TaCSLD1 gene related detection kit, recombinant expression vector and application

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and in particular to TaCSLD1 gene-related detection kits, recombinant expression vectors, and their applications. Background Technology

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

[0003] Drought resistance in wheat is a crucial aspect of its adaptation to arid environments, involving complex physiological and molecular mechanisms. In recent years, with advancements in genomics and transcriptomics, researchers have gradually revealed the response mechanisms of plants under abiotic stresses. In particular, studies of the CSL (Cellulose Synthase-Like) family of genes have demonstrated their important role in plant stress resistance and adaptation. CSL family genes have been found in plants such as Arabidopsis thaliana and rice to regulate cell wall synthesis, thereby influencing plant growth and stress resistance. However, research on wheat... TaCSLD1 The specific functions of genes in drought resistance have not yet been systematically studied and reported.

[0004] Wheat's drought resistance depends not only on its root growth and water absorption, but also on its leaf water retention capacity, stomatal regulation, and changes in physiological metabolism. Studies have shown that gene editing technology offers new ideas and methods for improving wheat drought resistance. Through gene editing technologies such as CRISPR / Cas9, relevant genes can be effectively knocked out or modified, thereby exploring their roles in drought resistance.

[0005] Our prior research (see Chinese Patent No. CN116121435 B) has revealed the function and potential application value of the TaCSLD gene family in regulating wheat root hair length, and disclosed a method for regulating wheat root hair length by knocking out or downregulating the expression of genes in the wheat genome. TaCSLD3B The gene reduces the length of wheat root hairs; a method for identifying or assisting in the identification of wheat root hair length is also disclosed, which detects genes in the wheat genome such as... TaCSLD3BThe polymorphism at the 89 bp site of the gene sequence was used to identify or assist in the early identification of wheat root hair length traits during breeding. This invention provides a foundation for the agricultural application of specific wheat genes. Furthermore, the discovery of STARP molecular marker primers based on STARP technology to identify genes related to wheat root hair development is of great significance for marker-assisted selection breeding of wheat roots. Building upon this, our further research discovered... TaCSLD1 Three homologous genes play a role in regulating the function and efficacy of wheat in responding to drought stress. This invention relates to Chinese Patent No. CN116121435 B, which is authorized by patent publication number CN116121435 B. TaCSLD1 The genes belong to the CSLD gene family, but this study aims to construct wheat... TaCSLD1 CRISPR gene-edited lines of wheat were systematically evaluated for their drought resistance performance under drought stress. TaCSLD1 New functions of genes reveal TaCSLD1 The potential role of genes in wheat drought resistance and the development of gene 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 its application.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows.

[0008] A kit for regulating the ability of plants to tolerate drought stress, the kit containing molecular biological elements capable of regulating the expression of specific genes; the specific genes being those related to the plant's ability to respond to drought stress.

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

[0010] As a preferred embodiment of the present invention, the molecular biological element is a combination of elements that knock out the expression of the specific gene.

[0011] As a preferred embodiment of the present invention, the specific gene is TaCSLD1 The three homologous genes, with CDS sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or equivalent genes with the same plant physiological functions.

[0012] This invention also includes the following technical solutions: comprising TaCSLD1 Three homologous genes or their homologous genes recombinant expression vectors.

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

[0014] A method to enhance plant drought stress tolerance by blocking the plant's... TaCSLD1 Expression of homologous genes can enhance plant drought stress tolerance.

[0015] A method to increase plant yield by blocking the plant TaCSLD1 The expression of three homologous genes enhances the drought stress tolerance of the corresponding plants, thereby increasing the yield of the corresponding plants.

[0016] The present invention also includes TaCSLD1 The three homologous genes are used to regulate the drought resistance and water retention capacity of plants.

[0017] In a preferred embodiment of the present invention, the plant is wheat.

