TaCSLD1 gene related detection kit, recombinant expression vector and application

Abstract

The invention discloses a TaCSLD1 gene related detection kit, a recombinant expression vector and application, relates to the technical field of molecular biology, and aims to enhance the drought stress tolerance of plants by blocking the expression of homologous genes of three TaCSLD1 parts in the plants. The method has important scientific research and practical value in the aspect of cultivating drought-resistant plant varieties.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a TaCSLD1 gene-related detection kit, a recombinant expression vector and applications. Background Art

[0002] wheat( Triticum aestivum L.) is one of the most important food crops in the world, feeding more than 33% of the world's population. In the context of global climate change, frequent droughts and unstable precipitation patterns pose a serious threat to wheat growth and yield. Drought not only affects the physiological growth of wheat, but is also directly related to food security and the sustainability of agricultural production. Therefore, improving wheat's drought resistance has become an important topic in global agricultural research.

[0003] Drought resistance of wheat is an important manifestation of its adaptation to drought environments, involving complex physiological and molecular mechanisms. In recent years, with the development of genomes and transcriptomes, researchers have gradually revealed the response mechanisms of plants under abiotic stress. In particular, the study of CSLs (Cellulose Synthase-Like) family genes has shown their important role in plant stress resistance and adaptability. CSLs family genes have been found in plants such as Arabidopsis and rice to regulate cell wall synthesis, thereby affecting plant growth and stress resistance. However, regarding CSLs family genes in wheat, there is still a lot of research on them. TaCSLD1 The specific functions of genes in drought resistance have not yet been systematically studied and reported.

[0004] The drought resistance of wheat depends not only on the growth of its root system and the absorption of water, but also on the water retention capacity of its leaves, the regulation of stomata, and changes in physiological metabolism. Studies have shown that gene editing technology provides new ideas and methods for improving the drought resistance of wheat. Through gene editing technologies such as CRISPR / Cas9, related genes can be effectively knocked out or modified to explore their role in drought resistance.

[0005] The previous research of the research group of this application (see Chinese patent with authorization announcement number CN116121435 B) revealed the function and potential application value of the TaCSLD gene family in regulating the length of wheat root hairs, and disclosed a method for regulating the length of wheat root hairs by knocking out or down-regulating the expression of the gene in the wheat genome. TaCSLD3B Gene, reducing the length of wheat root hairs; also disclosed is a method for identifying or assisting in identifying the length of wheat root hairs, detecting such genes in the wheat genome TaCSLD3BThe polymorphism of the 89bp position of the sequence shown in the gene can be used to identify or assist in the identification of the root hair length trait of wheat in the early stage of breeding. This invention provides a basis for the agricultural production application of specific wheat genes. At the same time, based on the STARP technology, the STARP molecular marker primers that mine and identify wheat root hair development-related genes are of great significance to the molecular marker-assisted selection breeding of wheat roots. On this basis, our further research found TaCSLD1 The functions and effectiveness of three partially homologous genes in regulating wheat's response to drought stress. TaCSLD1 The genes belong to the CSLD gene family, but this study aims to construct wheat TaCSLD1 CRISPR gene-edited strains of the gene were systematically evaluated for their drought resistance under drought stress, belonging to wheat TaCSLD1 New functions of genes revealed TaCSLD1 The potential role of genes in wheat drought resistance and the development of genetic tools at the molecular level that can regulate plant tolerance to drought stress have important scientific research and application value. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a TaCSLD1 gene related detection kit, a recombinant expression vector and applications.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0008] A kit for regulating the ability of a plant to tolerate drought stress, wherein the kit comprises molecular biological elements capable of regulating the expression of a specific gene; the specific gene is a gene related to the ability of a plant to tolerate drought stress.

[0009] As a preferred technical solution of the present invention, the molecular biological elements may optionally include: an element combination for overexpressing the specific gene, and / or an element combination for inhibiting or reducing the expression amount of the specific gene, and / or an element combination for silencing the expression of the specific gene, and / or an element combination for knocking out the expression of the specific gene.

[0010] As a preferred technical solution of the present invention, the molecular biological element is a combination of elements for knocking out the expression of the specific gene.

[0011] As a preferred technical solution of the present invention, the specific gene is TaCSLD1 The three partially homologous genes, the CDS sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or equivalent genes having equivalent plant physiological functions.

[0012] The present invention also includes the following technical solutions: TaCSLD1 The invention relates to a recombinant expression vector of three partially homologous genes or homologous genes thereof.

[0013] As a preferred technical solution of the present invention, the recombinant expression vector is a gene knockout vector.

[0014] A method for enhancing plant drought stress tolerance, comprising blocking TaCSLD1 Expression of homologous genes to enhance drought stress tolerance in plants.

