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Method for in-vitro verification of self-incompatibility function of S-RNase in citrus and pomelo

A citrus, in vitro technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problem of plant loss and rejection, and achieve the effect of avoiding transgenic verification

Pending Publication Date: 2019-01-25
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

In 1994, Leed et al. proved the function of this glycoprotein for the first time through transgenic technology. 2 S 3 Antisense suppression of S in Solanaceae plants 3 -RNase expression, eventually causing plants to lose rejection of S 3 pollen capacity

Method used

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  • Method for in-vitro verification of self-incompatibility function of S-RNase in citrus and pomelo
  • Method for in-vitro verification of self-incompatibility function of S-RNase in citrus and pomelo

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Embodiment Construction

[0033] The principles and features of the present invention are described below in conjunction with the accompanying drawings, and the examples given are only used to explain the present invention, and are not intended to limit the scope of the present invention.

[0034] 1. Shatian pomelo CgRNS 1 and CgRNS 2 expression and purification of

[0035] 1.1 Construction of expression vector

[0036] Get-CgRNS 1 and CgRNS 2 According to the amino acid sequence analysis, it was found that the first 21 amino acids were hydrophobic signal peptides. Designing primers to amplify CgRNS with truncated signal peptide using Shatian pomelo style cDNA as template 1 and CgRNS 2 gene, and add adapters with restriction sites to the primers. The final synthetic primers are as follows:

[0037] 6P-RNS 1 -F: 5'--GGGCCCCTGGGATCCAACAATTCTGGTTTTGACCAC--3' (SEQ ID NO: 1)

[0038] 6P-RNS 1 -R: 5'--GTCGACCCGGGAATTC TTAAGGCGGGGGAAAGGTA--3' (SEQ ID NO: 2)

[0039] 6P-RNS 2 -F: 5'--GGGCCCCTGGGAT...

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Abstract

The invention relates to a method for in-vitro verification of self-incompatibility function of S-RNase in citrus and pomelo. The method comprises that follow steps: step 1, obtaining an S-RNase protein; 2, collecting pollen from that donor plant; step 3: treating pollen obtained in the step 2 with S-RNase protein obtained in the step 1, and measuring the effect of the treatment of S-RNase proteinon the pollen germination pollen tube. By adopting the method of the invention, the self-incompatibility style gene of citrus can be rapidly verified in an in vitro culture mode, and the proper pollen can be treated with a recombinant protein with an appropriate concentration, which can cause a self-compatibility or incompatibility reaction, thereby avoiding the problem of transgenic verificationof the citrus S gene.

Description

technical field [0001] The invention relates to the field of citrus planting and breeding, and more particularly relates to a method for in vitro verification of the self-incompatibility function of citrus pomelo S-RNase. Background technique [0002] The citrus pomelo (Citrus grandis) belongs to the genus Citrus of the family Rutaceae, which has the phenomenon of self-incompatibility. The trait of self-incompatibility plays an important role in the evolution of species, which promotes outcrossing, inhibits selfing, and increases population diversity. Since the 1990s, research on self-incompatibility has mainly focused on Brassicaceae, Poppyaceae, Solanaceae, Rosaceae, and Scrophulariaceae. There are three different types of self-incompatibility mechanisms in these plants, and each type of mechanism is regulated by different genes. Among them, the most widely distributed self-incompatibility mechanism in plants is based on the S-RNase-mediated gametophyte mechanism represe...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/44C12Q1/02
Inventor 柴利军梁梅曹宗洪邓秀新
Owner HUAZHONG AGRI UNIV
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