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Functional Analysis Of Insects-Related Proteinase Inhibitor Gene SpCYS In Solanum Tuberosum

Posted on:2015-03-29Degree:MasterType:Thesis
Country:ChinaCandidate:X BaiFull Text:PDF
GTID:2180330482968848Subject:Biochemistry and Molecular Biology
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Potato(Solarium tuberosum L.) is the fourth important food crop after rice, wheat and corn. During its growth period, potato is vulnerable to all kinds of pest invasion, resulting in lower quality and yield. So in recent years, Researchers has been paying more and more attention to how to improve potato pest resistance.Potato, like other plants, has its own natural defense systems against the exogenous invasion and proteinase inhibitors (PI) serves as an important member in this system. Previously, our laboratory has already cloned a thiol protease inhibitor gene SpCYS from Solanum pinnatisectum Dun. This study focused in the analysis of the resistance function of this gene to pests. The main results are as follows:1 Construction of expression vectors and genetic transformation Over-expression vector 1304-SpCYS and anti-sense vector 1304-FSpCYS were constructed using pCAMBIA1304. The recombinant vectors were introduced into potato Desiree by Agrobacterium-mediated transformation, and six over-expression transgenic potato lines and two anti-sense transgenic potato lines were demonstrated to be positive by PCR.by PCR2 Expression analysis of SpCYS. The transcriptional expression of the transgene was analyzed by semi-quantitative RT-PCR and qRT-PCR and its coding protein level was detected by SDS-PAGE method. The results showed that expression level of SpCYS in the overexpression transgenic lines was significantly higher than that in the control plants, while in antisense transgenic lines the expression level was lower.3 Identification of transgenic insect-resistant lines. The insect resistance of the transgenic lines was identified through Spodoptera Larvae feeding experiment and feeding choice experiment, respectively. Larvae fed with overexpression transgenic leaves showed a significant decrease in food consumption, compared with the other two groups. And Spodoptera larvae also showed a tendency feeding choice, that is, compared to over-expression in transgenic plants they prefered to be fed with the leaves of other two plants.
Keywords/Search Tags:potato, SpCYS, genetic transformation, expression analysis, insect bioassays analysis
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