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Compositions and methods for delivery of nucleic acid to plant cells

A plant and nucleic acid technology, applied in the field of nucleic acid delivery to plant cells, to achieve the effect of improving operational efficiency

Pending Publication Date: 2020-10-16
UNIV OF BRISTOL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most biochemical processes involved in photosynthesis are estimated to be more than 80% efficient, meaning that improving any biochemical component would be very challenging

Method used

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  • Compositions and methods for delivery of nucleic acid to plant cells
  • Compositions and methods for delivery of nucleic acid to plant cells
  • Compositions and methods for delivery of nucleic acid to plant cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0182] Example 1 - Passivated carbon nanodot core formation

[0183] core synthesis

[0184] Combine glucosamine hydrochloride (1.00 g, 4.63 mmol) and 4,7,10-trioxa-1,13-tridecanediamine (TTDDA) (1.35 mL, 5.09 mmol) with 20 ml of ultrapure H 2 O mix. The mixture was then heated under microwave irradiation (3 minutes, 700 watts). 20mL CHCl 3 Added to the oil obtained and sonicated for 10 minutes. and discard the CHCl 3 . This process was repeated until the supernatant was clear.

[0185] The oil obtained after washing was then excluded using different conditions discussed below, depending on the scale of the reaction.

[0186] small scale

[0187] Then dissolve this brown oil in 20ml ddH 2 O and centrifuged through a 10,000 MWCO spin filter (GE Healthcare Life Sciences VIVASPIN 20, 5000 rpm, 1 hour).

[0188] The sample was then passed through a 200 nm syringe filter and ddH-treated with a Biotech cellulose ester membrane at 0.5 to 01 kDa MWCO 2 O dialyzed overni...

Embodiment 2

[0195] Example 2 - CND Core Functionalization and Nanocomplex Formation

[0196] CND-L-COOH

[0197] Succinic anhydride (5 g) was added to CND-L-NH 2 (1 g) (as obtained in Example 1) in distilled water (50 mL). The mixture was reacted overnight. The reaction volume was reduced under vacuum and CND-L-COOH was purified by dialysis against ultrapure water in 500 to 1000 MWCO overnight; or by eluting a size exclusion column (G15) with ultrapure water.

[0198] CND-L-PEG-NH 2

[0199] PEG1000-diamine (0.5 g) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (0.2 g) were added to CND-L-COOH (40 mg / mL) in distilled water ( 250 mL) and reacted overnight. The reaction volume was reduced under vacuum and CND-L-PEG1000-NH was purified by dialysis against ultrapure water in 1000 to 3000 MWCO overnight; or by eluting a size exclusion column (G15) with ultrapure water 2 . [PEG 1000 is a weight average molecular weight of 1000g.mol -1 of polyethylene glycol].

[0200] In separat...

Embodiment 3

[0207] Example 3 - Preparation of CND-plasmid polymer complex

[0208] hybrid approach

[0209] 10 μL of CND-L-PEG1000-NH as obtained in Example 2 2 (20 mg / mL), 10 μL of YFP229 plasmid, 130 μL of phosphate buffered saline (PBS) and 400 μL of ultrapure water, vortexed for 30 seconds and allowed to stand for 5 minutes before any analysis or experiment was performed.

[0210] In separate examples, the YFP229 plasmid was replaced with 131Y (7Kbp), PART27 (9Kbp), 127T (7Kbp) and 1T (7Kbp).

[0211] Precipitation

[0212] 10 μL CND-L-NH 2 (20 mg / mL) and 10 μL of Cas9 plasmid were vortexed for 30 seconds. The mixture was concentrated to dryness using a high speed vacuum concentrator and stored. The solids were resuspended in ultrapure water prior to any analysis or experiment.

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Abstract

The present proposals relate to the delivery of nucleic acids to plant cells and particularly, although not exclusively, to bioengineering of plants by delivery of DNA and expression in a host plant.The proposals also relates to enhancement of photosynthesis in plants and to algaecidal compositions. In these proposals, the CND compound has the formula: CND-[(L)y-X] in which: CND is a carbon nanodot having a diameter in the range 0.5-20 nm; L is a linker selected from C1-20 alkylene, or C1-20 alkenylene groups optionally including 1-5 heteroatoms selected from -0-, -N(H)-, and -S-; X is selected from: a) PEG-(NR2)q, in which each R is independently H or C1-6 alkyl and the integer q is 0 or 1 and in which the PEG-(NR2)q is optionally associated with a nucleic acid; b) a sugar moiety; and c)an NR2 group, in which each R is independently H or C1-6 alkyl wherein PEG is a polyethylene glycol having a weight average molecular weight (Mw) greater than 200 the integer y is 0 or 1; wherein when y is 1, each of the CND and X moieties is attached to the L group either directly or via an amide bond; and z is an integer greater than or equal to 1.

Description

technical field [0001] The present invention relates to the delivery of nucleic acids to plant cells, and in particular, although not exclusively, to plant bioengineering by delivery of DNA and expression in host plants. The present invention also relates to enhancing photosynthesis in plants. The present invention also relates to algaecidal compositions. Background technique [0002] Transforming plants enables a fundamental understanding of both plant biology through modification of model species and revolutionizes commercial agriculture, and will continue to be central to future technologies such as plant synthetic biology, molecular agriculture, and genome editing. However, the main bottleneck to fully exploiting the potential of these technological advances is that for most species transformation and plant regeneration, this remains a slow, arduous, inefficient and expensive process (Altpeter, F., et al. The Plant Cell 28.7(2016):1510-1520). Currently, there is no on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/87B82Y5/00
CPCB82Y5/00C12N15/8282C12N15/87Y02A40/146C01B32/168A01H1/06A01H3/04C09K11/65C01B2202/22C01B32/174C12N15/8213C12N15/8261C12N15/8269C12N15/8279Y10S977/704
Inventor M·卡门·加兰大卫·贝尼托-阿利方索托马斯·A·斯威夫特希瑟·M·惠特尼
Owner UNIV OF BRISTOL
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