Pear hexokinase gene PbHXK1 and application thereof

A technology of gene and hexose catalysis, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of decreased photosynthetic rate, decreased chlorophyll content of leaves, and decreased electron transfer efficiency

Active Publication Date: 2014-12-03
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Increased catalytic activity of AtHXK1 was accompanied by decreased leaf chlorophyll content, decreased photosynthetic rate, and decreased electron transfer efficiency in photosystem II reaction centers

Method used

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  • Pear hexokinase gene PbHXK1 and application thereof
  • Pear hexokinase gene PbHXK1 and application thereof
  • Pear hexokinase gene PbHXK1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the cloning of pear PbHXK1 gene

[0036] Total RNA was extracted and reverse transcribed from 'Yali' pulp 50 days after full flowering, and the obtained first-strand cDNA was used to amplify the PbHXK1 gene. Extract total RNA by CTAB method (CTAB extraction buffer includes 2% CTAB, 2% PVP K-30, 0.05% spermidine, 10mM Tris HCl (pH=8.0), 25mM EDTA, 2M NaCl), and take 1 μg RNA sample After being incubated with 1U DNase I (purchased from Fermentas) at 37°C for 30min, 1μLEDTA (25mM) was added and incubated at 65°C for 10min. The TOYOBO reverse transcription kit (purchased from TakaRa Company, operated according to the instructions of the kit) was used for the synthesis of the first-strand cDNA.

[0037] The full-length cDNA sequence of the PbHXK1 gene was amplified by nested PCR technology, and the nucleotide sequence of the first round of ordinary PCR primers for amplifying the PbHXK1 gene is as follows:

[0038] Forward primer PbHXK1-F1: 5'-CGTATCCCTCCCCCGA...

Embodiment 2

[0047] Example 2, Correlation analysis between PbHXK1 gene expression and hexokinase activity during pear fruit development

[0048] 1. qRT-PCR analysis of pear PbHXK1 gene during pear fruit development

[0049] The extraction of pear pulp total RNA, the method for cDNA synthesis are the same as in Example 1. Using pear tubulin (AB239681) as an internal control, the nucleotide sequence of the primer is as follows:

[0050] Forward primer PbHXK1-F3: 5'-TGGGCTTTGCTCCTCTTAC-3',

[0051] Reverse primer PbHXK1-R3: 5'-CCTTCGTGCTCATCTTACC-3'(.

[0052] Use Primer Premier 5.0 to design gene-specific qRT-PCR primer pairs within the open reading frame of the PbHXK1 gene. The nucleotide sequences of the primers are as follows:

[0053] Forward primer PbHXK1-F4: 5'-TCCTTGAGTTTGCTCCCGAC-3',

[0054] Reverse primer PbHXK1-R4: 5'-TGGAGTGGGGTAACTTTCGC-3'.

[0055]qRT-PCR used SYBR Green kit (purchased from TaKaRa Company, operated according to kit instructions). The 20 μL qRT-PCR reacti...

Embodiment 3

[0062] Embodiment 3, construct the plant overexpression vector of pear PbHXK1 gene

[0063] The multiple cloning site of the pCAMBIA1301 vector and the nucleotide sequence of the pear PbHXK1 gene were analyzed, and enzyme cutting sites NcoI and BstE II were added to the 5' ends of the primers PbHXK1-F2 and PbHXK1-R2, respectively, to obtain the corresponding The nucleotide sequences of primers PbHXK1-F5 and PbHXK1-R5 are as follows:

[0064] Forward primer PbHXK1-F5: 5'-CATG CCATGG CTCACTACCCAAACTTTCTCACTCAT-3' (SEQ ID NO. 11),

[0065] Reverse primer PbHXK1-R5: 5'-CG GGTAACC CACTTCATTCATCTACCTGGTCTTG-3' (SEQ ID NO. 12).

[0066] Containing 100mg·L -1 E. coli DH5α successfully transformed with the recombinant plasmid 'PbHXK1-pMD19-T' was suspended in liquid LB medium with ampicillin and cultured at 37°C and 220rpm for 12h. Extract the 'PbHXK1-pMD19-T' recombinant plasmid as a template for PCR. The 25 μL PCR reaction system includes: 1×LA PCR Buffer II (Mg 2+ free) (pur...

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Abstract

The invention discloses a pear hexokinase gene PbHXK1 and an application thereof. A nucleotide sequence of the gene is shown as a sequence table SEQ ID No.1, wherein an amino acid sequence corresponding to the nucleotide sequence of the gene is shown as a sequence table SEQ ID No.2. The gene PbHXK1 is inoculated into tomato to carry out functional verification; the expression quantity of the gene PbHXK1 and the activity of the hexokinase of a transgenosis tomato plant which is obtained by taking a wild tomato plant as a reference are obviously improved, the growth of the plant is obviously inhibited and the content of soluble sugar is obviously reduced so as to show that the cloned gene PbHXK1 is a functional structure gene for coding the hexokinase, has the function of phosphorylating hexose, plays a regulation role in the fruit sugar accumulation process and also takes part in regulation of the growth and the development of the plant.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering. It specifically relates to pear hexokinase gene PbHXK1 and its application. Background technique [0002] Pear is one of the main fruit tree species planted in the world. The quality of pear fruit determines the commodity value, and fruit soluble sugar is an important economic trait that constitutes the fruit quality. The regulation of sugar metabolism can directly affect the content and composition of sugar. The sweetness of fructose is twice that of glucose and 1.8 times that of sucrose. The phosphorylation of hexose is closely related to the content of sucrose and hexose. Therefore, hexose metabolism is an important part of sugar metabolism. Therefore, screening key gene resources in the process of hexose metabolism in pear fruit will help to understand the molecular physiological mechanism of sugar metabolism involved in plant hexokinase and the process of sugar signal transduction ...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/12C12N15/11C12N15/84C12N1/21A01H5/00
Inventor 张绍铃赵碧英黄小三齐开杰
Owner NANJING AGRICULTURAL UNIVERSITY
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