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Pyrus sugar transport gene PbSWEET4 and applications thereof

A gene and pear-derived technology, applied in the field of pear sugar transport gene PbSWEET4 and its recombinant expression vector, can solve the problems of lack of research on the regulation mechanism, achieve the effect of regulating soluble sugar and promoting leaf senescence

Active Publication Date: 2020-05-15
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on the regulatory mechanism of the effect of leaf senescence on fruit quality is still relatively lacking.
There is no related report on the function of SWEET in pear

Method used

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  • Pyrus sugar transport gene PbSWEET4 and applications thereof
  • Pyrus sugar transport gene PbSWEET4 and applications thereof
  • Pyrus sugar transport gene PbSWEET4 and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1, analysis of expression pattern of pear PbSWEET4 gene during pear leaf development.

[0026] RNA was extracted from the leaves of 'Dangshan' pear. The RNA was extracted using the CTAB method (Gasic et al., 2004), digested with DNase I (Invitrogen) to remove genomic DNA contamination in the RNA, and then reverse-transcribed by TOYOBO kit (purchased First-strand cDNA was synthesized using 1 μg of RNA from TakaRa Company (operated according to the kit instructions). The first-strand cDNA obtained by reverse transcription was used for real-time fluorescent quantitative PCR (qRT-PCR) of PbSWEET4. Use pear PbTublin (Pbr042345.1) as an internal reference, and the nucleotide sequences of the primers are as follows:

[0027] Forward primer TUB-F: 5'-TGGGCTTTGCTCCTCTTAC-3' (SEQ ID No.5)

[0028] Reverse primer TUB-R: 5'-CCTTCGTGCTCATCTTACC-3' (SEQ ID No.6)

[0029] A gene-specific qRT-PCR primer pair was designed within the open reading frame of the PbSWEET4 gene usi...

Embodiment 2

[0034] Example 2, Cloning and vector construction of pear PbSWEET4 gene and its promoter

[0035] 1. the extraction of pear leaf total RNA, the method for cDNA synthesis are with embodiment 1. The forward primer sequence for amplifying PbSWEET4 is PbSWEET4-F1: 5'-ATGGCTACAGTAGCAGACAGTCAC (SEQ ID No. 3), and the reverse primer sequence is PbSWEET4-R1: 5'-TCACACTGCTGATGGTGTTTCAT (SEQ ID No. 4). High-fidelity DNA polymerase for gene cloning ( Super-Fidelity DNA Polymerase (P505-d1)) was purchased from Novozyme Biotechnology Company. The amplification reaction system is 50 μL, including 200 ng of cDNA, 25 μL of 2×Phanta Max Buffer, 1 μL of 10 mM dNTP, 1 μL of Phanta Max Super-Fidelity DNA Polymerase, 2 μL of each of the above primers at 10 μM, plus ddH 2 0 to 50 μL. The PCR reaction was completed on the Eppendorf amplification instrument according to the following procedures: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 15 ...

Embodiment 3

[0039] Example 3, Qualitative analysis of GUS staining of transgenic Arabidopsis under the control of pear PbSWEET4 gene 2kb promoter at different developmental stages

[0040] The construction method of the PbSWEET4 promoter vector pMDC107-pPbSWEET4 is the same as that in Example 1. The final recombinant vector was transformed into Agrobacterium strain GV3101 by freeze-thaw method, then cultured in LB solid medium with 50 μg / mL kanamycin, 100 μg / mL rifampicin, and then sterilized with 10 mL centrifugation Tubes will be identified with the correct Agrobacterium strain for expansion until OD 600 The value is about 1-1.2, and the bacterial liquid is collected by centrifugation at 6000rpm for 10min. This vector was then transformed into wild-type Arabidopsis plants by the floral dip method (Clough and Bent, 1998). Using GUS staining solution (purchased from Suo Laibao, China), according to the instructions, four stages of Arabidopsis from fully developed rosette leaves (14 days...

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Abstract

The invention discloses a pyrus sugar transport gene PbSWEET4 and applications of a recombinant expression vector of the gene. The nucleotide sequence of the structural gene PbSWEET4 separated from Dangshan pears and having sugar efflux functions is shown as SEQ ID No. 1, and the amino acid sequence is shown as SEQ ID No. 2. The gene PbSWEET4 is converted into diploid forest strawberries to perform function verification; wild strawberries are taken as reference, the sucrose content of the obtained transgenic strawberry leaves can be significantly reduced, and the leaves show the phenomenon ofpremature aging; and therefore, the cloned PbSWEET4 gene is the functional structure gene encoding sugar transporters and has the functions of the efflux of soluble sugar, so that negative regulationeffects in leaf sugar accumulation can be achieved, and the gene also takes part in the aging process of the leaves.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and relates to pear sugar transport gene PbSWEET4 and its recombinant expression vector and application. Specifically, it involves the isolation and cloning of a SWEEET family member PbSWEET4 gene involved in pear sugar transport from 'Dangshan Suli' and its application. Background technique [0002] Pear (Pyrus) is a perennial deciduous fruit tree belonging to the genus Pyrus L. of the Rosaceae family. It is widely planted all over the world and has important economic and social values. The edible quality of pear fruit is an important factor to determine its value, so it is of great significance to improve the edible quality of pear fruit. The edible quality of pear fruit is affected by many factors, among which sugar is one of the important indicators of fruit quality. Increasing the sugar content of pear fruit is very important to improve the quality of pear. In recent years, the mai...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/74
CPCC07K14/415C12N15/8205C12N15/8245
Inventor 吴俊泥江萍李甲明张绍铃朱荣香刘海楠薛程张明月刘月园李晓龙
Owner NANJING AGRICULTURAL UNIVERSITY
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