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The Study on Screening and Identification of Houttuynia Cordata Thunb. Transformed with Foreign Antimicrobial Peptides

Author: ZhangYing
Tutor: WangPeiXun
School: Guangzhou University of Traditional Chinese Medicine
Course: Chinese and Western medicine combined with the basis
Keywords: Houttuynia Antimicrobial Peptides Trangene Agrobacterium tumefaciens Screening Molecular hybridization
CLC: R282.7
Type: Master's thesis
Year: 2010
Downloads: 71
Quote: 4
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Abstract

Houttuyniae is a perennial plant which comes from the entire plants of Houttuynia cordata. It can clear heat and detoxicate, diminish carbuncle, eliminate purulence, induce diuresis for treating strangurtia. Using for curing asthma, heat dysentery, heat stranguria, and carbuncle. Because of the artificial cultivation, Houttuynia growth environment of change, reduce the capacity of plant diseases and insect pests, plant diseases and insect pests in general and much more serious, directly affecting the quality and yield of Houttuynia. Foreign antibacterial peptide gene transfered into Houttuynia can improve its anti-pest’s ability and enhance its antibacterial activity.Cecropin is a small molecule antibacterial activity of cationic peptides, on a strong anti-bacterial effect, some Cecropin have anti-viral, anti-fungal activity, and kill cancer cells, which are, or even can enhance immunity, speed up wound healing process. Its unique mechanism of broad-spectrum anti-microbial activity, known as natural antibiotics. At present, the Cecropin has been transferred to tobacco、rice、vegetables、fruit trees、herbs and other plants successfully.Plant Genetic Engineering achieved remarkable results after more than 20 years of development, so far, the world has isolated more than 100 used target gene for the purpose of genetic engineering, transgenic plants were nearly 200. Nearly 1,000 cases of transgenic plants were approved to enter field trials, involving more than 50 plant species, has approved 48 transgenic plants for commercial production. In the aspect of transgenic medicinal plants, China has realized the foreign gene expression in the Chinese wolfberry, pinellia tuberifera tenore, and other Chinese herbal medicine.This paper aims to optimize the method of Agrobacterium-mediated transformation on Houttuynia, screen and identify the resistant buds of transgenic Houttuynia and identify the plants which were identified as positive results on DNA/RNA level. The results are list blow:1. Transformation of Houttuynia by Sonication-assisted Agrobacterium-mediatedThe Agrobacterium tumefaciens EHA105 has powerful virulence. The transformation plants can express stably, but the effect is not very well when use it mediate the transformed on Houttuynia. In order to solve this issue, we optimize the method of transformation based on the system our laboratory has already built. In this study, sonication direct conversion method, Agrobactium-mediated transformation method, constant-frequency sonication instrument 15min after the treatment of plants by Agrobacterium-mediated infection of plants and in the process of Agrobacterium infection by sonication-assisted four ways to deal with 10s Transgenic Research carried out on the Houttuynia. The result shows that sonication instrument to deal with a constant frequency of plants by Agrobacterium-mediated 15min infected plants and in the process of Agrobacterium infection secondary to sonication treatment 10s two kinds of transformation transient expression of gus gene in the highest rate shows that sonication-assisted Agrobacterium-mediated genetic transformation method is an efficient Houttuynia cordata thumb. transgenic method, which enables foreign gene expression in plants efficient.2. Peptide transgenic screening and rapid identification of HouttuyniaThe laboratory has been to construct expression vectors LBA4404/pBI121-CN that harbor kanamycin resistance plant selection marker gene and the exogenous peptide genes, the experimental application of Agrobacterium-mediated transformation of Houttuynia by kanamycin Screening for resistance were resistant shoots, and acquired resistance to shoot rapid PCR screening, resulting in eight in the resistant medium resistant shoots that grow well in six plants were positive.3. Identification on integration level of the transgenic antimicrobial peptide of HouttuyniaIdentification on the DNA level of transgene Houttuynia, that is genomic PCR and Southern blotting. The results showed that six genes can be amplified to the target peptide fragments, then these six PCR products were detect by Southern blot hybridization. The analysis have indicated that foreign peptide gene was integrated into genomic DNA of Houttuynia.4. Identification on transcription level of the transgenic antimicrobial peptide of HouttuyniaIdentification on the RNA level of transgene Houttuynia, that is genomic RT-PCR and molecular hybridization. The results showed that transgenic Houttuynia from RNA extracted can be amplified in a length of about 250bp of DNA area band with the theoretical value 243bp match and then use these PCR products to have hybridization, it comes out positive result. This shows that foreign peptide can be expressed in the genome of Houttuynia.Conclusion:Through the identification on integration and transcription level of the transgenic Houttuynia, the foreigner peptide gene has insert the gene of Houttuynia and to achieve foreign gene expression for further study of transgenic Variety of antimicrobial peptides in the antibacterial activity and laid the foundation for further evaluation for the expression of antimicrobial peptides in the Chinese herbal plants to provide theoretical and applied basis.

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