Method for improving lily anti-aging property by means of antisense RNA technology

A lily flower and antisense technology, applied in the direction of recombinant DNA technology, botany equipment and method, biochemical equipment and method, etc., can solve the problems of prolonging the life of lily flower

Inactive Publication Date: 2008-11-12
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there has been no report on prolonging the lifespan of lilies using antisense RNA technology of the ACC oxidase gene.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Obtaining the Antisense Fragment of Liaco Gene in Lily by Gene Cloning Method

[0019] 1. Extraction of lily total RNA

[0020] Take a small amount of lilium musk lily "Snow Queen" (Lilium longiflorum Thunb'Snow Queen') petal tissue, freeze it with liquid nitrogen, quickly grind it with a mortar, add 1.5mL Eppendorf containing 1mL TRIzol (TRIzolReagents, GIBCO BRL, USA) In the tube, after fully shaking, place it at room temperature for 5 minutes, add 200 μL chloroform, shake vigorously for 15 sec, place it at room temperature for 2-3 minutes, then centrifuge at 4°C and 12,000 g for 15 minutes; pipette the supernatant (about 600 μL) into a clean 1.5 mL Add an equal volume of isopropanol to the Eppendorf tube, mix it upside down, let it stand at room temperature for 10 minutes, then centrifuge at 12,000 g for 10 minutes at 4°C; discard the supernatant, add 1 mL of 75% ethanol to wash, shake, and place Centrifuge at g for 5 min; dry at room temperature for 15-20 min and d...

Embodiment 2

[0025] The Liaco antisense gene antiLlaco is operably linked to the expression control sequence to form a plant expression vector containing antiLlaco

[0026] Using pCAMBIA1304 as the expression vector, the Liaco antisense gene (antiLlaco) in Example 1 was inserted between the CaMV35S promoter and the gfp gene of the pCAMBIA1304 vector. Specifically, PMD18-T+antiLlaco and pCAMBIA1304 were digested with BglII / NcoI, and then large fragments of antiLlaco and pCAMBIA1304 were recovered, ligated and transformed, single clones were picked, and plasmids were extracted for PCR detection and enzyme digestion verification. The constructed plant expression vector CAMBIA1304::p35S-antiLlaco-nos containing the antiLlaco gene contains the hygromycin resistance gene hpt, which can be screened with hygromycin in the process of genetic transformation and regeneration, and also contains gfp and gus. A reporter gene is conducive to the rapid detection of the results of genetic transformation. ...

Embodiment 3

[0029] Transforming the plant expression vector containing the antiLlaco gene into Agrobacterium tumefaciens to obtain the Agrobacterium tumefaciens strain containing the antiLlaco gene plant expression vector for transforming lily

[0030] The plant expression vector CAMBIA1304::p35S-antiLlaco-nos that contains antiLlaco gene in embodiment 2 is transformed into Agrobacterium tumefaciens (such as EHA105, has the biological material that publicly sells for the market, can buy from Australia CAMBIA company, bacterial strain number is Gambar 1), and PCR verification. The results showed that the plant expression vector containing antiLlaco gene had been successfully constructed into the Agrobacterium tumefaciens strain.

[0031] This embodiment has obtained the plant expression vector CAMBIA1304::p35S-antiLlaco-nos containing antiLlaco gene Agrobacterium tumefaciens strain EHA105 (CAMBIA1304::p35S-antiLlaco-nos), this bacterial strain can be used to improve Lily's aging resistance b...

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Abstract

The invention provides a method for improving anti-aging capacity of lily by means of an antisense RNA technology, belonging to the biologic technical field. The invention comprises the following steps of: cloning an antisense fragment of Liaco gene from lily; building a plant expression vector containing antiLiaco of a Liaco antisense gene; transferring the antiLiaco gene into lily to re-grow a plant by mediation of agrobactrium tumefaciens; detecting an integration condition of outside source purpose gene antiLiaco by PCR; testing anti-aging performance of transgenic plant based on aging test; and finally obtaining transgenic lily plant with prolonged service life. The invention provides a method for improving lily service life and has an important meaning for culturing new variety of lily with improved service life.

Description

technical field [0001] The invention relates to a method for improving aging resistance in the field of biotechnology, in particular to a method for improving lily's aging resistance by using antisense RNA technology. Background technique [0002] Many species and varieties of Lilium spp. are collectively called "lily". Liliaceae (Liliaceae) plants are perennial open field bulb flowers. There are many wild species or artificially cultivated varieties, and the genetic variation of germplasm resources is very rich. According to incomplete statistics, there are more than 100 species of Lilium plants in the world (130 species recorded in literature). China is one of the natural distribution centers of Lilium plants. According to literature records, there are about 55 species of Lilium plants native to China. Among them, 36 species and 18 varieties are unique to China. Many species in the genus have huge flowers, bright and colorful flowers, various postures, and pleasant fra...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C12Q1/68A01H1/00A01H5/00
Inventor 唐克轩唐东芹许洁婷王月
Owner SHANGHAI JIAO TONG UNIV
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