Application of tomato SLMYB42 gene in regulation and control of plant height

Abstract

The invention discloses application of a tomato SLMYB42 gene in regulation and control of plant height, and belongs to the technical field of plant genetic engineering. According to the tomato SLMYB42 gene, a rice OsMYB42 protein sequence is taken as a reference, and an MYB42 gene is cloned from a tomato sequencing variety Micro-Tom and is named as the tomato SLMYB42 gene. The gene number of the tomato SLMYB42 gene is Solyc08T002208.1, the CDS sequence is as shown in SEQ ID NO: 1, genetic transformation identification shows that the gene is a new gene for regulating and controlling plant height, overexpression of the gene can significantly improve the plant height of tomato in multiple periods, and the gene can be applied to regulation and control of plant height and can be applied to the field of plant growth. And an effective gene resource and a breeding improvement strategy are provided for genetic improvement of plant height traits and creation of new varieties.

Description

Technical Field

[0001] The present invention relates to the application of plant genes, in particular to the application of tomato SLMYB42 gene in regulating plant strain growth, and belongs to the technical field of plant gene engineering. Background Art

[0002] Tomato (Solanum lycopersicum L.) is one of the most consumed and widely cultivated vegetable crops worldwide, playing a crucial role in the nutritional structure of the human diet. Plant height is a key agronomic trait of crops, closely related to lodging resistance and the degree of mechanization, and directly impacting the economic benefits of crops. However, few studies have identified genes or QTLs associated with plant height in tomatoes. Therefore, identifying and cloning genes associated with plant height 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) family of transcription factors is one of the most diverse transcription factors, found across all eukaryotic plants. MYB-related genes have been reported in sorghum, maize, and rice to regulate plant height. While 127 MYB-related genes have been identified in tomato, no SLMYB42 gene has been reported. Summary of the Invention

[0004] The purpose of the present invention is to use existing plant genetic engineering technology to clone the SLMYB42 gene from tomato, construct an overexpression vector of the SLMYB42 gene, transform the overexpression vector into plants (such as tomatoes), directionally improve the plant genetic background, and cultivate tall plants.

[0005] In order to achieve the above objectives, the present invention adopts the following technical solutions: The invention relates to an application of a tomato SLMYB42 gene in regulating plant height. The CDS sequence of the tomato SLMYB42 gene is shown in SEQ ID NO: 1. The application is to increase plant height by overexpressing the tomato SLMYB42 gene in plants.

[0006] Preferably, the plant is tomato.

[0007] Preferably, the method for increasing plant height by overexpressing the tomato SLMYB42 gene in a plant comprises the following steps: (1) Using the cDNA of tomato variety Micro-Tom as a template, PCR amplification was performed with primers qSLMYB42-F and qSLMYB42-R. The total reaction volume was 10 μL, including 3 μL cDNA, 1 μL qSLMYB42-F, 1 μL qSLMYB42-R, and 5 μL TB Green Premix Ex Taq II. After purification, the target gene fragment was obtained. The nucleotide sequences of qSLMYB42-F and qSLMYB42-R are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. (2) The target gene fragment was homologously recombined with the vector fragment to obtain the positive clone p35S::SLMYB42; (3) The tomato SLMYB42 gene was genetically transformed into the recipient material using the Agrobacterium-mediated method to obtain transgenic plants.

[0008] The benefits of the present invention lie in that: using existing plant genetic engineering technology, the SLMYB42 gene was cloned from tomatoes 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 in multiple stages (flowering stage, fruiting stage); the tomato SLMYB42 gene can be used to regulate plant height, providing effective genetic resources and breeding improvement strategies for the genetic improvement of plant height traits and the creation of new varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a phenotypic comparison of transgenic tomatoes at different stages; Figure 2 This is the result of fluorescence quantitative PCR detection of the relative expression level of SLMYB42 gene in transgenic tomatoes; Figure 3 This is a comparison of the plant height of transgenic tomatoes at the seedling stage; Figure 4 This is a comparison of the plant height of transgenic tomatoes during the flowering period; Figure 5 This is a comparison of the plant height of transgenic tomatoes during the fruiting period; Figure 6 This is a comparison of the length of the first internode of transgenic tomatoes; Figure 7 This is a comparison of the length of the second internode of transgenic tomatoes; Figure 8 This is a comparison of the length of the third internode of transgenic tomatoes; Figure 9 This is a comparison of the length of the fourth internode of transgenic tomatoes; 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 DESCRIPTION

[0010] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0011] 1. Tomato SLMYB42 gene cloning, vector construction and genetic transformation

[0012] Using the rice OsMYB42 protein sequence as a reference, the present invention cloned a MYB42 gene from the sequenced tomato variety Micro-Tom through bioinformatics analysis and PCR amplification and sequencing verification. This gene, named the tomato SLMYB42 gene, is designated Solyc08T002208.1. Its CDS sequence is shown in SEQ ID NO: 1, and its encoded amino acid sequence is shown in SEQ ID NO: 2.