[0018] The beneficial effects of adopting the above technical solution are as follows: The theoretical and experimental research of the research group of this invention have confirmed that by blocking in wheat TaCSLD1 The expression levels of the three homologous genes can be used to cultivate wheat plants with strong drought resistance and water retention capacity. This has important theoretical research value and broad practical application prospects for cultivating drought-resistant wheat plants. Attached Figure Description

[0019] Figure 1 Under drought stress TaCSLD1 Expression analysis of the three homologous genes in the transgenic recipient Fielder root;

[0020] Figure 2 for TaCSLD1 Schematic diagram of drought resistance phenotype identification of Crispr gene-edited lines under different drought conditions;

[0021] Figure 3 In drought resistance tests TaCSLD1 Statistical chart of survival rate data for Crispr gene-edited lines and transgenic recipient Fielder;

[0022] Figure 4 In drought resistance tests TaCSLD1 Statistical chart of water loss rate data for Crispr gene-edited lines and transgenic receptor Fielder;

[0023] Figure 5 In drought resistance testsTaCSLD1 Statistical chart of relative water content data for Crispr gene-edited lines and transgenic receptor Fielder. Detailed Implementation

[0024] Our prior research (see Chinese Patent No. CN116121435 B) revealed the function and potential application value of the TaCSLD gene family in regulating wheat root hair length. Based on this scientific discovery, we constructed a method for regulating wheat root hair length by knocking out or downregulating the following gene family members in the wheat genome. TaCSLD3B The study also disclosed a method for identifying or assisting in the identification of wheat root hair length, namely, detecting genes in the wheat genome that reduce wheat root hair length. TaCSLD3B The polymorphism at the 89bp site of the gene sequence enables the identification or auxiliary 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 by using STARP technology to identify STARP molecular marker primers related to wheat root hair development, it has extremely important significance for molecular marker-assisted selection breeding of wheat roots.

[0025] TaCSLD3B Genes and the present invention TaCSLD1 The genes belong to the CSLD gene family, but this study focuses on wheat. TaCSLD1 The gene was designed to construct its CRISPR gene-edited lines and comprehensively evaluate its drought resistance performance under drought stress. This belongs to wheat. TaCSLD1 The exploration of novel gene functions aims to reveal... TaCSLD1 The potential efficacy of genes in wheat drought resistance, and the development of gene tools at the molecular level that can regulate plant tolerance to drought stress, undoubtedly have extremely important scientific research value and broad application prospects.

[0026] Example 1 TaCSLD1 Gene

[0027] TaCSLD1 The gene numbers of the three partially homologous genes are as follows: TaCSLD1-A :TraesCS1A02G196100; TaCSLD1-B :TraesCS1B02G210700; TaCSLD1-D TraesCS1D02G199500 TaCSLD1 The CDS sequences of the three partial homologous genes are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0028] Example 2 TaCSLD1Obtaining Crispr gene-edited lines (KO#1 and KO#2)

[0029] To verify TaCSLD1 The drought resistance function of the gene was determined by knocking it out using the wheat variety Fielder as the transgenic recipient and the CRISPER / Cas9 gene editing system. TaCSLD1 The three homologous genes on the surface were obtained TaCSLD1 Gene mutants KO#1 and KO#2. In KO#1... TaCSLD1-A Gene, TaCSLD1-B Gene, TaCSLD1-D The CDS sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; in KO#2 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.

[0030] Example 3: Real-time quantitative detection of fluorescence TaCSLD1 Analysis of expression patterns of three partial homologous genes under drought stress

[0031] Using the wheat transgenic receptor Fielder (WT) as material, plump and uniform seeds were selected and placed in petri dishes lined with moistened germination paper. The petri dishes were then incubated at 4°C for 24 hours, followed by 12 hours of dark treatment at room temperature to induce germination. After germination, the petri dishes were placed in an artificial climate chamber for 4 days (the artificial climate chamber was set at 23°C / 16 hours of light and 18°C / 8 hours of darkness). After 4 days of culture, WT seedlings with uniform growth were selected and transferred to hydroponic boxes, with 1 / 2 Hoagland nutrient solution added. When the seedlings reached the two-leaf-one-heart stage, one group was subjected to drought treatment (20% PEG6000). Wheat root tissues under normal conditions and at 1h, 2h, 4h, 6h, 12h, and 24h under drought treatment were collected in 2ml centrifuge tubes and quickly frozen in liquid nitrogen. Wheat rootlets (WT) were ground using a tissue homogenizer, and total RNA was extracted from the root tissue using the Trizol method. cDNA was then obtained by reverse transcription using the Nanjing Novizan HiScript II QRT Super Mix for qPCR (+gDNA wiper). The obtained cDNA was used as a template. TaActin For internal reference gene pair TaCSLD1 Three homologous genes were detected by qRT-PCR.

