Application of AcTPS1 gene and related biological material thereof in regulation and control of plant drought resistance

Abstract

The invention discloses an application of an AcTPS1 gene and a related biological material thereof in regulation and control of plant drought resistance, and belongs to the technical field of gene engineering. The nucleotide sequence of the AcTPS1 gene disclosed by the invention is as shown in SEQ ID NO. 13. Researches find that the AcTPS1 gene can positively respond to drought stress and can increase the content of ABA, sucrose, glucose and trehalose in a plant body; the AcTPS1 gene is transferred into a plant body, so that the plant shows excellent drought resistance after stable overexpression of the AcTPS1 gene.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of AcTPS1 gene and related biological materials in regulating plant drought resistance. Background Art

[0002] Chinese kiwifruit is a woody vine of the Actinidiaceae family, native to China, and one of the most economically important fruits in the world. However, global climate change has led to uneven rainfall throughout the year and severe degradation of drylands, which has damaged the growing environment of Chinese kiwifruit and severely restricted the sustainable development of the Chinese kiwifruit industry.

[0003] Drought stress can adversely affect the growth, development, yield and fruit quality of Actinidia chinensis; the most significant effects of drought stress on plants are stunted growth, severe damage to the photosynthetic apparatus, reduced photosynthesis, reduced seed germination, reduced nutrient absorption, and reduced yield and quality. In order to maintain growth and productivity, plants must adapt to adverse conditions and exercise various tolerance mechanisms by activating and manipulating specific stress-related genes. These genes are involved in cell signaling pathways and cellular responses, such as transcriptome control and the production of stress proteins, including quaternary amines, amino acids, and polyols (such as trehalose). Trehalose is a non-reducing disaccharide that is widely present in various organisms. Among the various known compounds, trehalose plays an important role in regulating various biotic and abiotic stresses, including plants. The trehalose content in plants is usually low, but it may be changed by environmental stress. Transgenic plants overexpressing microbial trehalose-synthesizing genes have been shown to contain higher trehalose levels and show drought, salt and cold tolerance. And sugar plays an active role in the regulation of growth, photosynthesis, carbon allocation, carbohydrate and lipid metabolism, osmotic homeostasis, and protein synthesis and gene expression during various abiotic stresses. Trehalose-6-phosphate synthase (TPS) gene plays a key role in the synthesis and metabolism of trehalose. Overexpression of TPS gene can enhance the tolerance of plants to environmental stresses such as drought, cold, high salt, and pests and diseases. Therefore, studying the related genes in the synthesis and metabolism of trehalose will help accelerate the discovery and development of drought-resistant genes. However, there are currently few studies on the response of Chinese kiwifruit to adverse environments, and there is also little development of drought-resistant genes. Summary of the invention

[0004] The purpose of the present invention is to provide the application of AcTPS1 gene and related biological materials in regulating plant drought resistance to solve the problems existing in the above-mentioned prior art. The present invention has found that AcTPS1 gene can actively respond to drought stress and can increase the content of ABA, sucrose, glucose and trehalose in plants; after the AcTPS1 gene is transduced into the plant body and stably overexpressed, the plant shows excellent drought resistance.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the use of the AcTPS1 gene or its related biological materials, including any of the following uses:

[0007] A1. Regulate plant drought resistance;

[0008] A2. Cultivate plant varieties with strong drought resistance;

[0009] A3. Early identification of plant strains with high drought resistance;

[0010] The nucleotide sequence of the AcTPS1 gene is shown in SEQ ID NO.13;

[0011] The regulation refers to increasing the expression level of the AcTPS1 gene, which can enhance the drought resistance of plants.

[0012] Optionally, the relevant biological material includes any one of the following:

[0013] B1. An expression cassette comprising the AcTPS1 gene;

[0014] B2. A recombinant vector comprising the AcTPS1 gene;

[0015] B3. A recombinant microorganism comprising the AcTPS1 gene.

[0016] Optionally, the plants include Arabidopsis thaliana and Actinidia chinensis.

[0017] The present invention also provides a method for improving plant drought resistance, comprising the steps of using genetic transformation technology to transduce the AcTPS1 gene or its related biological materials into the plant body, so that the AcTPS1 gene is stably overexpressed, thereby improving the plant drought resistance;

[0018] The nucleotide sequence of the AcTPS1 gene is shown in SEQ ID NO.13.

