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Introduction:

Introduction:

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Introduction:

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  1. Introduction: Use of conserved plant sequence as primer, the DNA sequences in the ribosomal ITS1-5.8S- ITS2 region have been amplified. The rDNA of eight species of Lycium genus were initially subcloned into PUC18 vector and sequenced using thermo sequenced Cy5.5 dye terminator cycle sequencing kit and SEQ4X4 personal sequencer. The sequences were aligned manually and the percenage identity was calculated by the computer program DNASIS. Result show that sequences of lycium species is a very high degree of homology in the 5.8S gene. The DNA sequence in the two ITS regions are more variable. This method directly study DNA polymorphisms by determination of the nucleotide sequence of a defined region. It is more robust confirmed that broader application of this approach to authenticate other morphologically similar and closely relaed of chinese medicinal material is rationalised.

  2. Background The ribosomal DNA of plant exist as an array of repeating units. Each unit consists of coding region namely 18S, 5.8Sand 26S. They are highly conserved. The coding region 18S and 5.8S are separated by internal transcribed region 1 (ITS1) while 5.8S and 28S are separated by internal transcribed region 2 (ITS2). These two regions are more variable. So it may be used as a tool to separated at various taxonomic levels within families. Consequently, it behaves like a single copy gene which allows comparison between different species. Initial, the use of ribosomal RNA genes as genetic marker were employed to differentiate species and strains of yeasts, parasites and soybean.

  3. The application of DNA sequencing in a defined DNA region in authentication of Chinese medicinal material have been reported. For example, 5S rRNA gene fragment of the Augelica acutiloba (Danggui) (Mizukami, 1995). Buplenrum falcatum (Shandaochaihu) (Mizukami, 1993) and Atractylodes (Changzhu) (Mizukami, 1996). 12S rRNA gene fragment of 12 samples of Chinemys reevesii gray (turtle shells) and 4 sample of species Pelodiscus sinensis (Wiegmann) (Wu, 1998). Comparison of these sequences indicated that the technique was found to be effective and reliable for the identification of turtle shells and wiegmann. Other sequence such as rbh choroplast DNA or cytochrome b gene can also be used in identification. For example, Cyt b gene sequence analysis of Zaocys dhumades (Wushaoshe) from its substitutes (Wang, 1999) in the crude snake drugs. The ITS1-5.8S-ITS2 of Panax species has been also sequenced (F. Ngan, 1999), four panax species and two common adulterants were differentiated. In the part of this study, We will study the ITS1-5.8S-ITS2 sequence among eight Lycium species and varieties in order to reveal their relatedness. The phylogenetic tree generated from sequence data would be compared with the data revealed by RAPD.

  4. Figure The gene-spacer-gene arrangement of plant ribosomal RNA genes. The 3 subunits: 18S, 5.8S and 26S are separated by internal transcribed spacers (ITS 1 and 2). The repeat unit is separated by intergenic spacer (IGS). The sizes of ITS1 & ITS2 in L. barbarum species are about 250bp and 250bp respectively. A number of primers are used for the study of Lycium speciesrDNA. 18B.RW: 5'TAG AGG AAG GAG AAG TCG TA3'; ITS5.RW: 5’GGA AGT AAA AGT CGT AAC AAG G3’; 5.8A.RW: 5'TGA AGA ACG TAG CGA AAT GCG3'; 5.8B.RW: 3’GTA GCT CAG AAA CTT GCG TT5’; ITS4.RW: 3’CGT ATA GTT ATT CGC CTC CT5’; 28CC.RW: 3’AAGGGG ATCATTGCCG CTCA5’.

  5. Work in progress Samples Origin Lycium barbarum (LB)Zhongning, Ningxia L. barbarum var. auranticarpum (LBA)Yinchuan, Ningxia L. barbarum cv. tianjinense (LBT)Jinghai, Tianjin L. Chinense (LC) Nanjing, Jiangsu L. Chinense var. potaninii (LCP)Yuncheng, Shanxi L. ruthenicum (LR)Yinchuan, Ningxia L. Truncatum (LT)Yan'an, Shaanxi L. dasystemum var. rubricaulium(LDR)Xining, Qinghai

  6. Purification of plasmid 3 ml LB 50g Amp./per ml Agar plate with clony 37C overnight in a rotary shaking incubator

  7. ATTAGACGTCCG A T G C A ATTA ATTAGA ATTAG ATTAGACG ATTAGACGTCCG AT ATT ATTAGACGT ATTAGAC ATTAGACGTC ATTAGACGTCC A T G C ATTAGACGTCCG ATTAGACGTCC ATTAGACGTC ATTAGACGT ATTAGACG ATTAGAC ATTAGA ATTAG ATTA ATT AT A

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