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Method for cultivating long-storage tomato plants by virtue of silent tomato gene SlNZG

A tomato and plant technology, applied in botany equipment and methods, genetic engineering, plant genetic improvement, etc., can solve the problems of tomato rot and waste, and achieve the effects of long cycle, good market application prospects, economic value, and high cost

Inactive Publication Date: 2015-07-15
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the process of tomato ripening, transportation, resale and storage, there will be a lot of tomato rot and waste

Method used

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  • Method for cultivating long-storage tomato plants by virtue of silent tomato gene SlNZG
  • Method for cultivating long-storage tomato plants by virtue of silent tomato gene SlNZG
  • Method for cultivating long-storage tomato plants by virtue of silent tomato gene SlNZG

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, clone tomato gene SlNZG core fragment

[0027] Design specific primers (NZGi-F and NZGi-R) according to the sequence of tomato gene SlNZG (AK327648) and add an enzyme cleavage site at the 5' end, the sequence of which is as follows:

[0028] NZGi-F: 5'-cggggtacc atcgat cactttcgtctcgcgacata-3' (SEQ ID NO.1), the underline is the Cla I restriction site, and the bold is the Kpn I restriction site;

[0029] NZGi-R: 5'-ccg ctcgag tctagaacttctcatccaatggaccac-3' (SEQ ID NO.2), the underline is the Xba I restriction site;

[0030] The total RNA of tomato was extracted, and the total RNA was used as the template. According to the instructions of the RNA PCR Kit (AMV) Ver.3.0, oligodT was used as the primer to reverse-transcribe and synthesize cDNA. -R is a specific primer for PCR amplification. The PCR amplification program is 94°C pre-denaturation for 5min; 94°C denaturation for 30s; 56°C annealing for 30s; The PCR amplification products were identified by a...

Embodiment 2

[0031] Embodiment 2, the construction of SlNZG silencing vector

[0032] The pMD18-SlNZG vector was double digested with Cla I and Xba I, and then connected with the pHANNIBAL vector that had undergone the same double digestion to the PHANNIBAL vector containing the CaMV35S promoter and terminator to obtain the forward recombinant plasmid pHANNIBAL::SlNZG; The pMD18-SlNZG vector was double digested with Kpn I and Xho I, and then ligated with the recombinant plasmid that had undergone the same double digestion to obtain the RNAi intermediate vector pHANNIBAL::SlNZG(i), which was double digested with Sac I and Spe I pHANNIBAL:: The SlNZG (i) intermediate vector is then connected with the pBin19 vector double digested by Sac I and Xba I to obtain the final RNAi vector, named pBIN19::SlNZG, and pBin19::SlNZG is transformed into Escherichia coli DH5α to obtain pBin19:: Escherichia coli DH5α of SlNZG.

Embodiment 3

[0033] Example 3. Transformation of Agrobacterium with RNAi expression vector containing tomato SlNZG core fragment

[0034] Agrobacterium tumefaciens LBA4404 was streaked on YEB solid medium containing 1.2% (w / w) agar, 50 mg / L rifampicin, and 500 mg / L streptomycin, and cultured at 28±2°C in the dark for 2-2.5 days To grow a single colony; E. coli DH5α containing pBin19::SlNZG and E. coli containing motile plasmid pRK2013 were streaked on LB solid medium containing 50 mg / L kanamycin, under the condition of 37±2℃ Inverted culture for 14 to 16 hours until a single colony grows; a single colony of Agrobacterium tumefaciens LBA4404, a single colony of Escherichia coli containing the motile plasmid pRK2013 and a single colony of Escherichia coli DH5α containing the recombinant plasmid pBin19::SlNZG were overlapped and evenly coated on the The YEB solid medium (pH 7.2) containing 1.2% (w / w) agar was placed in a circle with a diameter of 1 cm in the center, and was co-cultured upside...

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Abstract

The invention discloses a method for cultivating long-storage tomato plants by virtue of a silent tomato gene SlNZG. The method comprises the following steps: reversely connecting a nucleotide sequence shown in SEQ ID NO. 3 to a pBin19 vector Sac I and Xba I restriction enzyme cutting sites to obtain an RNAi vector pBin19::SlNZG; then transforming agrobacterium by using a pBin19::SlNZG vector to obtain agrobacterium engineering bacteria, then performing transfection on a tomato explant which is cut and pre-cultured by using the agrobacterium engineering bacteria, performing co-culture, induced germination and rooting culture, and finally screening transgenic tomato plants to obtain long-storage tomato plants; and performing SlNZG gene silencing expression on the obtained long-storage tomato plants. Fruit storage tolerance experiments show that the storage tolerance of tomato fruits of which SlNZG genes are silent can be greatly improved; and the method disclosed by the invention can be used for cultivating long-storage tomato varieties.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for cultivating storable tomato plants by silencing the tomato gene SlNZG. Background technique [0002] Tomato is a worldwide favorite vegetable, and tomato is rich in nutrients and has a special flavor. It has the effects of losing weight, eliminating fatigue, increasing appetite, improving protein digestion, and reducing stomach bloating and food accumulation. In nature, fruit softening is beneficial for seed dispersal and species stabilization; in agriculture and commercial production, fruit softening is beneficial for improving taste and nutritional value. However, the fruit is prone to rot and deterioration after excessive softening, resulting in huge economic losses. Under natural conditions, the immature tomato fruit can be kept at room temperature for about 2 months, while the ripe red fruit can only be kept for about 30 days. In the process of tomato...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/29A01H5/00
Inventor 胡宗利陈国平李安洲郭旭虎朱智国王玲玲
Owner CHONGQING UNIV
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