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Molecular Detection of Viruses in Fructus Trichosanthis Kirilowii and Their Effects on Photosynthesis Characteristics of Cucurbitaceae Plant

Author: ZhangYuChuan
Tutor: ChenJiShuang；ChenShaoNing
School: Zhejiang University of Technology
Course: Biochemistry and Molecular Biology
Keywords: Fructus Trichosanthis Kirilowii Molecular detection Papaya leaf distortion mosaic virus Electron microscopic observation Photosynthetic characteristics
CLC: S436.429
Type: Master's thesis
Year: 2011
Downloads: 17
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Abstract

Fructus Trichosanthis Kirilowii is an economic plant developed from civil area having edible and medicinal varieties. For economic benefits, it was introduced to cultivate in China. Virus disease on Fructus Trichosanthis Kirilowii is very common recently and affects both quality and yield seriously. Therefore molecular detection of virus on this plant, as well as analyzing effects of virus on photosynthesis characteristics is essential.Samples were collected from ChangShan area of Zhejiang province and inoculated cucurbit and N. benthamiana. Symptoms on cucurbit are mosaic, vein-clearing, chlorotic spot, puckered, leaf roll and shrink. Mosaic, chlorosis, necrosis and puckered were occured on N. benthamiana. No symptoms on N. glutinosa and N. tabacum were observed.Crooked rhabditiform virus particles, Pinwheel Inclusion, Bundles and Cylinderical Inclusion bodies were found in the leaf by electron microscopic observation.DsRNA was extracted and tested by electrophoresis. A band of 8000～10000bp was found, corresponding with the number of potyvirus’genome. RT-PCR using degenerate primer also showed the the virus in Fructus Trichosanthis Kirilowii belonged to potyvirus. RT-PCR using degenerate primer also showed the the virus in Fructus Trichosanthis Kirilowii belonged to potyvirus. RT-PCR products were cloned and sequenced. Zucchini yellow mosaic virus （ZYMV）, Papaya ring spot virus （PRSV） and Papaya leaf distortion mosaic virus （PLDMV） were shown to be single or double infect the Fructus Trichosanthis Kirilowii.The PLDMV detected in Fructus Trichosanthis Kirilowii in the main land of China was reported for the first time. Part sequence of PLDMV from 4461 to 10154nt was sequenced by RACE method and functional genomics of CI、6K2、NIa-VPg、NIa-Pro、Nib、Coat protein、3′UTR was contained.Compared with the sequence uploaded to NCBI, maximum diversity of PLDMV-dg is 27.2%～27.5%, and homology of the CI parts is 83.1%～83.3%, the 6K2 parts is 78.2%～78.3%, the NIa-VPg is 84.1%～84.5%, NIa-Pro part is 85.3%～87.0%, 83.7%～84.3% for Nib, Coat Protein for 88.8%～89.2%, and the 3′UTR part is 87.4%～89.9% respectively. Analysis of phylogenetic sequence showed that all the sequences of CI、6K2、NIa-VPg、NIa-Pro、NIb、Coat protein and 3′UTR of PLDMV-dg are on the same side of the phylogenetic tree and far away from the reported sequence AB088221.1、BD171712.1、NC₀05028.1、EA012042.1 and EU233272.1.The Taiwan isolates （EU233272.1） is relatively far from the above sequences but the diversities less than PLDMV-dg contrast to other sequences the diversity of PLDMV-dg maybe due to the difference of regional and host.Effects of ZYMV and CMV on Photosynthesis and physiological characteristic of the Cucurbitaceae plant zucchini was recorded and analyzed. Zucchini plants were siglely or doublely inoculated with ZYMV and CMV. The data of Leaf area, Height, Photosynthesis, Volume of Chlorophyll a and Chlorophyll b of the leaf were analyzed. The results show that ZYMV and CMV-Fny were synergied on Cucurbitaceae plant zucchini.Differences of biomass and photosynthetic characteristics between healthy and disease plants were obviously before symptoms occurred. Compared with the infected plants, values of the Leaf area, plant height, plant weight, chlorophyll content, stomatal conductance （Gs）, net photosynthetic rate （A） and transpiration （E） were higher in the healthy ones. However, intercellular CO2 concentration （Ci） was higher in the infected plants.

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