[0015] A method for increasing plant yield by blocking TaCSLD1 The expression of three partially homologous genes is enhanced to enhance the drought stress tolerance of the corresponding plants, thereby increasing the yield of the corresponding plants.

[0016] The present invention also includes TaCSLD1 Use of three partially homologous genes for regulating drought resistance and water retention in plants.

[0017] As a preferred technical solution of the present invention, the plant is wheat.

[0018] The beneficial effects of the above technical solution are as follows: The theoretical research and experimental research of the research group of the present invention have confirmed that by blocking TaCSLD1 The expression levels of the three partially homologous genes can cultivate wheat plants with strong drought resistance and water retention capacity. It has important theoretical research value and broad practical application prospects for cultivating drought-resistant wheat plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Under drought stress TaCSLD1 Expression analysis of three partial homologues of the gene in the roots of the transgenic recipient Fielder; Figure 2 for TaCSLD1 Schematic diagram of drought resistance phenotype identification of Crispr gene-edited strains under different drought conditions; Figure 3 For drought resistance test TaCSLD1 Statistical graph of survival rates of Crispr gene-edited strains and transgenic receptor Fielder; Figure 4 For drought resistance test TaCSLD1 Statistical graph of water loss rate of Crispr gene-edited strains and transgenic receptor Fielder; Figure 5 For drought resistance test TaCSLD1 Statistical chart of relative water content data of Crispr gene-edited strains and transgenic receptor Fielder. DETAILED DESCRIPTION

[0020] The previous research of the research group of this application (see the Chinese patent with authorization announcement number CN116121435 B) revealed the function and potential application value of the TaCSLD gene family in regulating the length of wheat root hairs, and based on this scientific discovery, a method for regulating the length of wheat root hairs was constructed by knocking out or downregulating the gene in the wheat genome. TaCSLD3B Gene, thereby reducing the length of wheat root hairs; also disclosed is a method for identifying or assisting in identifying the length of wheat root hairs, that is, detecting TaCSLD3B The polymorphism of the 89bp site of the gene sequence is used to identify or assist in the identification of wheat root hair length traits in the early stages of breeding. This patent lays the foundation for the application of specific wheat genes in agricultural production, and with the help of STARP technology, STARP molecular marker primers related to wheat root hair development are mined and identified, which is of great significance to molecular marker-assisted selection breeding of wheat roots.

[0021] TaCSLD3B Gene and the present invention TaCSLD1 The genes belong to the CSLD gene family, and this study focuses on wheat TaCSLD1 gene, aiming to construct its CRISPR gene-edited strain and comprehensively evaluate the drought resistance performance of this gene under drought stress. TaCSLD1 The purpose of exploring new gene functions is to reveal TaCSLD1 The potential effectiveness of genes in wheat drought resistance and the development of genetic tools at the molecular level that can regulate plant tolerance to drought stress undoubtedly have extremely important scientific research value and broad application prospects.

[0022] Embodiment 1, TaCSLD1 Gene TaCSLD1 The gene numbers of the three homologous genes are as follows: TaCSLD1-A :TraesCS1A02G196100; TaCSLD1-B :TraesCS1B02G210700; TaCSLD1-D :TraesCS1D02G199500, TaCSLD1 The CDS sequences of the three partially homologous genes are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0023] Embodiment 2, TaCSLD1 Acquisition of CRISPR gene-edited strains (KO#1 and KO#2) To verify TaCSLD1The drought resistance function of the gene was knocked out using the CRISPER / Cas9 gene editing technology system using the wheat variety Fielder as the transgenic recipient TaCSLD1 The three homologous genes on TaCSLD1 Gene mutants KO#1 and KO#2. TaCSLD1-A Gene, TaCSLD1-B Gene, TaCSLD1-D The CDS sequences of KO#2 are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; TaCSLD1-A Gene, TaCSLD1-B Gene, TaCSLD1-D The CDS sequences are shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0024] Example 3: Real-time fluorescence quantitative detection TaCSLD1 Analysis of expression patterns of three partially homologous genes under drought stress Using wheat transgenic recipient Fielder (WT) as the material, seeds with full and uniform grains were selected and placed in a culture dish covered with soaked germination paper. The culture dish was placed in a 4℃ refrigerator for 24 hours, and the seeds were treated in the dark at room temperature for 12 hours to germinate. After germination, the culture dish was placed in an artificial climate chamber for 4 days (the artificial climate chamber was set at 23℃ / 16 hours of light and 18℃ / 8 hours of darkness). After 4 days of cultivation, WT seedlings with consistent growth were selected and transferred to a hydroponic box, and 1 / 2 Hoagland nutrient solution was added at the same time. When the seedlings were cultured to the two-leaf and one-heart stage, one group was subjected to drought treatment (20% PEG6000). The wheat roots under normal conditions and the wheat root tissues at 1h, 2h, 4h, 6h, 12h, and 24h under drought treatment conditions were taken in 2ml centrifuge tubes and quickly frozen in liquid nitrogen. The wheat WT root system was ground with a tissue crusher, and the total RNA of the root tissue was extracted by Trizol method. The cDNA was obtained by reverse transcription using Nanjing Novozyme Reverse Transcription Kit Hi Script II Q RT Super Mix for qPCR (+gDNA wiper). The obtained cDNA was used as a template. TaActin Internal reference gene pair TaCSLD1 The three homologous genes were detected by qRT-PCR.

