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Gene regulating sugar content in tomato fruit and its application

A tomato, gene technology, applied in the field of biotechnology and genetic breeding

Active Publication Date: 2022-07-19
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few studies reported in tomato, the mechanism of action of cytoplasmic invertase on the sugar metabolism of tomato fruit

Method used

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  • Gene regulating sugar content in tomato fruit and its application
  • Gene regulating sugar content in tomato fruit and its application
  • Gene regulating sugar content in tomato fruit and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Construction of RNAi Vector and Recombinant Agrobacterium

[0029] The SlCIN2-RNAi vector was constructed by gateway technology.

[0030] 1. Extract tomato RNA, reverse transcribed into cDNA, and amplify the target fragment SlCIN2 gene with cDNA as a template, and the target fragment size is 170bp ( figure 1 A), its nucleotide sequence is as shown in SEQ ID NO.1;

[0031] The primer sequences are as follows:

[0032] SlCIN2-F: 5'-CGCACCTGGTCCATTGTGTCGTCTT-3', shown in SEQ ID NO.2.

[0033] SlCIN2-R: 5'-GGTGTGTTCTTAGGGTCGCT-3', shown in SEQ ID NO.3.

[0034] The amplification reaction system is as follows:

[0035] High-fidelity DNA polymerase 10 μL, SlCIN2-F 1 μL, SlCIN2-R 1 μL, cDNA 2 μL, RNase-FreeddH2O 6 μL.

[0036] The amplification reaction procedure is as follows:

[0037] 98°C for 30s, 1 cycle; 98°C for 10s, 55°C for 20s, 72°C for 10s, 35 cycles; 72°C for 7min.

[0038] 2. Through the TOPO reaction, take 4 μL of the amplification product of the target fra...

Embodiment 2

[0040] Recombinant Agrobacterium transformation tomato

[0041] Using Micro-Tom tomato as the test material, the recombinant vector plasmid was transformed into tomato by leaf disk method to obtain stable genetic transformation material.

[0042] When the tomato seedlings in the medium grow until the cotyledons are fully stretched ( figure 2 A), cut cotyledons with a length of 0.5 cm and place them in the pre-culture medium for 2 days in the dark, using OD 600 Agrobacterium reaching 0.6-0.8 infects tomato cotyledons, and the infected leaves are placed in the co-culture medium with the back of the leaves facing up ( figure 2 B), after dark culture for 2 days, the explants were transferred to bacteriostatic medium for culture. When the explants were grown in it for two weeks, they were transferred to sprout medium ( figure 2 C), after which the medium was changed at two-week intervals. When the adventitious buds grow to 2-3cm, the adventitious buds are cut from the stem b...

Embodiment 3

[0044] Identification of SlCIN2-silenced transgenic tomato plants

[0045] The DNA of transgenic tomato leaves was extracted, and the screened silenced SlCIN2 transgenic plants were identified by PCR. SlCIN2 silenced plants were detected using the specific primers designed from the Bar gene locus on the pB7GWIWG2 vector plasmid.

[0046] The specific primer sequences are as follows:

[0047] Bar-F: 5'-GAAGTCCAGCTGCCAGAAA-3' as shown in SEQ ID NO.4.

[0048] Bar-R: 5'-CACCATCGTCAACCACTACA-3'T, as shown in SEQ ID NO.5.

[0049] PCR amplification was performed with DNA as a template and electrophoresis was performed to detect the specific fragment length of 439bp. The results showed that a specific fragment of 439 bp was detected in 10 transgenic lines ( image 3 ), while the negative controls (ddH2O and untransformed wild-type Micro-Tom) did not amplify any bands. These 10 lines are all transgenic positive plants, which can be used for subsequent experimental analysis.

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Abstract

The invention relates to the fields of biotechnology and genetic breeding, in particular to a gene for regulating sugar content in tomato fruit and its application. The gene for regulating tomato fruit sugar content provided by the present invention is SlCIN2, and its nucleotide sequence is shown in SEQ ID NO.1. The present invention first extracts tomato RNA, reverse transcribes cDNA, designs amplification primers, uses cDNA as a template, and amplifies SlCIN2 gene; uses gateway technology to construct SlCIN2 gene into pB7GWIWG2 vector, screen correct positive clones, and obtain positive clones Transform into LBA4404 Agrobacterium competent to obtain recombinant Agrobacterium, and transform the recombinant Agrobacterium into tomato to obtain tomato plants with increased fruit sugar content.

Description

technical field [0001] The invention relates to the fields of biotechnology and genetics and breeding, in particular to a gene for regulating the sugar content of tomato fruit and its application. Background technique [0002] The fruit quality of tomato consists of appearance quality, flavor quality, nutritional quality and processing and storage quality. Among them, flavor qualities including sweetness, sourness, aroma, etc., were related to soluble sugars, organic acids and volatile aromatic compounds in fruits. The loss of flavor can seriously affect the organoleptic quality of the fruit, which in turn affects its economic value. Improving the sweetness of tomato fruit is an important part of improving the quality of tomato. The content of sucrose in the fruit of most tomato varieties is very low, mainly glucose and fructose. The content of glucose and fructose in tomato fruit is positively correlated with the activity of invertase. During tomato fruit development, suc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/24C12N15/84A01H5/08A01H6/82
CPCC12N9/2402C12N15/8245
Inventor 姜晶张琼琼刘欣
Owner SHENYANG AGRI UNIV
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