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Gene segment for regulating synthesis of solanaceae flavonoids and caffeoyl quinic acid, and uses thereof

A technology of caffeoylquinic acid and gene fragments, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2014-02-19
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, how to increase the content of flavonoids and caffeoylquinic acid in tomato has always been a problem that cannot be solved by existing conventional technologies.

Method used

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  • Gene segment for regulating synthesis of solanaceae flavonoids and caffeoyl quinic acid, and uses thereof
  • Gene segment for regulating synthesis of solanaceae flavonoids and caffeoyl quinic acid, and uses thereof
  • Gene segment for regulating synthesis of solanaceae flavonoids and caffeoyl quinic acid, and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Cloning of embodiment 1 fruit-specific E8 promoter and SlMYB12 gene full-length cDNA, construction of plant expression vector:

[0044] 1. Cloning of Tomato Fruit-Specific E8 Promoter and S1MYB12 Gene cDNA

[0045] Using the genome DNA of tomato variety Zhongshu No. 4 as a template, primers E8PF1, whose gene sequence is shown in SEQ ID NO: 4, and E8PR1, whose gene sequence is shown in SEQ ID NO: 5, were amplified in combination to obtain the expected 1.1kb fragment ( figure 2 shown). Furthermore, the combined amplification product of E8PF1 / R1 was cloned by TA, and connected to the PCR product cloning vector pGEM-T Easy vector. Transform Escherichia coli DH5α to obtain a large number of clones. The 12 randomly selected clones were identified by colony PCR using the specific primer combination E8PF1 / R1, and 8 clones with the target fragment insertion were obtained. Two clones were randomly selected to extract plasmids and verified by EcoR I digestion to confirm that...

Embodiment 2

[0053] Example 2 Agrobacterium-mediated tomato genetic transformation and PCR detection of transgenic plants

[0054] 1. Genetic transformation of tomato mediated by Agrobacterium

[0055] Using tomato cotyledons as explants, the plant expression vector pX6-E8::SlMYB12 was transformed into Micro-Tom, CSl09-03, and cherry tomatoes by Agrobacterium (AGL I strain)-mediated leaf disk transformation method. body material. All operations are carried out in a strict aseptic environment, and the basic procedures for transgenic operations are as follows:

[0056] 1) The seeds were sterilized with 75% ethanol for 1 min, treated with 2% NaOCl available chlorine for 15 min, washed 4-5 times with sterile distilled water, sowed in 1 / 2MS medium with appropriate spacing, and then transferred to Cultivate under 16h light / 8h dark until the cotyledons are fully unfolded.

[0057] 2) Cut off the front and back sections of the cotyledon with a blade, and take a leaf about 0.5 cm in the middle,...

Embodiment 3

[0065] Example 3 HPLC analysis of tomato fruit flavonoids and caffeoylquinic acid content

[0066]There was no significant difference between transgenic tomato and non-transgenic plants in the vegetative growth period. During the reproductive growth period, the fruit shape of a few lines changed, and the umbilical tip and fruit swelled (mainly occurred in Micro-Tom grown in natural light greenhouses). From the discoloration stage, the skin of the transgenic fruit gradually tends to orange red or dark yellow, while the control fruit still maintains the corresponding red (Micro-Tom, CSL09-03) or light yellow (cherry tomato), Micro-Tom, CSl09-03 background The color of the transgenic fruit pulp was only slightly changed, and the cherry tomato was not obvious. The follow-up HPLC analysis proved that the color change of the transgenic fruit was related to the increase of flavonoid substances such as rutin and the content of caffeoylquinic acid ( Figure 8 , Figure 9 , Figure 10...

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Abstract

The invention relates to the technical field of plant genetic engineering, and provides a gene segment DNA capable of assigning capacities to solanaceae accumulated flavonoids such as rutin, kaempferol rutin and naringenin chalcone, and caffeoyl quinic acid, wherein the gene segment has a gene sequence shown in SEQ ID NO:1, and is obtained from tomato, and comprises a control gene S1MYB12 for controlling the synthesis of solanaceae flavonoids and caffeoyl quinic acid. The inventor provides a specific method of separation cloning, function verifying and applying of the gene segment. A tomato endogenous gene S1MYB12 is driven to be specifically expressed in the tomato fruit by using a tomato fruit specificity expression promoter E8, thus the contents of the flavonoids and the caffeoylquinic acid in the tomato fruit are greatly increased, and the function of the tomato endogenous gene S1MYB12 of regulating the synthesis of the flavonoids and the caffeoyl quinic acid is verified.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, and provides a gene fragment for regulating the synthesis of Solanaceae flavonoids and caffeoylquinic acid and its specific application method. The gene sequence of the fragment is shown in SEQ ID NO: 1, which can be endowed with Plants accumulate flavonoids including rutin, kaempferol rutin, and naringenin chalcone, as well as caffeoylquinic acid. Background technique [0002] Flavonoids, also known as vitamin P, often exist together with vitamin C, and refer to a large class of substances with α or β-phenylbenzene-condensed pyrone (Zhang Ganliang et al., 2005). Widely distributed in nature, it often exists in the roots, stems, leaves, flowers, and fruits of higher plants and ferns in the form of free or glycosides, and is an active ingredient of many Chinese herbal medicines (Graf et al., 2005; Hertog et al., 1995). Due to various physiological activities, flavonoids have at...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/84A01H5/00
Inventor 储朝辉丁新华周萌于丹丹
Owner SHANDONG AGRICULTURAL UNIVERSITY
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