[0025] The detection primers are: TaCSLD1-AF: 5'-GAAGGATCAAGCGTGAGTATGAT-3' (SEQ ID NO: 10) TaCSLD1-AR: 5'-GAATCTTCACTGGCTCAAATTG-3' (SEQ ID NO: 11) TaCSLD1-BF: 5'-GGTGAAGGCCGACTTTGTC-3' (SEQ ID NO: 12) TaCSLD1-BR: 5'-GCCTTGGGAATCTTCACTG-3' (SEQ ID NO: 13) TaCSLD1-DF: 5'-AGTTCAAGGTCCCGCGTTAAC-3' (SEQ ID NO: 14) TaCSLD1-DR: 5'-CGTGCATGATCTTGTGACG-3' (SEQ ID NO: 15) The results are as follows Figure 1 As shown, TaCSLD1-A , TaCSLD1-B and TaCSLD1-D The expression of all genes was down-regulated by drought.

[0026] Embodiment 4, wheat TaCSLD1 Phenotypic identification of drought resistance in CRISPR gene-edited strains Selection of wheat transgenic recipient Fielder (WT), stable inheritance TaCSLD1 50 seeds of the Crispr gene-edited strains (KO#1 and KO#2) with the same size and full grains were placed in a culture dish covered with germination paper, 10 ml of distilled water was added, and the seeds were cultured in a 4°C refrigerator for 2 days, and then cultured in the dark at room temperature for 12 h. The seeds of Fielder, KO#1, and KO#2 with the same germination were selected and transferred to the culture pots, where the culture pots were filled with mixed culture matrix (nutrient soil: vermiculite = 3:1), and each culture pot was filled with the same weight of nutrient soil and vermiculite, and the same amount of water was added to the tray to allow the matrix to absorb water until the surface was moist. Four seeds were sown in each culture pot and transferred to the artificial climate chamber for further culture. A control group and a drought treatment group were set up. After culturing to the two-leaf and one-heart stage, the control treatment was watered normally, and the treatment group was subjected to drought stress treatment.

[0027] The results are as follows Figure 2 As shown, after 8 days of drought treatment, the transgenic receptors WT and TaCSLD1 The leaves of mutants KO#1 and KO#2 were both green and upright, with no significant difference. After 8 days of drought treatment, WT showed severe wilting of leaves and bending of stems, while leaves of KO#1 and KO#2 showed mild wilting and upright stems. After 20 days of drought treatment, WT and TaCSLD1 All the Crispr gene-edited strains were subjected to extreme drought, and the seedlings wilted severely. After 10 days of rehydration, TaCSLD1 The Crispr gene-edited lines have more green leaves than the WT and have a better recovery rate.TaCSLD1 The Crispr gene-edited strains showed improved drought resistance.

[0028] Example 5: Survival rate determination after drought treatment The wheat transgenic receptor Fielder, TaCSLD1 The Crispr gene-edited strains (KO#1 and KO#2) were used as materials. When they reached the two-leaf and one-heart stage, they were subjected to drought treatment. After 20 days of drought treatment until the leaves turned yellow and wilted, they were rewatered. After 10 days of normal watering, photos were taken and the survival rate of the plants was calculated.

[0029] The results are as follows Figure 3 As shown, after 10 days of normal watering, TaCSLD1 The survival rate of seedlings of Crispr gene-edited strains (KO#1, KO#2) was higher than that of WT, with the survival rate of WT being 43%. TaCSLD1 The survival rates of mutants KO#1 and KO#2 were 85.5% and 91.8%, respectively. TaCSLD1 The survival rate of mutants was significantly higher than that of WT, indicating TaCSLD1 Gene knockout can improve wheat drought resistance.

[0030] Example 6, Determination of water loss rate WT, TaCSLD1 The second leaf of the Crispr gene-edited strains (KO#1, KO#2) was cut off and weighed for fresh weight, and then placed at room temperature for natural water loss. The fresh weight of the in vitro plants was measured at 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h and 12h, respectively, and the water loss rate of the plants was reflected by the proportion of the reduction in fresh weight of the plants.

