Application of tomato slmyb42 gene in regulating plant height

Abstract

The application discloses application of a tomato SLMYB42 gene in regulation of plant plant height and belongs to the technical field of plant genetic engineering. The present application takes a rice OsMYB42 protein sequence as a reference, clones a MYB42 gene from a tomato sequencing variety Micro-Tom, and names the gene as a tomato SLMYB42 gene. The gene number of the tomato SLMYB42 gene is Solyc08T002208.1, the CDS sequence is shown as SEQ ID NO:1, and genetic transformation identification indicates that the gene is a new plant height regulation gene, overexpression of the gene can significantly increase the plant height of tomatoes at multiple periods, and the gene can be applied to regulation of plant plant height, thereby providing effective gene resources and breeding improvement strategies for genetic improvement and new variety creation of plant height traits.

Description

Technical Field

[0001] This invention relates to the application of plant genes, specifically the application of the tomato SLMYB42 gene in regulating plant height, and belongs to the field of plant genetic engineering technology. Background Technology

[0002] Tomato (Solanum lycopersicum L.) is one of the most consumed and widely cultivated vegetable crops globally, playing a vital role in the nutritional structure of the diet. Plant height is an important agronomic trait, closely related to lodging resistance and mechanization levels, directly impacting economic benefits. However, research reports on tomato plant height-related genes or QTLs are currently scarce. Therefore, identifying and cloning tomato plant height-related genes and elucidating their biological functions will provide effective genetic resources and breeding strategies for cultivating ideal tomato plant types.

[0003] The MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factor family is one of the most diverse transcription factors, widely distributed in all eukaryotic plants. Currently, MYB-related genes regulating plant height have been reported in sorghum, maize, and rice. 127 MYB-related genes have been identified in tomato; however, the tomato SLMYB42 gene has not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to clone the SLMYB42 gene from tomatoes using existing plant genetic engineering technology, construct an overexpression vector of the SLMYB42 gene, transform the overexpression vector into plants (e.g., tomatoes), and directionally improve the plant's genetic background to cultivate tall plants.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The application of the tomato SLMYB42 gene in regulating plant height, wherein the CDS sequence of the tomato SLMYB42 gene is shown in SEQ ID NO: 1, and the application is to increase plant height by overexpressing the tomato SLMYB42 gene in plants.

[0007] Preferably, the plant is a tomato.

[0008] Preferably, a method for increasing plant height by overexpressing the tomato SLMYB42 gene in plants includes the following steps:

[0009] (1) Using the cDNA of the tomato variety Micro-Tom as a template, PCR amplification was performed using primers qSLMYB42-F and qSLMYB42-R. The total volume of the reaction system was 10 μL, specifically: 3 μL cDNA, 1 μL qSLMYB42-F, 1 μL qSLMYB42-R, and 5 μL TB Green Premix Ex Taq II. After purification, the target fragment of the target gene was obtained. The nucleotide sequences of qSLMYB42-F and qSLMYB42-R are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0010] (2) Homologous recombination reaction was performed between the target fragment of the target gene and the vector fragment to obtain the positive clone p35S::SLMYB42;

[0011] (3) The tomato SLMYB42 gene was genetically transformed into recipient material using Agrobacterium-mediated transformation to obtain transgenic plants.

[0012] The advantages of this invention are: using existing plant genetic engineering technology, the SLMYB42 gene was cloned from tomato for the first time. Genetic transformation identification showed that the tomato SLMYB42 gene is a new gene that regulates plant height. Overexpression of the tomato SLMYB42 gene can significantly increase the plant height of tomatoes at multiple stages (flowering stage and fruiting stage). The tomato SLMYB42 gene can be used to regulate plant height, providing effective gene resources and breeding improvement strategies for the genetic improvement of plant height traits and the creation of new varieties. Attached Figure Description

[0013] Figure 1 A comparative phenotypic diagram of genetically modified tomatoes at different stages;

[0014] Figure 2 The image shows the results of real-time quantitative PCR detection of the relative expression level of the SLMYB42 gene in transgenic tomatoes.

[0015] Figure 3 A comparison chart of the plant height of genetically modified tomatoes during the seedling stage;

[0016] Figure 4 A comparison chart of plant height during the flowering period of genetically modified tomatoes;

[0017] Figure 5 A comparison chart of plant height during the fruiting stage of genetically modified tomatoes;

[0018] Figure 6 A comparison chart of the first internode lengths in genetically modified tomatoes;

[0019] Figure 7 A comparative diagram of the second internode length in genetically modified tomatoes;

[0020] Figure 8 A comparative diagram of the length of the third internode in genetically modified tomatoes;

[0021] Figure 9 A comparative diagram of the fourth internode length in genetically modified tomatoes;

[0022] Among them, the recipient material is CK (Micro-Tom), and SLMYB42-OE#1, SLMYB42-OE#2, and SLMYB42-OE#3 are three independent transgenic lines. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0024] I. Cloning, vector construction, and genetic transformation of the tomato SLMYB42 gene

[0025] Using the rice OsMYB42 protein sequence as a reference, this invention cloned a MYB42 gene from the tomato cultivar Micro-Tom through bioinformatics analysis and PCR amplification sequencing verification, and named it the tomato SLMYB42 gene. The gene number of the tomato SLMYB42 gene is Solyc08T002208.1, the CDS sequence is shown in SEQ ID NO: 1, and the encoded amino acid sequence is shown in SEQ ID NO: 2.