[0019] Optionally, the relevant biological material includes any one of the following:

[0020] B1. An expression cassette comprising the AcTPS1 gene;

[0021] B2. A recombinant vector comprising the AcTPS1 gene;

[0022] B3. A recombinant microorganism comprising the AcTPS1 gene.

[0023] Optionally, the plants include Arabidopsis thaliana and Actinidia chinensis.

[0024] The present invention also provides a method for cultivating a plant variety with strong drought resistance, comprising using genetic transformation technology to transfer the AcTPS1 gene or its related biological materials into the plant body to construct a plant variety with strong drought resistance;

[0025] The nucleotide sequence of the AcTPS1 gene is shown in SEQ ID NO.13.

[0026] Optionally, the relevant biological material includes any one of the following:

[0027] B1. An expression cassette comprising the AcTPS1 gene;

[0028] B2. A recombinant vector comprising the AcTPS1 gene;

[0029] B3. A recombinant microorganism comprising the AcTPS1 gene.

[0030] Optionally, the plant varieties include Arabidopsis thaliana and Actinidia chinensis.

[0031] The present invention discloses the following technical effects:

[0032] The present study found that the AcTPS gene family in Actinidia chinensis showed a positive response attitude under drought stress. Under drought stress, the ABA expression and soluble sugar (sucrose, glucose and trehalose) content of Actinidia chinensis increased significantly, indicating that Actinidia chinensis can respond to drought stress by increasing the content of ABA, sucrose, glucose and trehalose, revealing the mechanism of the AcTPS gene family of Actinidia chinensis responding to drought stress.

[0033] The research results of the present invention reveal the response mechanism of Chinese kiwifruit to adverse environments and the changes in gene expression in adverse environments, laying a theoretical foundation for in-depth research on the drought resistance of Chinese kiwifruit, providing new gene resources for improving the drought resistance of Chinese kiwifruit, and providing technical support for the breeding of highly resistant Chinese kiwifruit. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 The figure shows the results of the physical and chemical property analysis of the AcTPS gene family; A is the fat solubility index; B is the instability index; C is the molecular weight; and D is the number of amino acids.

[0036] Figure 2 is the phylogenetic tree of the TPS gene family of Actinidia chinensis, rice, Arabidopsis thaliana, apple and watermelon; A is the phylogenetic tree; B is the number of subfamily I and subfamily II TPS genes in Actinidia chinensis, Arabidopsis thaliana, rice, watermelon and apple;

[0037] Figure 3 Figure 1 is a diagram of the conserved motifs and gene structure of the AcTPS gene family; A is the members and classification of the AcTPS gene family; B is the exon-intron structure of the AcTPS gene, green represents UTR, yellow represents CDS, and the middle line represents intron; C is the distribution of the conserved motifs of the AcTPS gene, and the black line represents the relative length of the protein;

[0038] Figure 4 are the cis-acting elements of the AcTPS gene; A is the number of cis-acting elements of each TPS gene, and the numbers in the heat map boxes represent the number of different elements in these AcTPSs; B is the category, number and proportion of cis-acting elements; C is the position of each cis-acting element;

[0039] Figure 5 Figure 1 is the AcTPS gene localization and colinearity map; A is the chromosome localization map of the AcTPS gene, the gray lines represent all the repeated genes in the Chinese kiwifruit genome, and the blue lines represent the TPS fragment repeated genes in the Chinese kiwifruit; the gray graph represents the chromosome of the Chinese kiwifruit; B is the colinearity map between different species of Chinese kiwifruit, rice, Arabidopsis thaliana and apple, the blue lines represent the repeated blocks, the pink lines represent the colinear TPS gene pairs, the yellow represents the chromosome of rice, the orange represents the chromosome of Chinese kiwifruit, the green represents the chromosome of Arabidopsis thaliana, and the rose red represents the chromosome of apple;

[0040] Figure 6 This is the AcTPS protein interaction diagram;

[0041] Figure 7The following are the results of phenotypic observation, growth and physiological index detection of Actinidia chinensis under drought stress; a is the phenotypic observation diagram of Actinidia chinensis before and after drought stress; b is the soil moisture statistics of the drought treatment group and the control group; c is the height statistics of Actinidia chinensis in the drought treatment group and the control group; d is the comparison diagram of relative water content of leaves in the drought treatment group and the control group; N: P>0.05**: P<0.01;

