Pear laccase gene PbLAC1 and vector, host cell and application thereof

A technology of host cell and pear laccase, which is applied in the field of plant molecular biology and genetic engineering, can solve problems such as unclear function, achieve the effect of improving fruit quality, improving internal and appearance quality, and increasing lignification degree

Active Publication Date: 2019-01-15
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, there are few reports on the cloning and functional analysis of pear laccases, and it is not clear which members of the pear laccase family play a role in lignin biosynthesis

Method used

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  • Pear laccase gene PbLAC1 and vector, host cell and application thereof
  • Pear laccase gene PbLAC1 and vector, host cell and application thereof
  • Pear laccase gene PbLAC1 and vector, host cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] A pear laccase gene PbLAC1 in this example, the cloning steps are as follows: select the fruit of Dangshansu pear about 39 days after flowering, extract the total RNA and reverse transcribe it into cDNA, and then design specific amplification primers according to the published pear genome sequence PbLAC1-F and PbLAC1-R, and use cDNA as a template for PCR amplification, followed by recovery of PCR products and connection to pMD-18T vector; wherein, the nucleotide sequences of specific amplification primers PbLAC1-F and PbLAC1-R They are respectively shown in SEQ ID NO.3 and SEQ ID NO.4.

[0037] The specific cloning process is as follows:

[0038] (1) Take 1 μg (2 μL) of total RNA, add 2 μL 5×gDNA Buffer, 2 μL 10× Fast RT Buffer, 1 μL RT Enzyme Mix, 2 μL FQ-RT Primer Mix, 11 μL RNase-Free ddH 2 O, mix carefully, incubate at 42°C for 15 minutes, then incubate at 95°C for 3 minutes, and place on ice for 5 minutes to obtain the corresponding reverse transcription product c...

Embodiment 2

[0043] Construction of a PbLAC1 overexpression vector and host cell of the present embodiment:

[0044] (1) Design primers PbLAC1-ZH-F and PbLAC1-ZH-R with Bgl II and Nco I restriction sites according to the sequence of the target gene PbLAC1 that has been successfully cloned, and use the constructed pMD-18T-PbLAC1 plasmid as a template Amplify, and after the PCR product is detected by 1% agarose gel electrophoresis, the target fragment is recovered and connected to the vector pMD-18T for sequencing; among them, the primer PbLAC1 with Nco I and Bgl II restriction sites The nucleotide sequences of -ZH-F and PbLAC1-ZH-R are respectively shown in SEQ ID NO.5 and SEQ ID NO.6.

[0045] (2) Use restriction endonucleases Bgl II and Nco I to carry out double enzyme digestion on the pMD-18T-PbLAC1 plasmid and the plant expression vector pCambia1304 plasmid respectively; use 1% agarose gel electrophoresis to recover the target fragment PbLAC1 and plant expression vector pCambia1304; wi...

Embodiment 3

[0048] Construction of a PbLAC1 overexpression Arabidopsis strain of this embodiment:

[0049] (1) Prepare the infection solution:

[0050] After the host cells obtained in Example 2 are activated, they are resuspended with a buffer to obtain an infection solution; wherein, the buffer is specifically a 1 / 2MS liquid containing 5% sucrose and 0.02% Silwet L-77 by mass ratio Medium, OD of infection solution 600 The value is 0.5 to 0.8.

[0051] (2) Genetic transformation of Arabidopsis:

[0052] This was accomplished using the Agrobacterium-mediated flower dip method.

[0053] (3) Screening of transgenic plants:

[0054] Will T 0 The generation seeds were sterilized and inoculated into MS solid screening medium containing 50mg / L hygromycin, cultured in a light incubator, and the light time was controlled to be 16h / d; the results showed that the non-transgenic plants turned yellow and stopped growing, Only transgenic plants can grow.

[0055] (4) Identification of positive ...

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Abstract

The invention relates to the technical fields of plant molecular biology and genetic engineering, and provides a pear laccase gene PbLAC1, the nucleotide sequence of which is shown in SEQ ID NO. 1. The invention also provides a plant over-expression vector, wherein the plant over-expression vector comprises pMD-18T-PbLAC1 plasmid constructed by PbLAC1. The invention also provides a genetically engineered host cell comprising a PbLAC1 gene sequence. The invention also provides the application of the pear laccase gene PbLAC1 in regulating plant lignin synthesis and cell wall development. The pear laccase gene PbLAC1 of the present invention provides new evidence for perfecting the plant lignin synthesis pathway, has important theoretical and practical significance for improving fruit qualityand other key agronomic characteristics of crops, and also provides a new way for regulating the content of pear stone cells by genetic engineering.

Description

technical field [0001] The invention relates to the technical fields of plant molecular biology and genetic engineering, in particular to a pear laccase gene PbLAC1 and its carrier, host cell and application. Background technique [0002] Dangshansu pear (Pyrus bretshneideri cv. DangshanSu), a white pear (Pyrus bretshneideri) pear variety, originated in Dangshan County, Anhui Province, and is now the main cultivated variety in North China, Northwest my country, and the Yellow River Basin. Dangshan pear has a long history of cultivation and has a good reputation. During the Warring States period, Zhuangzi recorded in "Human World": Fuqu pear orange pomelo, a genus of fruits and vegetables, is peeled when it is ripe, and humiliated when peeled. Big branches are broken and small branches are leaked. This is the earliest written description of Dangshan Suli that can be tested. Emperor Gaozong of the Qing Dynasty once visited the south of the palace, and after eating Dangshan pe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/82C12N1/21C12R1/01
CPCC12N9/0061C12N15/8255C12Y110/03002
Inventor 蔡永萍程曦李国辉马晨辉金青孙燕铭林毅张金云
Owner ANHUI AGRICULTURAL UNIVERSITY
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