[0032] The detection primers are:

[0033] TaCSLD1-AF: 5'-GAAGGATCAAGCGTGAGTATGAT-3' (SEQ ID NO: 10)

[0034] TaCSLD1-AR: 5'-GAATCTTCACTGGCTCAAATTG-3' (SEQ ID NO: 11)

[0035] TaCSLD1-BF: 5'-GGTGAAGGCCGACTTTGTC-3' (SEQ ID NO: 12)

[0036] TaCSLD1-BR:5'-GCCTTGGGAATCTTCACTG-3' (SEQ ID NO: 13)

[0037] TaCSLD1-DF: 5'-AGTTCAAGGTCCCGCGTTAAC-3' (SEQ ID NO: 14)

[0038] TaCSLD1-DR: 5'-CGTGCATGATCTTGTGACG-3' (SEQ ID NO: 15)

[0039] The results are as follows Figure 1 As shown, TaCSLD1-A , TaCSLD1-B and TaCSLD1-D All genes were downregulated in expression due to drought.

[0040] Example 4, Wheat TaCSLD1 Identification of drought resistance phenotypes in Crispr gene-edited lines

[0041] Select wheat transgenic receptor Fielder (WT), stable genetic... TaCSLD1 Fifty seeds each of the Crispr gene-edited lines (KO#1 and KO#2), of uniform size and plumpness, were placed in petri dishes lined with germination paper, 10 ml of distilled water was added, and the dishes were incubated at 4°C for 2 days. Afterward, they were incubated in the dark at room temperature for 12 hours. Seeds from Fielder, KO#1, and KO#2 with consistent germination were transferred to culture pots. Each culture pot contained a pre-mixed culture medium (nutrient soil: vermiculite = 3:1), with equal weights of nutrient soil and vermiculite in each pot. An equal amount of water was added to the tray to allow the substrate to absorb water until the surface was moist. Four seeds were sown in each culture pot, and the dishes were transferred to an artificial climate chamber for further cultivation. A control group and a drought treatment group were set up. After the two-leaf-one-heart stage, the control group received normal watering, while the treatment groups were subjected to drought stress.

[0042] The results are as follows Figure 2As shown, after 8 days of drought treatment, under normal conditions, the transgenic receptor WT and TaCSLD1 Mutants KO#1 and KO#2 both exhibited bright green leaves and an upright posture, with no significant difference. After 8 days of drought treatment, WT showed severe leaf wilting and stem bending, while KO#1 and KO#2 showed mild leaf wilting and upright stems. After 20 days of drought treatment, WT and... TaCSLD1 The Crispr gene-edited lines were all subjected to extreme drought, and the seedlings wilted severely; after rehydration for 10 days... TaCSLD1 The Crispr gene-edited lines had more green leaf area and better recovery than the WT lines. This indicates that under drought stress... TaCSLD1 Crispr gene-edited lines showed better drought resistance.

[0043] Example 5: Survival rate determination after drought treatment

[0044] With wheat transgenic receptor Fielder, TaCSLD1 Using the Crispr gene-edited lines (KO#1 and KO#2) as materials, the plants were cultured to the two-leaf-one-heart stage and then subjected to drought treatment. After 20 days of drought treatment until the leaves turned yellow and wilted, the plants were rehydrated. After 10 days of normal watering, photos were taken and the survival rate of the plants was counted.

[0045] The results are as follows Figure 3 As shown, after 10 days of normal watering recovery, TaCSLD1 Crispr gene-edited lines (KO#1, KO#2) had a higher survival rate than WT seedlings, with WT having a survival rate of 43%. TaCSLD1 The survival rates of mutants KO#1 and KO#2 were 85.5% and 91.8%, respectively, and TaCSLD1 The survival rate of the mutant was significantly higher than that of the WT mutant, indicating that TaCSLD1 Gene knockout can improve wheat's drought resistance.