[0042] Figure 8 The figure is a comparison of ABA expression and soluble sugar content in Actinidia chinensis leaves under drought stress; A is a comparison of ABA expression between the drought treatment group and the control group; B is a comparison of sucrose, glucose and trehalose content between the drought treatment group and the control group; *: P<0.05, **: P<0.01;

[0043] Fig. 9 The expression of AcTPS gene under drought stress; A is the expression heat map of AcTPS gene under drought stress; B is the qRT-PCR detection result of 5 AcTPS genes; N: P>0.05, *: P<0.05, **: P<0.01;

[0044] Fig.10 The GO enrichment analysis diagram of AcTPS gene; A is the GO functional enrichment of TPS gene in Actinidia chinensis. From the inside to the outside, the first circle of bar graphs indicates the proportion of TPS genes with significant differences enriched in the same GOterm to the total number of TPS genes; the second circle indicates the number of TPS genes enriched in the difference between up-regulated and down-regulated GOterms, green indicates up-regulated, and yellow indicates down-regulated; the third circle of heat map indicates the total number of genes enriched in the corresponding GOterm; the fourth circle represents the GO number, and different categories are represented by different colors; B is the name of the GO corresponding to the GO ID;

[0045] Fig.11 This is the KEGG enrichment map of TPS genes in Actinidia chinensis;

[0046] Fig.12 is the qRT-PCR result of AcTPS1 gene; Ta, Tb, Tc, Td, Te and Tf respectively represent 0h, 12h, 24h, 36h, 48h and 60h after drought stress; N indicates P>0.05, ** indicates P<0.01;

[0047] Fig.13A is a graph showing changes in phenotype and growth indicators of Arabidopsis thaliana overexpressing the AcTPS1 gene before and after drought stress; A is a graph showing PCR identification results of Arabidopsis thaliana positive plants (hygromycin resistance gene HPT, 598 bp); B is a graph showing changes in phenotypes of wild-type Arabidopsis thaliana and transgenic Arabidopsis thaliana before and after drought; C is a graph showing changes in plant height, root length and fresh weight of wild-type Arabidopsis thaliana and transgenic Arabidopsis thaliana before and after drought; N indicates P>0.05, * indicates P<0.05, and ** indicates P<0.01. DETAILED DESCRIPTION

[0048] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0049] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0050] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0051] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0052] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0053] Example 1 Identification and analysis of the TPS gene family in Actinidia chinensis

[0054] 1. Identification and physicochemical properties of the TPS gene family in Actinidia chinensis

[0055] Kiwifruit gene data (Actinidia chinensis (HongYang V3)) were downloaded from the Kiwifruit Genome Database KGD (https: / / kiwifruitgenome.org / ftp / A_chinensis / Hongyang / v3.0 / ), and sequence information of the conserved domain of the TPS gene family was downloaded from the Pfam website (http: / / pfam.xfam.org / ), and the hidden Markov model file was constructed using the HMMER3 software. The protein sequences and genome data of 11 Arabidopsis TPS genes were downloaded from the Arabidopsis genome website TAIR (https: / / www.Arabidopsis.org / ). The rice genome data was downloaded from the China Rice Data Center (https: / / ricedata.cn). The protein sequences and genome data of 13 apple TPS genes and 7 watermelon TPS genes were downloaded from the Rosaceae Genome Database (https: / / www.rosaceae.org / ) and the watermelon genome database (http: / / cucurbitgenomics.org / ), respectively.

[0056] The protein sequences of the Arabidopsis TPS gene family were used as controls, and the protein sequences of the whole genome of Actinidia chinensis were searched by Blast and hmmsearch. By predicting the conserved domains of the TPS gene family of Actinidia chinensis, the sequences with incomplete or no TPS and TPP domains were deleted, and the sequences with E-value=1×10 -5 The protein sequences of Actinidia chinensis were analyzed and 13 TPS genes were identified. Finally, they were named AcTPS1-AcTPS13 according to their locations on the chromosome.

