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Application of tomato mir156e-3p gene in improving tomato low temperature resistance and plant overexpression vector

An overexpression vector, tomato technology, applied to the application of tomato miR156e-3p gene in improving low temperature resistance of tomato, in the field of plant overexpression vector, can solve the problems affecting tomato yield, etc., to improve tomato low temperature resistance, low temperature resistance sex-reducing effect

Active Publication Date: 2021-12-07
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the embodiment of the present invention is to provide an application of tomato miR156e-3p gene in improving tomato low temperature resistance, so as to solve the existing problem that low temperature stress seriously affects tomato yield in winter and spring

Method used

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  • Application of tomato mir156e-3p gene in improving tomato low temperature resistance and plant overexpression vector
  • Application of tomato mir156e-3p gene in improving tomato low temperature resistance and plant overexpression vector
  • Application of tomato mir156e-3p gene in improving tomato low temperature resistance and plant overexpression vector

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Effect test

Embodiment 1

[0026] Example 1: Construction of miR156e-3p gene overexpression vector

[0027] To understand the molecular mechanism of plant response to low temperature, the miR156e-3p precursor sequence was cloned from the tomato genome. According to the sequence analysis of the coding region, design specific primers pre-F and pre-R, and respectively add restriction enzyme sites (Asc I and BamH I) on the primers, the sequences are shown in SEQ ID NO.3 and 4 . The DNA fragment of the miR156e-3p precursor sequence was amplified by PrimerSTAR high-fidelity enzyme PCR, and then the PCR amplified fragment and the vector were digested, and the precursor sequence was connected to pFGC1008-HA to obtain an overexpression vector. The recombinant plasmid was sequenced and confirmed, and the obtained gene miR156e-3p precursor nucleotide sequence is shown in SEQ ID No.2. The results showed that the cloned sequence was consistent with the published sequence (MI0029110) in miRBase.

Embodiment 2

[0028] Example 2: Construction of miR156e-3p gene interference vector

[0029] Short tandem target mimic Short tandem target mimic (STTM), which effectively reduces the activity of endogenous microRNA by simulating the target, is a powerful tool for studying endogenous microRNA. Specific primers tgt-F and tgt-R were designed using STTM technology, and a restriction enzyme cutting site Eco31I (BsaI) was added to the primers respectively, and the sequences are shown in SEQ ID NO.5 and 6. Use PrimerSTAR high-fidelity enzyme PCR amplification to recover a 100bp electrophoresis fragment, then digest the PCR amplified fragment, and connect it to the Eco31I (BsaI) restriction site of the pBWA(V)HS-amiRNA vector. The above recombinant plasmids were sequenced and confirmed.

Embodiment 3

[0030] Example 3: Construction and detection of tomato miR156e-3p gene overexpression and silenced plants

[0031] The overexpression vector pFGC1008::miR156e-3p-HA and the gene interference vector pBWA(V)HS-amiRNA(miR156e-3p) were transformed into Agrobacterium GV3101, and tomato cotyledons were infected to induce callus and induce differentiation of resistance And rooting culture, obtain tissue culture seedling, carry out the test of chloramphenicol resistance and kanamycin resistance respectively to T2 generation overexpression seed and silent seed, select 3 / 4 to have resistance and remaining 1 / 4 has no resistance The strain indicates that the overexpression vector or interference vector of the target gene is connected in the strain and inserted in a single copy form. These plants were removed and harvested individually.

[0032] Positive plants for overexpression and gene silencing were verified by qRT-PCR technology. The results showed that 4 lines were detected in the ...

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Abstract

The invention discloses tomato miR156e-3p gene and its application in plant low temperature resistance and plant overexpression vector. The mature body sequence of the gene miR156e-3p involved in tomato low temperature regulation is shown in SEQ ID NO.1, and its precursor sequence is shown in SEQ ID NO.2. Tomato miR156e‑3p overexpression plants and gene silencing plants were constructed by transgenic means, and the expression level of the gene miR156e‑3p was regulated to study its regulatory mechanism for tomato low temperature resistance. The results showed that miR156e-3p gene silencing plants could improve the low temperature resistance of tomato, and the low temperature resistance of overexpressed plants decreased. The invention provides genetic resources for cultivating new varieties of low-temperature-resistant tomato, has good potential application value, and lays a theoretical foundation for studying the mechanism of tomato plants responding to adversity signals and the molecular mechanism of tolerance to unfavorable environments.

Description

technical field [0001] The invention relates to the fields of genetic engineering, molecular biology, physiology and the like, and in particular relates to an application of tomato miR156e-3p gene in improving low temperature resistance of tomato and a plant overexpression vector. Background technique [0002] Tomato (Solanum lycopersicum), a horticultural crop, originated in tropical and subtropical regions and is warm-loving. Tomato fruit is rich in vitamins and minerals. It can be eaten fresh as a vegetable or as a fruit. It plays an important role in the protection of the human cardiovascular system and the repair of the skin after sun exposure. It is a well-deserved "fruit in the dish". As an off-season vegetable, tomato is mainly cultivated in winter and spring, and the loss caused by low temperature stress in its growth and development stage is particularly serious. Therefore, it is of great practical significance to improve the ability of tomato to cope with adverse ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H5/00A01H5/10A01H6/82
CPCC12N15/8218C12N15/8273
Inventor 周艳虹张露月喻景权
Owner ZHEJIANG UNIV
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