Novel watermelon sugar transporter as well as coding gene ClVST1 and application thereof

A technology of transgenic plants and proteins, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of low selection efficiency, high cost, and heavy workload.

Active Publication Date: 2022-06-24
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] For many years, people have adopted methods such as traditional conventional breeding to solve the problem of resistance to diseases and insect pests of watermelon varieties, but the cost is often large, the workload is heavy and the selection efficiency is not high.

Method used

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  • Novel watermelon sugar transporter as well as coding gene ClVST1 and application thereof
  • Novel watermelon sugar transporter as well as coding gene ClVST1 and application thereof
  • Novel watermelon sugar transporter as well as coding gene ClVST1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] This example is used to illustrate the discovery, cloning and localization of a novel watermelon sugar transporter and its encoding gene CLVST1.

[0069] 1. The discovery of a novel sugar transporter in watermelon and its encoding gene CLVST1

[0070] "By using the ultra-high-density SNP genetic map and 326 natural population materials, the fine mapping and association analysis of the sugar content of watermelon fruit were completed, and the watermelon database was used to retrieve and compare the related gene sequences of other species, and one was obtained from the watermelon variety 97103. The new protein is named as watermelon novel sugar transporter, and its sequence is shown in sequence 1 in the sequence table (consisting of 470 amino acid residues). The gene encoding the watermelon novel sugar transporter is named CLVST1 gene, and its cDNA The open reading frame is shown in Sequence 2 in the Sequence Listing (consisting of 1413 nucleotides).

[0071] 2. Cloning ...

Embodiment 2

[0082] This example is used to illustrate the acquisition of CRISPR knockout CLVST1 plants.

[0083] 1. Construction of CRISPR-ClVST1 knockout vector

[0084] 1. pCBC-DT1T2 (Xing, H.L.#, Dong, L.#, Wang, Z.P., Zhang, H.Y., Han, C.Y., Liu, B., Wang, X.C., and Chen, Q.J. *(2014). A CRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14, 327.) PCR amplification as template. The sequence composition of the four primers is as follows.

[0085] Sequence composition of DT1-BsF-ClVST1:

[0086] 5'-ATATATGGTCTCGATTGAAATCAAAACCACTCCCTTACTGGGTT-3';

[0087] Sequence composition of DT1-F0-ClVST1:

[0088] 5'-TGAAATCAAACCACTCCCTTACTGGGTTTTAGAGCTAGAAATAGC-3';

[0089] Sequence composition of DT2-R0--ClVST1: 5'-AACGGTCGAGGCGAGGCCGGCGCAATCTCTTAGTCGACTCTAC-3';

[0090] Sequence composition of DT2-BsR--ClVST1:

[0091] 5'-ATTATTGGTCTCGAAAC GGTCGGAGGCGAGGCCGGCGCAA-3'.

[0092] 20μL PCR reaction system: pCBC-DT1T2 template, 10×Buffer 2μL, 10mmol / L dNTPs 2μL, MgSO 4...

Embodiment 3

[0137] This example is used to illustrate the disease resistance response of gene knockout homozygous lines to bacterial fruit spot.

[0138] 1. Disease resistance analysis of watermelon gene knockout homozygous lines

[0139] The CLVST1 gene knockout homozygous strain VST1-CRISPR (three groups, average value for each group) and receptor 97103 (three groups, average value for each group) obtained in Example 2 were primed respectively. The vermiculite after high temperature sterilization is used as the matrix, and the vermiculite is covered after sowing, and the film is covered with heat preservation and moisturizing. Referring to the identification method of artificial inoculation of watermelon bacterial fruit spot reported by Zhao Tingchang et al., the pathogenicity of the strain was determined by spray inoculation. The virulence of M. melon fruit spot mutant strain, wild type strain and complementary strain (Ren Zhengguang, Lin Min, Jiang Wenjun, et al.. quorum sensing syst...

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Abstract

The invention belongs to the field of gene engineering, and relates to a novel watermelon sugar transporter and a coding gene ClVST1 and application thereof. The novel watermelon sugar transport protein has a sequence composition shown in a sequence 1 in a sequence table, and the coding gene ClVST1 has a sequence composition shown in a sequence 2 in the sequence table. The invention also relates to application of the novel watermelon sugar transporter protein and the novel watermelon sugar transporter coding nucleic acid sequence in watermelon disease and insect pest resistance. The invention further provides an obtaining method of the watermelon plant disease and insect pest resistant strain. The watermelon novel sugar transporter and the coding gene ClVST1 provided by the invention are closely related to disease and insect pest resistance, and the higher the expression quantity is, the stronger the disease and insect pest resistance is; therefore, a stable watermelon strain resistant to bacterial fruit blotch and aphid can be obtained by knocking out novel sugar transport of watermelons in watermelon plants, and a quick and efficient way is provided for breeding of watermelons resistant to diseases and insect pests.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to a novel watermelon sugar transporter and its encoding gene CLVST1 and application. Background technique [0002] The sugar produced by photosynthesis in watermelon leaves is transported to the fruit through the stem phloem. In the fruit, it needs to go through four steps of metabolism, unloading, transport and storage, that is, oligosaccharides such as raffinose are hydrolyzed into sucrose, the phloem of the fruit unloads the sucrose, and the sugar in the intercellular space is composed of Sugar transport proteins are transported into pulp cells and ultimately store sugar in pulp cell vacuoles. The novel sugar transporter CLVST1 is a key factor regulating fruit sugar unloading, that is, a key gene that plays a role in the second link unloading. " [0003] Watermelon (Citullus lanatus (Thunb.) Matsum & Nadai) is an important horticultural crop in the world. my country's w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/84A01H6/34A01H5/00
CPCC07K14/415C12N15/8286C12N15/8281Y02A50/30
Inventor 许勇李茂营任毅张海英宫国义郭绍贵张洁
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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