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Application of lycium ruthenicum LrEIN3 gene in delaying fruit ripening

A technology of black wolfberry and fruit, which is applied in the field of biotechnology and genetic engineering, can solve the problems that restrict the development of black wolfberry industry, and achieve the effect of delaying natural ripening and extending shelf life

Active Publication Date: 2022-04-26
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002]Lycium ruthenicum (Lycium ruthenicum) is a small perennial deciduous shrub of the genus Lycium genus in the Solanaceae family. The fruit of Lycium ruthenicum softens rapidly after ripening, making the market of Lycium ruthenicum more dry and mature. Mainly, it greatly restricts the development of black wolfberry industry

Method used

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  • Application of lycium ruthenicum LrEIN3 gene in delaying fruit ripening
  • Application of lycium ruthenicum LrEIN3 gene in delaying fruit ripening
  • Application of lycium ruthenicum LrEIN3 gene in delaying fruit ripening

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction of CRIPSR-Cas9 gene editing vector pAGM4723::CR-LrEIN3 and transformation of Agrobacterium

[0046] In this example, the CRIPSR-Cas9 gene editing vector pAGM4723::CR-LrEIN3 was first constructed, and then the gene editing vector pAGM4723::CR-LrEIN3 was transformed into Agrobacterium GV3101 to obtain recombinant Agrobacterium pAGM4723::CR-LrEIN3 / GV3101, specifically including the following steps:

[0047] 1. Obtain the LrEIN3 gene CRIPSR-Cas9 gene editing target sequence

[0048] Obtain the genome information of LrEIN3 from the genome information of Lycium barbarum, analyze the structural domain of the transcription factor of the LrEIN3 gene (sequence is SEQID No.1), and the analysis results are as follows figure 1 shown.

[0049] Using the online software http: / / skl.scau.edu.cn / targetdesign / , on the LrEIN3 gene sequence, select the sequence with a high score, the exon region, and the 20bp length of the front end of the NGG sequence. Through th...

Embodiment 2

[0074] Example 2 Obtaining and identification of LrEIN3 gene-edited Lycium barbarum plants of T0 generation

[0075] In this example, T0 generation LrEIN3 gene-edited Lycium barbarum plants were obtained and identified. The specific method is as follows:

[0076] 1. Transform the recombinant bacterium pAGM4723::CR-LrEIN3 / GV3101 of Example 1 into the leaf disk explants of Lycium barbarum berries (transformation method refers to the University of Chinese Academy of Sciences, Xue Dinglei's master's degree thesis), and then contain 0.5mg / L6-benzylamino In the MS solid medium (pH 5.8) of purine and 0.4mg / L1-naphthaleneacetic acid, co-cultivate for 48 hours at 25°C under dark conditions; In MS solid medium (pH 5.8) of L1-naphthylacetic acid, 200mg / L cephalosporin and 50mg / L kanamycin, cultivate it under the conditions of 25°C, photoperiod 16h / d, and light intensity 3000lux until growth Regenerated buds: Cut off the regenerated buds when they grow to 2-3 cm, transfer them to MS soli...

Embodiment 3

[0087] Example 3 Research on Fruit Ripening of LrEIN3 Gene Edited Lycium barbarum Lycii Plants

[0088] Continuous observation of the phenotype of the fruit of the LrEIN3 gene-edited Lycium barbarum LrEIN3-4 and lrein3-9 plants obtained in Example 2 after flowering, the results are as follows Figure 5 shown.

[0089] From Figure 5 It can be seen that the fruit development of LrEIN3 gene-edited Lycium barbarum plants was obviously affected. The fruit of the wild-type Lycium barbarum plant suddenly turned color and matured at 32 days after flowering, and then the fruit suddenly swelled; while the fruit of the LrEIN3 gene-edited Lycium barbarum plant lrein3-4 turned slightly at 32 days after flowering, and the color was slightly darker at 34 days. The fruits of the LrEIN3 gene-edited Lycium barbarum plant lrein3-9 were always small after flowering, and did not change color until 34 days later, and the fruit size did not change significantly. After 37 days, the fruit color dee...

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Abstract

The invention discloses an application of a lycium ruthenicum LrEIN3 gene in delaying fruit ripening. The lycium ruthenicum LrEIN3 gene has a nucleotide sequence as shown in SEQ ID No. 1 or an amino acid sequence as shown in SEQ ID No. 2. The lycium ruthenicum LrEIN3 gene has a nucleotide sequence as shown in SEQ ID No. 2. In the invention, the lycium ruthenicum LrEIN3 gene is edited by a CRIPS-Cas9 technology, so that the lycium ruthenicum LrEIN3 gene is mutated and then is transferred into lycium ruthenicum, compared with wild lycium ruthenicum, the fruit is ripe and discolored after 32 days, the fruit of an LrEIN3 gene mutant plant starts to color after 34 days, and the ripening of the fruit is lagged. The LrEIN3 gene can delay natural ripening of the fruits, so that the shelf life of the lycium ruthenicum is prolonged.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and genetic engineering. More specifically, the invention relates to the application of a LrEIN3 gene of Lycium barbarum and its encoded protein in delaying the fruit ripening of Lycium barbarum. Background technique [0002] Lycium ruthenicum (Lycium ruthenicum) is a small perennial deciduous shrub belonging to the Solanaceae Lycium genus. The fruit of Lycium ruthenicum softens rapidly after ripening, which makes the market of Lycium ruthenicum mainly dry and ripe fruits, which greatly restricts the development of Lycium ruthenicum industry. [0003] Gene editing technology can achieve targeted knockout of specific genes to create artificial mutants. For non-model plants, gene editing technology can accelerate the process of species domestication, obtain mutants through short-term and efficient biotechnological means, and break the barriers of traditional breeding methods. For example, tar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/84C12N1/21C12N15/113C12N9/22C07K14/415A01H5/08A01H6/00C12R1/01
CPCC07K14/415C12N15/113C12N9/22C12N15/8218C12N15/8261C12N2310/20
Inventor 曾少华王瑛艾培炎
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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