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Clone and Transformation of Chitinase ClassⅢ Gene in Medicago Sative

Author: ZhangWenJuan
Tutor: LiCong
School: Chinese Academy of Agricultural Sciences
Course: Grassland
Keywords: The chitinase ClassIII gene Clone Bioinformatics Genetic transformation Tobacco Alfalfa
CLC: S541.9
Type: Master's thesis
Year: 2010
Downloads: 82
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Abstract

Alfalfa is one of the excellent forage legumes, high yield, palatability characteristics, known as the \However alfalfa diseases forage yield large degree of convergence can be reduced forage nutrients reduce palatability declined severely affect the yield and feeding value of alfalfa. The study showed that alfalfa disease is caused by pathogenic fungi, bacteria, viruses and other pathogens, the chitinase gene is a single gene encoding the degradation of chitin glycosidase can degrade the fungal cell wall, the main component of chitin and inhibit the growth and reproduction of the fungi, chitinase extensive research in plant breeding for disease resistance. The main findings are as follows: 1. Of alfalfa chitinase ClassIII gene cloning and bioinformatics analysis. Full-length primers were designed according to the cut-off-type alfalfa (Medicago trucatula) the chitinase ClassIII gene (GenBank: AY294484.1) was obtained by RT-PCR method chitinase ClassIII nucleic acid sequence of alfalfa (Medicago sative) NCBI Registry Number: FJ872918, named MsChiIII. Use of biological information learning software analysis alfalfa chitinase nucleic acid sequence and amino acid sequence, the isoelectric point of the predicted amino acid sequence, secondary structure and tertiary structure, and phylogenetic tree was constructed, and the results show the nucleic acid sequence of full-length 953bp including the complete open reading frame encoding 301 amino acids, such as isoelectric point of 8.212; the sequence of the encoded protein is the chitinase 18 family endonuclease, located in the cell gap, and containing the chitinase 18 family characteristic sequence-LGDVDFDIE, with the cut-off type alfalfa chitinase enzyme ClassIII nucleic acid sequence and amino acid sequence homology were 90.55% and 95.03%. Plant expression vector. Take advantage of even the and Nco Ⅰ (CCATGG) and Spe Ⅰ (ACTAGT) PCR amplification MsChiIII the ORF recycling purified ORF PMD18-T is connected to the cloning vector and sequenced, and extract The PMD-MsChiIII plasmid. The same restriction endonuclease the enzyme cutting the PMD-MsChiIII Plasmid and PCAMBIA1302 expression vector, and will be connected to the sticky ends of the carrier and the target fragment in T4 ligase, the ORF even into the plant expression vector to form a fusion with GFP genes. Freeze-thaw method the PCAMBIA1302-MsChiIII plasmids were transformed into Agrobacterium tumefaciens LBA4404. Genetic transformation of tobacco. Infect tobacco leaf discs the preculture tobacco leaf discs 2d, containing plasmid PCAMBIA1302-MsChiIII Agrobacterium tumefaciens total for 72h, 10 resistant plants after the Hyg (50mg / L) of the screening, PCR and RT-PCR detected positive tobacco plants to 8, the conversion was 80%. Genetic transformation of alfalfa. Set of five different the Hyg concentration, to determine an appropriate selection pressure to 30mg / L. 8 infection time and pre-incubation time gradient observed Agrobacterium infection alfalfa callus growth, results show the infection time 6 to 8min total incubation time 72 ~ 80h better infection conditions were set. Agrobacterium tumefaciens infection preculture 2d alfalfa explants after co-culture, after a screening of Hyg resistant plants 7, PCR and RT-PCR detected positive alfalfa plant 5 conversion rate of 71.5 %.

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