[0057] To further understand the AcTPS gene, the physicochemical properties of the gene were analyzed using the Expasy ProtParam (https: / / web.expasy.org / protparam / ) online tool, WoLF PSORT, and TBtools. The results showed that ( Figure 1, Table 1), the protein sequences of each TPS gene are quite different, ranging from 336 to 934; the molecular weight is between 38.22 and 105.92; the isoelectric point pI ranges from 5.52 to 9.48; the instability coefficient is between 34.03 and 44.84, among which the instability coefficients of AcTPS2, AcTPS6, AcTPS11, AcTPS12 and AcTPS13 are all less than 40, indicating that the proteins of these five genes are stable proteins; the lipid solubility index is between 75.15 and 92.33, which is conducive to the increase of thermal stability of globular proteins. The subcellular localization prediction of the Plant-mPloc tool shows that more than 80% of the gene expression products are located in the vacuole and chloroplast (Table 1).

[0058] Table 1 Results of physicochemical analysis of the AcTPS gene family

[0059]

[0060] 2. Phylogeny of the TPS gene family in Actinidia chinensis

[0061] In order to understand the evolutionary relationship between TPS genes in different species, the neighbor-joining method was used to construct a phylogenetic tree based on the TPS gene protein sequences of Chinese kiwifruit, rice, Arabidopsis, apple and watermelon. The results showed that ( Figure 2 ), the 13 AcTPS genes were divided into two categories, AcTPS1, AcTPS7 and AcTPS9 belonged to the class I subfamily, and AcTPS2, AcTPS3, AcTPS4, AcTPS5, AcTPS6, AcTPS8, AcTPS10, AcTPS11, AcTPS12 and AcTPS13 belonged to the class II subfamily, which was consistent with the classification of Arabidopsis, watermelon and apple.

[0062] Studies have shown that AtTPS1, AtTPS2, AtTPS3 and AtTPS4 genes belong to the Class I subfamily, and all of them except AtTPS3 have TPS activity; overexpression of AtTPS1 gene can slightly increase the trehalose content in Arabidopsis, increase seed germination rate, and improve its drought tolerance; overexpression of OsTPS1 can improve rice's tolerance to low temperature, salt and drought. AcTPS1, AcTPS7 and AcTPS9 of Chinese kiwifruit belong to the Class I subfamily TPS genes, indicating that these three genes may have similar functions to AtTPS1 and OsTPS1 genes.

[0063] 3. Gene structure and conserved motifs of AcTPS gene

[0064] The gene structure of each gene in Actinidia chinensis was analyzed using TBtools software. Then, the CD-search online tool of the NCBI database (http: / / www.ncbi.nlm.nih.gov / Structure / bwrpsb / bwrpsb.cgi) was used to predict the domain structure of the TPS protein in Actinidia chinensis, with default parameters. The online tool MEME (https: / / meme-suite.org / meme / ) was used to perform motif analysis on the protein encoded by AcShaker (the maximum number of conserved motifs retrieved was 10, and the other parameters were default) to analyze the differences between the members of the shaker K+ channel protein gene family. Finally, the above results were visualized using TBtools software and Chipolt (https: / / www.chiplot.online / ) online software. The results show ( Figure 3 A and C), the conserved motifs 1, 2, 3, 5 and 6 are highly conserved, but the same motifs are located in different positions in different proteins, which may be related to the structure of the gene family. The conserved motifs in the same type of TPS protein sequences are similar. For example, only three conserved motifs, Motif7, 8 and 9, were found in the Class I subfamily; Motif1, 2, 3, 5 and 6 appeared in the Class II subfamily. Motif7 and 8 only appeared in the Class I subfamily, indicating that the Class I subfamily may have specific functions.

[0065] The distribution of conserved motifs in the AcTPS gene family may also be affected by gene structure. The distribution of gene structure is the key to studying evolution within a gene family. The gene structure analysis of 13 AcTPS genes showed that ( Figure 3 The length of the genes of subfamily I was similar, and the distribution of conserved motifs was also similar. The number of introns and exons of the 13 AcTPS genes varied greatly, ranging from 7 to 19 introns and 8 to 22 exons.

