Application of tomato SlMPK20 gene in creating tomato genic male sterile line

A male sterile line and gene technology, applied in application, genetic engineering, introduction of foreign genetic material using vectors, etc., can solve the problems affecting plant vegetative growth, stress resistance and disease resistance, transgenic materials cannot be completely aborted, knockout In addition to tomato genes and other issues

Active Publication Date: 2018-09-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above technical means cannot completely inhibit the transcription and translation of MAPKs in crops, and the obtained transgenic materials cannot be completely aborted; and because the MAPK genes that have been identified and have definite functions in the model plants Arabidopsis and rice are ubiquitously expressed Therefore, interf

Method used

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  • Application of tomato SlMPK20 gene in creating tomato genic male sterile line
  • Application of tomato SlMPK20 gene in creating tomato genic male sterile line
  • Application of tomato SlMPK20 gene in creating tomato genic male sterile line

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1: qRT-PCR analysis of the spatiotemporal expression of SlMPK20 in different tissues and organs of tomato

[0023] Using qRT-PCR technology to study the spatiotemporal expression pattern of SlMPK20, it was found that SlMPK20 was expressed in different tissues, with the highest expression in stamens, and only a small amount of expression in roots, stems, leaves and young fruits. SlMPK20 was expressed in the six stages of tomato stamen development, and the expression level of SlMPK20 gene was the highest in the middle and late mononucleate stage to the binucleate stage ( figure 1 a, 1b). The specific method is as follows:

[0024]Using the cDNA of roots, stems, leaves, calyxes, petals, stamens, pistils, green mature fruits, flower buds (Bud I-Ⅴ) and fully open flowers (F) at different stages as templates, the SYBR Premix Ex Taq from TAKARA Company was used for quantification The kit was performed on a fluorescent real-time quantitative PCR instrument (CFX96, Bio...

Embodiment 2

[0025] Example 2: In situ hybridization analysis of the expression of SlMPK20 in tomato stamens at different developmental stages

[0026] In order to further accurately locate the position of the SlMPK20 transcript in stamens, the present invention cloned a specific fragment of 302bp in SlMPK20, and the upstream and downstream primers were Yw-SlMPK20-F: 5'-GCTCTAGAGCTTGGAACGCCTTCAATGG-3'; Yw-SlMPK20-R: 5 '-CGGGATCCCCTCCTTCGTCACTCTTCGC-3', using tissue in situ hybridization to analyze the expression of SlMPK20 in wild-type tomato 'Micro-Tom' Ⅵ stamens, that is, cell blast stage, tetrad stage, early mononucleate, middle and late mononucleate, Dual-core period, mature period. The results showed that the hybridization signal began to appear at the tetrad stage, and was weaker at the early and late uninucleate stages, and a stronger hybridization signal was observed at the binucleate stage. No hybridization signal was observed in the parental stage, mature stage, and the control ...

Embodiment 3

[0027] Example 3: Isolation and expression activity analysis of SlMPK20 promoter sequence

[0028] In this study, the promoter sequence of 2000bp upstream of SlMPK20 was isolated from tomato SGN database. Using tomato stamen cDNA as a template, specific primers were designed to amplify the 1964bp promoter sequence upstream of the SlMPK20 gene (as shown in SEQ ID No.2); Pro-SlMPK20-F: 5'-CCAAGCTTGACAATCACCTTTTCTACGTGTTC-3'; Pro-SlMPK20-R : 5'-CTAGTCTAGACCGAATCGAGTGATCCAAGT-3'), it was connected to pBI121, and the promoter vector pBI121-SlMPK20Pro-GUS was constructed. SlMPK20 can initiate expression in inflorescences ( figure 1 d). Further confirming the promoter activity of SlMPK20, it was found that the obvious signal first appeared in the tetrad stage, while in the uninucleate stage, the signal in the peripheral tissue was stronger and the signal in the pollen grain was weakened, and the obvious signal could also be detected in the pollen grain in the binucleate stage. GUS...

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Abstract

The invention discloses an application of a tomato SlMPK20 gene in creating a tomato genic male sterile line. The tomato SlMPK20 is subjected to fixed-point knockout via a CRISPR/Cas9 knockout system,so that a tomato transgenic line undergoing SlMPK20 gene specific knockout is obtained; by implementing the SlMPK20 knockout, complete abortion of pollen grains is caused, and just a few of parthenocarpic fruits are formed; however, the SlMPK20 knockout has no obvious influence to vegetative growth of plants and development of female reproductive organs; and the SlMPK20 can specially act to a development process of tomato anthers. The tomato genic male sterile line, which is created through the SlMPK20 knockout, can achieve an infertility rate of 100%; and based upon genetic analysis, it is indicated that the tomato genic male sterile line belongs to recessive genic male sterility, and the genic male sterile line can be propagated by taking corresponding wild-type tomatoes as a maintainerline. The sterile line, which is free from transgenic ingredients, can be obtained through offspring screening. The sterile line can hybridize with a great amount of tomato breeding materials (a restorer line), so that the first generation of hybrid can be obtained. With the application of the method provided by the invention, sterile traits can be rapidly led into normal tomato breeding materials; therefore, a broad application prospect is achieved in tomato cross-breeding.

Description

technical field [0001] The invention belongs to the fields of vegetable molecular breeding and plant genetic engineering, and in particular relates to the application of tomato S1MPK20 gene in establishing tomato nuclear male sterile lines. Background technique [0002] Tomato (Solanum lycopersicum L.) is a plant of the genus Tomato in the family Solanaceae. It has a large adaptability, high yield and rich nutrition, and plays an important role in the production and consumption of vegetables in my country. Utilization of heterosis is an important and effective way to increase crop yield, and the use of sterile lines can better exert heterosis. Therefore, the research on tomato male sterility is increasing day by day. The use of tomato male sterile lines for breeding can not only save manpower and material resources in breeding, but also improve breeding efficiency and ensure the purity of seeds. [0003] Nuclear male sterile breeding is one of the important ways to utilize ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H6/82A01H5/00
CPCC12N15/8213C12N15/8289
Inventor 卢钢陈丽妃杨丹丹潘长田
Owner ZHEJIANG UNIV
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