Malus hupehensis MhWRKY40a gene and applications thereof

A gene, crabapple technology, applied to the 'Hubei Begonia' MhWRKY40a gene and its application fields, can solve the problems of reducing production costs, squeezing living space, and threatening grain and oil security.

Inactive Publication Date: 2013-11-13
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the 1960s, genes Pl1 and Pl2 highly resistant to apple powdery mildew were found in Malus robusta and Malus zumi (Knight and Alston1968; Shu Huairui 1999), but until now For this there is no Pl 1 and Pl 2 Reporting of specific sequence information
At present, crop varieties with certain aspects of resistance (such as insect resistance, herbicide resistance, etc.) obtained through transgenic methods have greatly reduced their production costs, and their products have obvious competitive advantages in terms of price and occupy a large market share. Market share, foreign genetically modified soybeans and corn have severely squeezed the living space of corresponding domestic industries, and even threatened my country's grain and oil safety issues

Method used

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  • Malus hupehensis MhWRKY40a gene and applications thereof
  • Malus hupehensis MhWRKY40a gene and applications thereof
  • Malus hupehensis MhWRKY40a gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Gene Cloning

[0030] RNA was extracted from leaves of Begonia hubei by CTAB method, and then the RNA was reverse-transcribed into cDNA, and the target gene was cloned from the cDNA template of leaves by RT‐PCR. PCR reaction system is template cDNA 1μL (50ng·μL ‐1 ), 10×PCR Buffer2.5μL, MgCl 2 1.5μL (25mmol·L ‐1 ), dNTP2.0μL (2.5mmol L ‐1 ), upstream primer F1 (SEQ ID NO.2), downstream primer R1 (SEQ ID NO.3) each 1.0 μL (100ng·μL ‐1 ), 1.0U rTaq enzyme, made up to 25 μL with ultrapure water. The PCR program was 94°C for 4min; 94°C for 30s, 56°C for 30s, 72°C for 2min, 35 cycles; 72°C for 10min. Take 20 μL of the PCR product and analyze it by electrophoresis on a 1.0% agarose gel. After the target fragment was recovered and connected to the pMD19-TSimple (TaKaRa) vector, it was transformed into DH5α Escherichia coli, and more than 5 single colonies were randomly selected for sequencing. The gene sequence is shown in SEQ ID NO.1. After sequencing, the an...

Embodiment 2

[0031] Embodiment 2 vector construction

[0032] On the basis of restriction site analysis, BamHI and SacⅠ restriction sites were introduced at the 5′ end of the original gene cloning primer, and primers with restriction sites (underlined partial sequence) were designed, namely the upstream primer K-MhWRKY40a F (SEQ ID NO.4), downstream primer K‐MhWRKY40a R (SEQ ID NO.5). Using the correctly sequenced returned plasmid in Example 1 as a template, a gene fragment with a restriction site was amplified by PCR. The reaction system is as follows:

[0033]

[0034]The PCR program was 94°C for 4min; 94°C for 120s, 65°C for 30s, 72°C for 2min, 35 cycles; 72°C for 10min. All PCR products were analyzed by 1.0% agarose gel electrophoresis. The target fragment was recovered and connected to PMD19-T Simple, the ligated product was transformed into DH5α Escherichia coli, and more than 5 single colonies were randomly selected for sequencing. The recovery fragment connection system is a...

Embodiment 3

[0054] Embodiment 3 MhWRKY40a Gene Transformation Tobacco and Detection

[0055] The transformation process requires no bacterial contamination and is carried out on an ultra-clean workbench. Inoculate a single colony of transformed Agrobacterium EHA105 in a solution containing 50ug·mL ‐1 Rif, 50ug·mL ‐1 Shake culture in Km’s YEB liquid medium at 28°C and 250rpm until OD600 is about 0.5-1.0, divide into 50mL centrifuges, fill each tube with 40mL, centrifuge at 5000rpm for 10min, discard the bacteria solution, and use 40ml of MS solution for precipitation (bacteria) Resuspend the culture medium, shake and activate at 28°C and 250rpm for 1h; cut tobacco leaves into 1-2cm 2 (leaf edges also need to be cut off to form wounds on the edges), put them into the activated Agrobacterium solution, soak for 3 to 5 minutes, and shake gently several times; take out the materials, put them on sterile filter paper to absorb excess bacteria solution, inoculated in the co-cultivation medium ...

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Abstract

The invention discloses a malus hupehensis MhWRKY40a gene and applications thereof. The nucleotide sequence of the malus hupehensis MhWRKY40a gene is shown as the SEQ ID NO.1. The MhWRKY40a gene is capable of applying to the production of novel species, which are salt resistant and low temperature resistant, and/or plant variety improvement. A novel MhWRKY40a gene is cloned from malus hupehensis for the first time, and the homology between the gene sequence and the arabidopis thaliana, grape and barley is 50.89%. But the protein tertiary structure of the MhWRKY40a is prominently different from the homologous gene of the model plant arabidopis thaliana; the former has 5 beta-folds, while the latter has 4 beta-folds. The MhWRKY40a transgenetic tobacco has the properties of salt resistance and low-temperature resistance, raises the resistance-associated gene expression, and therefore the fact, which the MhWRKY40a gene has the function of improving the salt resistance and low-temperature resistance properties of the plants, is testified.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to the 'Hubei Begonia' MhWRKY40a gene and its application. Background technique [0002] Hubei crabapple (Malus hupehensis Rehd.) is a perennial deciduous tree of the genus Apple in the Rosaceae Pear subfamily. It has the characteristics of wide distribution and strong adaptability. It is a commonly used apple rootstock in my country. Mainly distributed in my country's Hunan, Hubei, Sichuan, Yunnan, Guizhou, Gansu, Shaanxi, Henan, Hebei, Shandong, Shanxi, Anhui, Jiangsu, Zhejiang, Jiangxi, Fujian, and Guangdong. From the perspective of taxonomic distribution, Hubei crabapple is also a unique plant resource of the genus Malus in my country. In addition, Hubei crabapple has important research value in terms of disease resistance, drought resistance, waterlogging resistance, and apomixis. Mining the resistance gene resources of crabapple in Hubei is of great significance for the forma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C12N1/21A01H5/00C12R1/01
Inventor 渠慎春罗昌国章镇乔玉山蔡斌华张仕杰辛璐
Owner NANJING AGRICULTURAL UNIVERSITY
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