[0066] 4. Cis-acting elements of promoter of TPS gene family in Actinidia chinensis

[0067] In order to understand the response mechanism of TPS gene in Chinese kiwifruit, TBtools software was used to extract the 2000bp base sequence upstream of each gene, and the promoter cis-acting elements were analyzed using the online software plantCARE. The results showed that ( Figure 4AC), a total of 265 promoter cis-acting elements were screened, which can be mainly divided into six categories: plant hormone response elements, environmental pressure response elements, light response elements, plant-specific regulatory elements, MYB binding sites and protein binding sites. The proportions from small to large are plant hormone response elements (39.62%) > light response elements (29.43%) > environmental pressure response elements (16.60%) > plant-specific regulatory elements (7.93%) > MY binding sites (5.66%) > protein binding sites (0.76%). In summary, in addition to light response elements and protein binding site elements, 69.81% of promoter action elements can regulate the expression of genes and material metabolism in plants to make plants resistant and improve the inadaptability of plants to adverse environments. Among them, plant hormone response elements are the most, indicating that when plants are disturbed by external factors, the expression of TPS genes is most likely to be achieved through plant hormone regulation.

[0068] 5. Chromosomal localization and colinearity of the AcTPS gene

[0069] The chromosome position information was obtained from the genome annotation file (GFF3), and the chromosome position was mapped using TBtools software. The chromosome length, position, and density information of the AcShaker gene were extracted using TBtools, and the intraspecific collinearity of the AcShaker family members was analyzed using the MCSanX program in TBtools software.

[0070] The results of chromosome localization showed that ( Figure 5 A), the 13 TPS genes of Actinidia chinensis are unevenly distributed on 11 chromosomes, with the largest number of genes located on chromosomes 4 and 7 (2 genes). Gene duplication is an important means of expansion of plant gene families. A total of 13 pairs of segmental duplication genes were found in the 13 AcTPS genes, of which 5 pairs of segmental duplication gene pairs were found on chromosome 7, accounting for the largest proportion, and the remaining chromosomes also had duplicated gene pairs. These results indicate that segmental duplication also plays an important role in the expansion of the AcTPS family.

[0071] In addition, the TPS collinearity between Actinidia chinensis and rice, Arabidopsis thaliana and apple was analyzed, and the results showed that ( Figure 5 B), there are more homologous gene pairs between Actinidia chinensis and Arabidopsis, and the least homologous gene pairs between Actinidia chinensis and rice. This shows that the evolutionary relationship between Actinidia chinensis and Arabidopsis is closer, and the evolutionary relationship between Actinidia chinensis and rice is farther.

[0072] 6. Protein network interactions of the TPS gene family in Actinidia chinensis

[0073] The network interaction analysis of 13 AcTPS proteins showed that ( Figure 6), 13 genes formed interacting proteins, indicating that these proteins are closely related to each other. Among them, AcTPS1, AcTPS7 and AcTPS 9 interacted with other proteins and belonged to the Class I subfamily proteins. The Class I subfamily has TPS activity and can regulate plant stress. The other proteins belong to the Class II subfamily TPS proteins and have related interactions with them. Among them, AcTPS1, AcTPS7 and AcTPS9 are the centers of the protein interaction network, indicating that these three genes may have special and multiple functions.

[0074] Example 2 Functional Identification of TPS Gene in Actinidia chinensis

[0075] 1. Experimental materials: The tissue culture seedlings of Actinidia chinensis cv. 'Hongyang' in the tissue culture room of Southwest Forestry University were used as research materials. The tissue culture seedlings were hardened and transplanted to obtain Actinidia chinensis seedlings.

[0076] 2. Phenotypic and physiological changes of Actinidia chinensis seedlings under drought stress

[0077] The humidity of all planting soils was maintained at 80-85%. 18 Chinese kiwifruit seedlings of the same size were selected and randomly divided into two groups, with 9 plants in each group. Among them, 9 plants were used as the control group, and the soil humidity was maintained at 80-85%. Three replicates were set up and numbered CK1, CK2 and CK3; the other 9 plants were used as the drought treatment group, and the soil humidity was 40-45%. The drought stress was maintained for 72 hours, and three replicates were set up and numbered T1, T2 and T3. The statistical results of soil humidity in the control group and the drought treatment group are shown in the figure. Figure 7 As shown in b. Each numbered group had three samples. Growth indicators such as plant height and physiological indicators such as relative water content of leaves were observed and recorded in each group. Leaves of each sample were taken out in turn as experimental materials, stored in liquid nitrogen, and handed over to Maiwei Metabolic Biology Company (Wuhan, China) for determination of transcriptome, plant hormone (ABA) and soluble sugar (sucrose, glucose and trehalose) content.

