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Cloning and Preliminary Function Analysis of AtTOM-like Genes from Nicotiana Benthamiana and Tomato

Author: TanZhong
Tutor: ZhouXuePing；QianYaJuan
School: Zhejiang University
Course: Biochemistry and Molecular Biology
Keywords: Benthamiana Tomato Tobacco mosaic virus Copy Virus-induced gene silencing
CLC: S432.41
Type: Master's thesis
Year: 2007
Downloads: 83
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Abstract


The virus itself only carry a small number of genes in the virus infected plant process, and its encoded protein is far from sufficient to complete the virus infection process, and therefore required the help of host factors to achieve infection. In recent years found that some host factors related to the replication of the virus, such as Arabidopsis genes in AtTOM1 with tobacco mosaic virus (TMV) replication, the the identification of NtTOM1 Sansei smoke and NtTOM3 benthamiana plants The identified NbTOM1 associated with TMV replication. This thesis is based on the reports Sansei the smoke of NtTOM1 and the NtTOM3 gene sequence (AB193039, AB193040) primers were designed from benthamiana the amplification to NbTOM1 and NbTOM3 gene fragment by RT-PCR. Sequence analysis showed that NbTOM1 and NbTOM3 Sansei smoke NtTOM1 and NtTOM3 the homology of 95.9% and 98.2%, respectively. The NbTOM1 and NbTOM3 Tobacco curly shoot virus (TbCSV) inserted into the satellite molecules DNA1 silence carrier, get DNA1: TOM1, DNA1: TOM3 and DNA1: the TOM1-TOM3 three recombinant plasmids, recombinant plasmid triparental mating method introduced into Agrobacterium benthamiana, and Chinese tomato yellow leaf curl virus (TYLCCNV) vaccination. 14 days after the inoculation of semi-quantitative PCR analysis showed that the inoculation of of DNA1: TOM1 plants in NtTOM1 gene was silent while NtTOM3 mRNA levels were not affected; while inoculation DNA1: TOM3 plants NtTOM3 gene is silent while NtTOM1 mRNA levels did not affected; vaccination DNA1: TOM1-TOM3 plants, two genes simultaneously silence. The silent plants inoculated of TMV: GFP, GFP observation and Northern blot analysis shows that separate silent NbTOM1 or NbTOM3 with both silenced plants TMV virus proliferation inhibition. The above results show that, NbTOM3 has a similar mechanism may be provided with NbTOM1 support TMV replication. Reported tomato AtTOM1-class gene sequence (AB193041, AB193042, AB193043) design specific primers amplified from tomato AtTOM1 gene fragments (LeTH1 LeTH2 LeTH3) and insert same TbCSV satellite molecules DNA1 silencing vector in the recombinant plasmid DNA1: LeTH1 LeTH2-LeTH3. The inoculated tomato recombinant plasmid was introduced into Agrobacterium by tri-parental mating method with TYLCCNV common. 20 days after the inoculation of semi-quantitative PCR analysis the reorganization plasmid and TYLCCNV of common tomato after inoculation, several genes simultaneously silence, silence after the plants were inoculated with TMV: GFP, GFP observation and Northern blot analysis showed that the TMV replication by inhibiting. Several genes affect TMV replication and whether there is functional redundancy remains to be further analyzed. Used in this thesis TbCSV DNA1 silencing system, its high silencing efficiency, inoculated plants asymptomatic, can be effectively used for the rapid identification of gene function. And the use of the VIGS system silence host plants TOM class factor regarded as a quick method of anti-TMV plants.

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Plant diseases and their prevention > Transgression ( pass ) an infectious agent harmful > Virus
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