[0031] The results are as follows Figure 4 As shown, under normal conditions TaCSLD1 The water loss rates of seedlings of the Crispr gene-edited strains (KO#1 and KO#2) were significantly lower than those of the WT, indicating that TaCSLD1 The Crispr gene-edited strain has a strong ability to retain water.

[0032] Example 7: Determination of relative water content under drought stress conditions WT, TaCSLD1The second leaf of the Crispr gene-edited strains (KO#1, KO#2) was cut and quickly weighed and recorded as fresh weight (FW); then the leaves were completely immersed in deionized water for 6 hours, and the moisture on the surface of the leaves was quickly absorbed. The weight at this time was the saturated fresh weight (TW); finally, the leaves were placed in a drying bag and dried in an oven at 85°C for 8 hours to a constant weight, i.e., dry weight (DW). The calculation formula for relative water content is: relative water content = (FW–DW) / (TW–DW)×100%.

[0033] The results are as follows Figure 5 As shown, under normal conditions, WT and TaCSLD1 There was no significant difference in the relative water content of the Crispr gene-edited strains (KO#1 and KO#2); after 10 days of drought treatment, the relative water content of KO#1 and KO#2 was significantly higher than that of WT, which was consistent with the lower water loss rate of KO#1 and KO#2, indicating that under a certain degree of drought stress, TaCSLD1 The Crispr gene-edited strains can enhance the plants' drought resistance through their stronger water retention ability.

[0034] Embodiment 8, related applications A kit for regulating the ability of wheat to tolerate drought stress, the kit comprising molecular biological elements capable of regulating the expression of specific genes; the specific genes are genes related to the ability of wheat to tolerate drought stress. The molecular biological elements may optionally include: an element combination for overexpression of the specific genes, and / or an element combination for inhibiting or reducing the expression of the specific genes, and / or an element combination for silencing the expression of the specific genes, and / or an element combination for knocking out the expression of the specific genes. The molecular biological elements are element combinations for knocking out the expression of the specific genes. The specific genes are three partially homologous genes of TaCSLD1, and the CDS sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or equivalent genes having equivalent physiological functions in wheat.

[0035] Include TaCSLD1 A recombinant expression vector of three partially homologous genes or homologous genes thereof, wherein the recombinant expression vector is a gene knockout vector.

[0036] A method for enhancing drought stress tolerance of wheat, comprising blocking the expression of three partially homologous genes of TaCSLD1 in the wheat to enhance the drought stress tolerance of the wheat.

[0037] A method for increasing wheat yield, comprising blocking the expression of three partially homologous genes of TaCSLD1 in the wheat to enhance the drought stress tolerance of the corresponding wheat, thereby increasing the yield of the corresponding wheat.

[0038] The use of three partially homologous genes of TaCSLD1 is used to regulate the drought resistance and water retention capacity of wheat.

[0039] In summary, in the present invention, by blocking TaCSLD1 The expression of genes can enhance the drought resistance and water retention capacity of wheat plants, which has important theoretical research value and broad practical application prospects for breeding wheat varieties with strong drought resistance and water retention capacity.

[0040] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0041] The embodiments described above 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, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included in the protection scope of the present invention.

Claims

1. A kit for regulating the ability of a plant to tolerate drought stress, characterized in that: The kit contains molecular biological elements capable of regulating the expression of specific genes; the specific genes are genes related to the plant's ability to respond to drought stress and tolerate.

2. The kit according to claim 1, characterized in that: The molecular biological elements may optionally include: an element combination for overexpressing the specific gene, and / or an element combination for inhibiting or reducing the expression level of the specific gene, and / or an element combination for silencing the expression of the specific gene, and / or an element combination for knocking out the expression of the specific gene.

3. The kit according to claim 1, characterized in that: The molecular biological elements are a combination of elements for knocking out the expression of the specific gene.

4. The kit according to claim 1, 2 or 3, characterized in that: The specific gene is TaCSLD1 The CDS sequences of the three partially homologous genes are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or equivalent genes having equivalent plant physiological functions.

5. Inclusion TaCSLD1 The invention relates to a recombinant expression vector of three partially homologous genes or homologous genes thereof.

6. The recombinant expression vector according to claim 5, characterized in that: The recombinant expression vector is a gene knockout vector.

7. A method for enhancing plant drought stress tolerance, characterized in that: Blocking the plant TaCSLD1 Expression of three homeologous genes to enhance drought stress tolerance in plants.

8. A method for increasing plant yield, characterized in that: Blocking the plant TaCSLD1 The expression of three partially homologous genes is enhanced to enhance the drought stress tolerance of the corresponding plants, thereby increasing the yield of the corresponding plants.

9. TaCSLD1 Use of three partially homologous genes for regulating drought resistance and water retention in plants.

10. The kit according to claim 4, the method according to claim 7, the method according to claim 8, or the use according to claim 9, characterized in that: The plant is wheat.

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