[0078] The results of physiological indicators of each group of Actinidia chinensis are as follows Figure 7 As shown in Tables 3 and 4, drought stress inhibits the growth of Actinidia chinensis. Compared with the control group, the leaves of Actinidia chinensis withered and grew poorly after drought treatment ( Figure 7 a). Before drought stress, the height of Actinidia chinensis was basically the same, but after drought treatment, the height of Actinidia chinensis varied greatly ( Figure 7 c, Table 2), which indicates that drought stress will slow down plant growth and reduce nutrient absorption. Compared with the control group and the drought treatment group, the fresh weight and relative water content of Actinidia chinensis leaves under drought stress were lower than those in the control group, and the difference was extremely significant (P<0.01)( Figure 7d, Table 3). This indicates that drought can accelerate the transpiration rate of leaves and reduce the relative water content of leaves.

[0079] Table 2 Changes in height of kiwifruit seedlings in the control group and drought treatment group

[0080]

[0081] Table 3 Leaf weight and relative water content of kiwifruit seedlings in the control group and drought treatment group

[0082]

[0083] The results of the determination of plant hormones and soluble sugar content in each group of Chinese kiwifruit are as follows Figure 8 , as shown in Table 4 and Table 5. Abscisic acid (ABA) can regulate plant growth and development and adapt to biotic and abiotic stresses. The results showed that the expression levels of ABA, ABA-GE and ABA-ald in Actinidia chinensis after drought stress were significantly higher than those in the control group ( Figure 8 A, Table 4). The correlation coefficient between ABA and trehalose calculated by SPASS software was 0.986, with a high correlation, indicating that the content of ABA and trehalose were positively correlated. Studies have shown that under abiotic stress, plants synthesize ABA in various organs and activate defense mechanisms, regulate stomatal aperture and express defense-related genes, thereby giving plants resistance to environmental stress.

[0084] Sugar substances are important regulators for promoting photosynthesis, flowering and resisting abiotic stress. The contents of sucrose, grape sugar and trehalose in Chinese kiwifruit after drought stress were higher than those in the control group, and the difference was extremely significant ( Figure 8 B, Table 5), indicating that plants, under environmental stress, will enhance their tolerance to abiotic stresses such as drought stress by accumulating soluble sugars.

[0085] Table 4 Expression levels of ABA analogs in the control group and drought treatment group

[0086] index Compound CK1 CK2 CK3 T1 T2 T3 ABA Abscisic acid 100.12 97.48 80.19 1155.61 1322.69 1037.88 ABA-GE Abscisic acid glucose ester 13126.39 14161.25 14810.04 33448.82 35722.10 23656.63 ABA-ald Abscisaldehyde 93.87 107.26 111.77 283.66 326.98 202.40

[0087] Table 5 Soluble sugar content in macaques of the control group and drought treatment group

[0088]

[0089] 3. Expression pattern and qRT-PCR results of AcTPS gene under drought stress

[0090] The expression pattern of AcTPS gene under drought stress was analyzed based on transcriptome data. The results showed that ( Fig. 9A, Table 6), compared with the control group, 7 of the 13 AcTPS genes were significantly differentially expressed genes (DEGs) under drought stress, of which only one gene (AcTPS4) was upregulated, and the expression levels of the remaining genes were downregulated. The number of downregulated genes was greater than the number of upregulated genes, indicating that the AcTPS gene may maintain its own life activities by negatively regulating the damage caused by drought stress to Chinese kiwifruit.

[0091] Table 6 FPKM value of AcTPS gene

[0092] ID CK1_fpkm CK2_fpkm CK3_fpkm T1_fpkm T2_fpkm T3_fpkm AcTPS1 23.35 17.54 16.01 5.06 5.12 2.91 AcTPS2 25.26 15.56 10.74 1.63 1.92 1.80 AcTPS3 16.27 17.70 12.90 23.08 22.81 19.22 AcTPS4 0.00 0.08 0.00 3.95 2.98 2.36 AcTPS5 0.00 0.00 0.00 0.00 0.00 0.00 AcTPS6 11.79 11.12 10.02 6.09 5.75 4.20 AcTPS7 3.11 2.63 2.20 1.86 1.92 1.58 AcTPS8 0.00 0.00 0.00 0.00 0.00 0.00 AcTPS9 19.41 16.80 19.88 6.11 4.84 4.06 AcTPS10 7.04 7.43 4.46 2.72 5.31 4.04 AcTPS11 3.54 6.32 2.68 1.43 1.55 1.46 AcTPS12 20.14 16.37 13.69 5.00 5.76 4.59 AcTPS13 0.00 0.00 0.00 0.00 0.00 0.00