[0046] Example 6: Determination of water loss rate

[0047] Select WT, which have been cultured to the two-leaf-one-heart stage. TaCSLD1 The second leaf of the Crispr gene-edited lines (KO#1, KO#2) was cut off and weighed fresh. The leaves were then left to dehydrate naturally at room temperature. The fresh weight of the detached plants was measured at 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, and 12h. The percentage decrease in plant fresh weight was used to reflect the plant's water loss rate.

[0048] The results are as follows Figure 4 As shown, under normal conditions TaCSLD1 The water loss rate of Crispr gene-edited lines (KO#1 and KO#2) seedlings was significantly lower than that of WT, indicating that...TaCSLD1 The Crispr gene-edited line has a strong water retention capacity.

[0049] Example 7: Determination of relative water content under drought stress

[0050] WT cells cultured to the two-leaf-one-heart stage under normal and drought conditions were selected. TaCSLD1 The second leaf of the Crispr gene-edited lines (KO#1, KO#2) was cut off and weighed quickly, and the weight was recorded as fresh weight (FW). The leaf was then completely immersed in deionized water for 6 hours, and the surface moisture was quickly absorbed. The weight at this point was recorded as saturated fresh weight (TW). Finally, the leaf was placed in a drying bag and dried in an oven at 85℃ for 8 hours until constant weight was achieved, which was recorded as dry weight (DW). The formula for calculating relative moisture content is: Relative moisture content = (FW – DW) / (TW – DW) × 100%.

[0051] The results are as follows Figure 5 As shown, under normal conditions, WT and TaCSLD1 The relative water content of the Crispr gene-edited lines (KO#1 and KO#2) showed no significant difference; after 10 days of drought treatment, the relative water content of KO#1 and KO#2 was significantly higher than that of WT, which is 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 line can enhance the drought resistance of plants through its strong water retention capacity.

[0052] Example 8, Related Applications

[0053] This kit is designed to regulate wheat's tolerance to drought stress. The kit contains molecular biological elements capable of regulating the expression levels of specific genes, which are genes associated with wheat's drought stress tolerance. The molecular biological elements may optionally include: combinations of overexpression elements for the specific gene, and / or combinations of elements that inhibit or reduce the expression level of the specific gene, and / or combinations of elements that silence the expression of the specific gene, and / or combinations of elements that knock out the expression of the specific gene. The molecular biological elements are combinations of elements that knock out the expression of the specific gene. The specific gene is a partial homolog of TaCSLD1, with CDS sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or an equivalent gene with the same physiological function as the specific gene in wheat.

[0054] Include TaCSLD1 Three partial homologous genes or their homologous genes recombinant expression vectors, wherein the recombinant expression vectors are gene knockout vectors.

[0055] A method to enhance wheat drought stress tolerance involves blocking the expression of three partial homologous genes of TaCSLD1 in wheat to enhance wheat drought stress tolerance.

[0056] A method to increase wheat yield involves blocking the expression of three partial homologous genes of TaCSLD1 in wheat to enhance the drought stress tolerance of the corresponding wheat, thereby increasing the yield of the corresponding wheat.

[0057] The three homologous genes of TaCSLD1 are used to regulate the drought resistance and water retention capacity of wheat.

[0058] In summary, according to the above embodiments, in this invention, by blocking in wheat TaCSLD1 Gene expression 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.

[0059] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0060] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A kit for enhancing wheat drought stress tolerance, characterized in that: The kit contains the simultaneous knockout of the three molecules shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

3. TaCSLD1 A combination of elements of a specific gene.

2. A method for enhancing the drought stress tolerance of wheat, characterized in that: Simultaneously blocking the three molecules shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the wheat. TaCSLD1 The expression of specific genes can enhance wheat's tolerance to drought stress.

3. A method for improving the survival rate of wheat after drought treatment, characterized in that: Simultaneously blocking the three molecules shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the wheat. TaCSLD1 The expression of specific genes can enhance the drought stress tolerance of the corresponding wheat species, thereby improving the survival rate of the wheat.

4. TaCSLD1 The purpose of these three specific genes is to simultaneously knock out or reduce the three genes shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the wheat. TaCSLD1 The expression of specific genes enhances the drought resistance and water retention capacity of wheat.

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