperature conditions. Figure 5 AC, Figure 6A-C); after high temperature stress, the staining degree of all materials was significantly enhanced, but the DAB ( Figure 5 A), NBT ( Figure 5 B) and trypan blue staining ( Figure 5 C) of StPIF4 overexpression lines were significantly lower than that of wild type, while the RNAi lines showed darker staining signals ( Figure 6 A-C). Enzyme activity assays showed that there was no significant difference between wild type and StPIF4 transgenic lines at normal temperature, while the activities of POD ( Figure 5 D), SOD ( Figure 5 E) and CAT ( Figure 5 F) of overexpression lines were significantly higher than that of wild type after high temperature treatment; on the contrary, the activities of RNAi lines were significantly lower than that of wild type ( Figure 6 D-F). In addition, MDA content analysis showed that there was no significant difference between wild type and StPIF4 transgenic lines at normal temperature, while the MDA content of overexpression lines was significantly lower than that of wild type (AT) after high temperature treatment ( Figure 5 G), while the RNAi lines were significantly higher ( Figure 6 G). The above results showed that StPIF4 effectively alleviated the oxidative damage induced by high temperature by positively regulating the antioxidant enzyme system, thereby enhancing the heat tolerance of potato.

[0039] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The application of the StPIF4 gene or its encoded protein in regulating potato heat tolerance, wherein the CDS sequence of the StPIF4 gene is shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID No.

2.

2. The application according to claim 1, characterized in that, The application involves using genetic engineering techniques to overexpress the StPIF4 gene, thereby enhancing the heat resistance of potatoes.

3. A method for enhancing the heat resistance of potatoes, characterized in that, The method includes using genetic engineering techniques to overexpress the StPIF4 gene, thereby enhancing the heat resistance of potatoes.

4. A preparation for enhancing the heat resistance of potatoes, characterized in that, The formulation includes a reagent that promotes the overexpression of the StPIF4 gene.

5. Application of the StPIF4 gene, the StPIF4 gene-encoded protein, and the recombinant expression vector of the StPIF4 gene in potato heat resistance improvement breeding.

6. A method for creating heat-resistant potatoes, characterized in that, The method involves using genetic engineering techniques to overexpress the StPIF4 gene, thereby obtaining a potato mutant with enhanced heat resistance.

7. The method according to claim 6, characterized in that, The method involves cloning the CDS sequence of the StPIF4 gene into the pCAMBIA1300 vector to obtain a 35S::StPIF4-GFP recombinant vector, and then using Agrobacterium-mediated potato chip genetic transformation to obtain a potato mutant with overexpression of StPIF4 and enhanced heat resistance.

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