[0093] Further randomly selected 5 AcTPS genes (AcTPS1, AcTPS3, AcTPS4, AcTPS9 and AcTPS12) from the transcription data for qRT-PCR verification. The leaves of Actinidia chinensis grown in soil moisture of 80-85% were used as the control group (CK1, CK2, CK3), and the leaves of Actinidia chinensis grown in soil moisture of 40-45% for 72h were used as the treatment group (T1, T2, T3), with three biological replicates in each group. The total RNA of Actinidia chinensis leaves was extracted using the Plant RNA Extraction Kit (DP432), and cDNA was synthesized using the Monad kit. The reverse transcribed cDNA was diluted 10 times and mixed for the quantitative PCR formal experiment. Actin was used as the internal reference gene, and qRT-PCR primers were designed using Primer 6.0 (Table 7). The QIAGEN kit was then used for standard qRT-PCR, and each gene was repeated at least three times. SPASS v21 software was used for data analysis.

[0094] Table 7 qRT-PCR primer sequence list

[0095]

[0096] The results show that Fig. 9 B, Table 8), compared with the control group (CK), the expression of AcTPS3 and AcTPS4 was up-regulated under drought stress, while the expression of AcTPS1, AcTPS9 and AcTPS12 was down-regulated. And the correlation coefficients of the FPKM value and qRT-PCR results in the transcriptome data (Table 9) were all greater than 0.75, indicating that the correlation between the qRT-PCR results and the transcriptome data was high. The accuracy of the transcriptome data was proved, and the reliability of the differentially expressed genes in the transcriptome data was verified. At the same time, it was shown that these genes were involved in the response of Chinese kiwifruit to drought stress.

[0097] Table 8 Original CT values ​​of qRT-PCR

[0098]

[0099] Table 9 Correlation between transcriptome data and qRT-PCR results

[0100]

[0101]

[0102] 4. GO enrichment

[0103] In order to further clarify the biological function of the AcTPS gene family, the EggNOG-MAPPERS database was used to annotate the gene functions of the proteins of 13 AcTPS genes, and GO functional enrichment analysis was performed in combination with transcriptome data. The results showed that ( Fig.10 ), 13 AcTPS genes were annotated into three categories: biological process, cellular component, and molecular function. In biological process, most of them were related to the biological process of sugar substances and osmotic regulation; in cellular components, most of them were related to biological membranes. This suggests that AcTPS genes may enable plants to adapt to adverse environments by regulating sugar levels.

[0104] 5. KEGG enrichment

[0105] KEGG enrichment analysis was performed on AcTPS gene under drought stress. The results showed that ( Fig.11 ), 9 AcTPS genes were enriched in metabolic pathways (ko01100), biosynthesis pathways of secondary metabolites (ko01110), and metabolic pathways of starch and sucrose (ko00500) under drought stress, and all AcTPS genes on the 9 KEGG pathways were DEGs. This indicates that AcTPS genes can enhance tolerance to drought stress by regulating the metabolism, synthesis of secondary metabolites, and metabolic pathways of starch and sucrose in Actinidia chinensis.

[0106] Example 3 Analysis of expression changes of the AcTPS1 gene in Actinidia chinensis under drought stress

[0107] To further verify the expression of AcTPS1 gene under drought stress, qRT-PCR was performed on AcTPS1 gene after drought stress for 0h, 12h, 24h, 36h, 48h and 60h (respectively denoted as Ta, Tb, Tc, Td, Te and Tf). The results showed that ( Fig.12, Table 10), after 0h, 12h and 24h of drought stress treatment, AcTPS1 gene was expressed but not significantly, and after 36h, 48h and 60h of drought stress, the expression of AcTPS1 was upregulated, and the expression of AcTPS1 gene was significantly upregulated after 36h and 60h of drought stress treatment. This shows that AcTPS1 gene is a key gene in Chinese kiwifruit responding to drought stress. It is worth mentioning that the previous research results showed that after 72h of drought stress, the expression of AcTPS1 gene was downregulated (see Fig. 9 B), but the trehalose content increased (see Figure 8 B), indicating that the accumulation of trehalose content has a time lag effect compared with the expression level of AcTPS1.

