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Cloning and Expression Analysis of Several Key PcG Genes in Camellia oleifera

Author: ZuoMengMeng
Tutor: HuXiaoYi
School: Anhui Agricultural University
Course: Ornamental Plants and Horticulture
Keywords: Camellia oleifera PcG gene tissue culture cDNA clone RNAi
CLC: S794.4
Type: Master's thesis
Year: 2013
Downloads: 11
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Abstract


As the principal species of woody oil plants in the South of China, Camellia oleiferais one of the four most famous edible oil woody plants including Elaeis guineensis, Oleaeuropaea and Cocos nucifera. Camellia oil seeds are the source of the main channel, butnot unique. In this study, somatic embryos can induce lipid biosynthesis genes in principle,the proposed embryonic genes through the inhibition of RNA silencing PcG genes toachieve increased oil content of tea callus purpose. Therefore, this study first Camelliacallus and callus material sources, such as bud proliferation system, and optimized. Thentea homologous genes as templates, cloned several key Camellia3’end of the PcG genesequence. Further to the resulting sequence information was designed based on thedouble-stranded RNA hairpin structure into Agrobacterium tumefaciens LBA4404, andLBA4404Camellia PDS hairpin transformants were engineered bacteria infect leaves, forthe purpose of PcG gene RNA interference experiments laid the foundation. Researchresults were obtained as follows:1. The cultivation of Camellia oleifera leaf tissue.1.0mg/L AgNO3,1000mg/L CH,100mg/L Glu,0.1mM Sputrescine,0.5mM Spermine can be efficient callus of Camelliacallus;1/2MS+2%sucrose+0.7%agar+0.5mg/L TDZ+1.0mg/L IBA+1.0mg/L6-BA+1.0mM GSSG/0.1mM spermidine can be differentiated shoots;1/2MS+1.0mg/L6-BA+0.05mg/L NAAfor bud proliferation.2. Cloning of CLF, PKL, EMF2gene fragment. Among them,get the full-length cDNAof EMF2gene, named CoEMF2. The gene open reading frame of1866bp, encoding621amino acids. The protein sequence and physicochemical properties are analyzed: theprotein molecular weight is70.54kD, hydrophobic unstable protein; coiled coil predictionresults show that, there may be8obvious spiral structure of CoEMF2proteins; subcellularlocalization showed that CoEMF2is located in nuclei; also found to contain two conservedregions in CoEMF2: VEFS-Box domain and type C2H2zinc finger protein.3. Camellia PcG gene RNA interference. Gateway technology use to pJawohl8-RNAivectors were constructed camellia CLF, PKL, EMF2and PDS gene RNA interferencepattern vector; freeze-thaw method to import into Agrobacterium LBA4404, by sequencingand PCR primers specific validation of these recombinant vectors and transformantscorrectness. 4. leaves of Camellia infection by preliminary evidence Camellia transgenic researchis feasible. Select the appropriate weather to LBA4404containing PDS hairpin structurewith partial wound infection Camellia young shoots/leaves, there was silence PDSwandering specific symptoms.5. for several key PcG genes in different tissues of quantitative PCR analysis foundthat embryogenic callus inhibitory gene chromatin mark H3K27me3mark (CLF) andmaintenance (LHP1) genes are maintained at a low level However, the level of PKL stillhigh, so in the future Camellia capture key technology of somatic embryos may be PKLphysiological effects of suppression.

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CLC: > Agricultural Sciences > Forestry > Forest tree species > Special use of broad-leaved class > Camellia
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