[0026] Using cDNA from the tomato variety Micro-Tom as a template and SlAcTin as an internal reference gene for tomato materials, PCR amplification was performed (the total reaction volume was 10 μL, specifically including: 3 μL cDNA, 1 μL forward primer qSLMYB42-F (SEQ ID NO: 3), 1 μL reverse primer qSLMYB42-R (SEQ ID NO: 4), and 5 μL TB Green Premix Ex Taq II). After purification, the target fragment of the target gene was obtained. The target fragment of the target gene was then subjected to homologous recombination with the vector fragment. Combined with colony PCR and sequencing verification, the positive clone p35S::SLMYB42 was finally obtained.

[0027] Using Agrobacterium-mediated transformation, the tomato SLMYB42 gene was genetically transformed into the tomato recipient material Micro-Tom. Positive transgenic lines were screened and their T2 generation transgenic lines were identified.

[0028] II. Expression analysis of SLMYB42 gene in transgenic positive tomato lines

[0029] This invention identified three independent T2 generation transgenic positive lines through DNA and RNA identification, designated SLMYB42-OE#1, SLMYB42-OE#2, and SLMYB42-OE#3, respectively. The control line was Micro-Tom (CK).

[0030] Total RNA was extracted from tomato leaves using the TaKaRa MiniBEST Plant RNA Extraction Kit. Reverse transcription was performed using the TransScript One-Step gDNA Removal and cDNA Synthesis Super Mix kit. PCR amplification was performed using the Bio-Rad CFX96 real-time quantitative PCR system, with SlAcTin as the internal reference gene for the tomato material. The total reaction volume was 10 μL, specifically containing: 3 μL cDNA, 1 μL forward primer qSLMYB42-F (SEQ ID NO: 3), 1 μL reverse primer qSLMYB42-R (SEQ ID NO: 4), and 5 μL TB Green Premix Ex TaqII.

[0031] Use 2 -ΔΔCT The relative expression level of the SLMYB42 gene was calculated using a method with three replicates per sample group. Figure 2 It can be seen that, compared with the control line Micro-Tom, the expression level of the tomato SLMYB42 gene in the leaves of the transgenic positive lines SLMYB42-OE#1, SLMYB42-OE#2 and SLMYB42-OE#3 was significantly increased by more than 22 times.

[0032] III. Identification of plant height trait in transgenic tomato lines

[0033] Seeds of transgenic positive lines SLMYB42-OE#1, SLMYB42-OE#2, and SLMYB42-OE#3, as well as the control line Micro-Tom, were selected and germinated at 28℃ for 2 days. They were then sown in seedling trays containing a mixture of nutrient soil and vermiculite (volume ratio 1:1) and placed in a greenhouse for cultivation. When the second true leaf had fully unfolded, the seedlings were transferred to round flowerpots with a diameter of 12cm containing the same substrate and watered with Hogland's nutrient solution every three days.

[0034] To investigate the effect of the tomato SLMYB42 gene on tomato plant height, plant height traits were measured at the seedling, flowering, and fruiting stages. Figure 1 , Figure 3 , Figure 4 and Figure 5It can be seen that, compared with the control line Micro-Tom, the plant height of the transgenic positive lines (SLMYB42-OE#1, SLMYB42-OE#2, SLMYB42-OE#3) was significantly increased after flowering. Specifically, during the flowering period, the plant height increased significantly by 7.21-9.63% (p<0.05); and during the fruiting period, the plant height increased significantly by 13.05-14.62% (p<0.01).

[0035] In addition, the internode lengths of the transgenic positive lines and control lines were measured during the flowering period. Figure 6 , Figure 7 , Figure 8 and Figure 9 It was found that during the flowering period, the lengths of the second, third, and fourth internodes of the transgenic positive lines were significantly increased. Specifically, the length of the second internode increased significantly by 7.20-8.65% (p<0.05), the length of the third internode increased significantly by 8.77-9.57% (p<0.05), and the length of the fourth internode increased significantly by 11.70-15.09% (p<0.05), while the length of the first internode did not change significantly (p>0.05).

[0036] It is evident that the tomato SLMYB42 gene may increase tomato plant height by increasing the length of each internode, making it an important gene regulating tomato plant height.

[0037] In summary, this invention significantly improves tomato plant height by overexpressing the tomato SLMYB42 gene. The tomato SLMYB42 gene can be used to regulate plant height, providing an effective gene resource and breeding improvement strategy for the genetic improvement of plant height traits and the creation of new varieties.

[0038] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. Application of tomato SLMYB42 gene in regulating plant height, characterized in that, The CDS sequence of the tomato SLMYB42 gene is shown as SEQ ID NO:1, the application is to increase plant height by overexpressing the tomato SLMYB42 gene in plants, and the plants are tomatoes.

2. Use according to claim 1, characterized in that, The method for increasing plant height by overexpressing the tomato SLMYB42 gene in plants comprises the following steps: (1) Taking the cDNA of tomato variety Micro-Tom as a template, performing PCR amplification by using primers qSLMYB42-F and qSLMYB42-R, and the reaction system is 3 μL of cDNA, 1 μL of qSLMYB42-F, 1 μL of qSLMYB42-R, and 5 μL of TB Green Premix ExTaq II, and after purification, the target gene is obtained, and the nucleotide sequences of qSLMYB42-F and qSLMYB42-R are shown as SEQ ID NO:3 and SEQ ID NO:4 respectively; (2) Homologous recombination is performed between the target gene and the vector fragment to obtain a positive clone p35S::SLMYB42; (3) The tomato SLMYB42 gene is genetically transformed into a receptor material by using an Agrobacterium-mediated method to obtain a transgenic plant.

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