[0108] Table 10 Original CT values ​​of qRT-PCR

[0109]

[0110]

[0111] Example 4 Functional analysis of AcTPS1 gene overexpression

[0112] After cloning, the AcTPS1 gene was transferred into the model plant Arabidopsis for overexpression analysis. The gene cloning and genetic transformation experiments were completed by Shanghai Pujie Biotechnology. The nucleotide sequence of the cloned AcTPS1 gene is shown in SEQ ID NO.13. After receiving the positive seeds, the seeds were placed in 1 / 2MS medium for culture. The hygromycin resistance gene HPT in the transgenic Arabidopsis resistant seedlings was detected by PCR. The results showed that ( Fig.13 A), the transgenic Arabidopsis resistant seedlings can be determined as positive, and the AcTPS1 gene has been integrated into the genome of the transgenic Arabidopsis positive seedlings.

[0113] The nucleotide sequence of the cloned AcTPS1 gene is as follows: (SEQ ID NO.13)

[0114]

[0115] Transgenic Arabidopsis thaliana (L1) and wild-type Arabidopsis thaliana (WT) were cultured in a medium containing 10% PEG6000 for one week, and the growth of Arabidopsis thaliana was observed and its growth indicators such as root length, plant height and fresh weight were measured. The results showed that ( Fig.13 B, C), in ordinary culture medium, MT type and L1 type grow in the same way, and the root length, plant height and fresh weight of Arabidopsis are slightly different. However, under PEG stress, L1 type grows better than WT type, and its root length, plant height and fresh weight are significantly higher than WT type. This shows that overexpression of AcTPS1 gene can enhance the drought resistance of Arabidopsis.

[0116] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of AcTPS1 gene or its related biological materials, characterized in that: Includes any of the following applications: A1. Regulate plant drought resistance; A2. Cultivate plant varieties with strong drought resistance; A3. Early identification of plant strains with high drought resistance; The nucleotide sequence of the AcTPS1 gene is shown in SEQ ID NO.13; The regulation refers to increasing the expression level of the AcTPS1 gene, which can enhance the drought resistance of plants.

2. The use according to claim 1, characterized in that: The relevant biological material includes any one of the following: B1. An expression cassette comprising the AcTPS1 gene; B2. A recombinant vector comprising the AcTPS1 gene; B3. A recombinant microorganism comprising the AcTPS1 gene.

3. The use according to claim 1, characterized in that: The plants include Arabidopsis thaliana and Actinidia chinensis.

4. A method for improving plant drought resistance, characterized in that: The method comprises the steps of using genetic transformation technology to transfer the AcTPS1 gene or its related biological materials into the plant body, so that the AcTPS1 gene is stably overexpressed, thereby improving the drought resistance of the plant; The nucleotide sequence of the AcTPS1 gene is shown in SEQ ID NO.

13.

5. The method according to claim 4, characterized in that The relevant biological material includes any one of the following: B1. An expression cassette comprising the AcTPS1 gene; B2. A recombinant vector comprising the AcTPS1 gene; B3. A recombinant microorganism comprising the AcTPS1 gene.

6. The method according to claim 4, characterized in that The plants include Arabidopsis thaliana and Actinidia chinensis.

7. A method for cultivating a plant variety with strong drought resistance, characterized in that: This includes using genetic transformation technology to transfer the AcTPS1 gene or its related biological materials into plants to construct plant varieties with strong drought resistance; The nucleotide sequence of the AcTPS1 gene is shown in SEQ ID NO.

13.

8. The cultivation method according to claim 7, characterized in that: The relevant biological material includes any one of the following: B1. An expression cassette comprising the AcTPS1 gene; B2. A recombinant vector comprising the AcTPS1 gene; B3. A recombinant microorganism comprising the AcTPS1 gene.

9. The cultivation method according to claim 7, characterized in that: The plant varieties include Arabidopsis thaliana and Actinidia chinensis.

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