[0013] PCR amplification was performed using the cDNA of the tomato variety Micro-Tom as a template and SlAcTin as an internal reference gene of the tomato material (total reaction volume of 10 μL, specifically: 3 μL cDNA, 1 μL forward primer qSLMYB42-F (SEQ ID NO: 3), 1 μL reverse primer qSLMYB42-R (SEQ ID NO: 4), 5 μL TB Green Premix Ex Taq II). After purification, the target gene fragment was obtained, and the target gene fragment was homologously recombined with the vector fragment. Combined with colony PCR and sequencing verification, the positive clone p35S::SLMYB42 was finally obtained.

[0014] The tomato SLMYB42 gene was genetically transformed into the tomato recipient material Micro-Tom using the Agrobacterium-mediated method. Positive transgenic lines were screened and their T2 generation transgenic lines were identified.

[0015] 2. Expression analysis of the SLMYB42 gene in transgenic tomato positive lines

[0016] The present invention obtained three independent T2 generation transgenic positive lines through DNA and RNA identification, which were designated as SLMYB42-OE#1, SLMYB42-OE#2, and SLMYB42-OE#3. The control line was Micro-Tom (CK).

[0017] 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 PCR Detection System, using SlAcTin as the internal reference gene. The total reaction volume was 10 μL and contained: 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.

[0018] Use 2 -ΔΔCT The relative expression level of SLMYB42 gene was calculated by the method, and 3 replicates were set for each group of samples. Figure 2 It can be seen that compared with the control strain Micro-Tom, the expression level of the tomato SLMYB42 gene in the leaves of the transgenic positive strains SLMYB42-OE#1, SLMYB42-OE#2 and SLMYB42-OE#3 was significantly increased by more than 22 times.

[0019] 3. Identification of plant height traits of transgenic tomato positive lines 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°C for 2 days. They were sown in seedling trays containing a mixed matrix of nutrient soil and vermiculite (volume ratio of 1:1) and cultured in a greenhouse. When the second true leaf was fully expanded, the seedlings were transferred to round pots with a diameter of 12 cm containing the same matrix and irrigated with Hoagland's nutrient solution every three days.

[0020] 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 5 It can be seen that compared with the control strain Micro-Tom, the plant height of the transgenic positive strains (SLMYB42-OE#1, SLMYB42-OE#2, SLMYB42-OE#3) increased significantly after flowering. Among them, during the flowering period, the plant height increased significantly by 7.21-9.63% (p<0.05); during the fruiting period, the plant height increased significantly by 13.05-14.62% (p<0.01).

[0021] In addition, the internode lengths of the transgenic positive lines and the control lines were measured during the flowering period. Figure 6 、 Figure 7 、 Figure 8 and Figure 9 It can be seen that during the flowering period, the lengths of the second internode, the third internode and the fourth internode of the transgenic positive lines were significantly increased, among which 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).

[0022] It can be seen that the tomato SLMYB42 gene may increase the height of tomato plants by increasing the length of each internode and is an important gene regulating tomato plant height.

[0023] In summary, the present invention significantly increased tomato plant height by overexpressing the tomato SLMYB42 gene. 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.

[0024] It should be noted that the above embodiments are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications may be made based on the above description. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. Application of the tomato SLMYB42 gene in regulating plant growth, characterized in that: 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.

2. The use according to claim 1, characterized in that The plant is tomato.

3. The use according to claim 1, characterized in that The method for increasing plant height by overexpressing the tomato SLMYB42 gene in a plant comprises the following steps: (1) Using the cDNA of tomato variety Micro-Tom as a template, PCR amplification was performed with primers qSLMYB42-F and qSLMYB42-R. The total reaction volume was 10 μL, including 3 μL cDNA, 1 μL qSLMYB42-F, 1 μL qSLMYB42-R, and 5 μL TBGreen Premix Ex Taq II. After purification, the target gene fragment was obtained. The nucleotide sequences of qSLMYB42-F and qSLMYB42-R are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. (2) The target gene fragment was homologously recombined with the vector fragment to obtain the positive clone p35S::SLMYB42; (3) The tomato SLMYB42 gene was genetically transformed into the recipient material using the Agrobacterium-mediated method to obtain transgenic plants.

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