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Separation and application of salicylic acid-induced citrus sinensis osbeck promoter GSTU19P

A salicylic acid and promoter technology, applied in the field of plant genetic engineering, can solve problems such as false spots, failing to achieve the expected purpose of transgenic, and violating the natural physiological activity rules of plants

Inactive Publication Date: 2012-07-25
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many genetic resources have become an important weapon for the improvement of crop varieties by transgenic technology, but these genes are often expressed by constitutive promoters in transgenic plants. This expression method violates the natural physiological activity rules of plants and not only fails to achieve the desired results. To the intended purpose of transgenic, may even cause great harm to the physiological activities of the transgenic plant itself
For example, the overexpression of the disease resistance gene Pto in tomato (Li, et al.2002) and the overexpression of the disease resistance-related gene NPR1 in Arabidopsis (Blanco, et al.2005) both have adverse effects on the growth of transgenic plants. There are side effects, such as false lesions and short plants.

Method used

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  • Separation and application of salicylic acid-induced citrus sinensis osbeck promoter GSTU19P
  • Separation and application of salicylic acid-induced citrus sinensis osbeck promoter GSTU19P
  • Separation and application of salicylic acid-induced citrus sinensis osbeck promoter GSTU19P

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Experimental program
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Effect test

Embodiment 1

[0031] Example 1 Selection and isolation of salicylic acid-induced candidate genes

[0032] Present embodiment uses 0mmol / L, 1mmol / L, 5mmol / L, the salicylic acid of 4 concentrations of 10mmol / L to process adult period red navel orange (kind is " early red ", comes from Huazhong Agricultural University citrus planting resource nursery, This kind has been awarded the Chinese plant variety right, the variety right number is CN20060194.6, the date of authorization: on May 1, 2008). Every two plants constituted a replicate. Processing time is 8:00 am. Observe every day, observe the leaf situation after one week, research has determined that 5mmol / L SA is the concentration of processing red navel orange tree body (see figure 2 ).

[0033]Search the citrus EST database (HarvEST: Citrus ver.0.51) with GSTU as the keyword, and get 5 similar sequences, which are composed of DNAstar (public software) to form a unigene, use the sequence to design primers with Primer Premier 5.0, and u...

Embodiment 2

[0034] Cloning and bioinformatics analysis of embodiment 2GSTU19 promoter

[0035] The promoter of GSTU19 gene was obtained by chromosome walking method (GenomeWalkerTM Universal Kit, Clontech, USA). According to the GSTU19 cDNA sequence of citrus in HarvEST-citrus database, two antisense primers were designed using it as a template: GSP1 and GSP2, two sense primers Primers AP1 and AP2 (see Table 2), with GSP1 and AP1 as a pair of primers, according to the reaction conditions in Table 1, the first round of PCR reaction was performed. The PCR system is: 2.5μl 10×LA Taq Buffer, 0.5μl 10mM dNTP, 0.5μl 10μM GSP1 and AP1 primers, 1U LA Taq enzyme, 0.5μl DNA, add deionized water to make up to 25μl. In order to increase the specificity of the reaction, the PCR product of the first round was diluted 50 times as the template, and GSP2 and AP2 were used as the primer pair, and the second round of amplification was carried out by nested PCR. The PCR reaction system is: 2.5 μl 10×LA Taq ...

Embodiment 3

[0039] Embodiment 3 utilizes GSTU19P promoter to carry out genetic transformation to Arabidopsis

[0040] 1) Vector construction: In order to verify the spatial expression of GSTU19P, according to the multiple cloning site and GSTU19P sequence of the pCAMBIA1391 vector (purchased from the CAMBIA laboratory in Australia), the amplification design was designed with Primer Premier 5.0 software according to the general principle of primer design 3 Vectors with different fragment sizes ( Figure 5 and Figure 6 ) are 329bp (coded as Del 1), 702bp (coded as Del 2), and 1856bp (coded as Full-length, respectively). The primers for the construction of the Del1 vector are F1 and R1, the primers for the construction of the Del2 vector are F2 and R1, and the primers for the construction of the Full-length vector are F3 and R1 (see Table 2). PCR amplification was carried out using the DNA of red navel orange (variety "Zaohong") as a template, and the DNA extraction method was according t...

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Abstract

The invention belongs to the technical field of plant genetic engineering. The invention specifically relates to separation and identification of a promoter. A salicylic acid-induced promoter GSTU19P can be separated from GSTU19 genes which are separated from citrus sinensis osbeck, the nucleotide sequence of the promoter is as shown in SEQ ID NO: 1 (sequence identity number: 1), the length of the nucleotide sequence is 249bp, and the promoter has obvious salicylic acid induced activity. The induced promoter GSTU19P can be used for plant stress resistance or secondary metabolism and other physiological processes.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. It specifically relates to the cloning and spatial expression of a salicylic acid (SA)-inducible promoter and a GSTU19 gene promoter in navel orange. The isolation of the promoter provides a controllable expression regulatory element for agricultural production and biological research. The inducible promoter plays an important role in physiological processes such as plant resistance to stress and secondary metabolism. Background technique [0002] Agriculture is a country's basic industry, and the supply of food has become particularly important as the world's population continues to grow. Traditional agricultural production mainly depends on fertilization and pesticide weeding and pest control, which not only increases the cost of agricultural products, but also brings a great burden to the environment. The improvement of crop quality is now more and more dependent on the co...

Claims

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Application Information

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IPC IPC(8): C12N15/113A01H5/00
Inventor 张金智徐晓玲艾小艳胡春根
Owner HUAZHONG AGRI UNIV
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