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Cloning of Proteinase Inhibitor Gene CYS and API from Wild Potatoes and Preliminary Study of Potato and Tobacco Transformation with Spcys

Author: LiGenTao
Tutor: YangQing
School: Nanjing Agricultural College
Course: Biochemistry and Molecular Biology
Keywords: SpCYS ScCYS SpAPI ScAPI Gene cloning Genetic transformation
CLC: S532
Type: Master's thesis
Year: 2010
Downloads: 5
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Abstract


Potato(Solanum tuberosum L.) is the fourth important food crop in the world following maize, rice and wheat. When potato grows under field conditions, it is continually threatened by many insects, which could cause yield and quality losses of potato. As a result, researchers are increasingly focusing on raising its resistance against these insects.As all plants, potato has its own natural defense systems against the exogenous invasion and the proteinase inhibitors (PI) serve as an important member in this system. It has been reported that plant proteinase inhibitor genes had more ressisitance against the insects, so that they have been used as the useful insect-resistant genes in genetic engineering. In this paper, we cloned proteinase inhibitor genes SpCYS and SpAPI from Solanum. pinnatisectum and ScCYS and ScAPI from S. cardiphyllum subsp After that, we generated the over-expreession SpCYS transgenic potato and tabacoo plants by transformation. The main results are as follows:1. Two full-length cDNAs of cystatin genes SpCYS and ScCYS were cloned from wild potato. They all contained an open reading frame of 705 bp and encoded a same-size protein (234 amino acid residues) named SpCYS and ScCYS. The proteins all processed the amino acid sequences L55ARFAVDEHN64, Q-X-V-Y-G(Q82TVAG86) and E105AKVWVKPW113 conserved in most members of the plant cystatin superfamily. SpCYS had a calculated molecular weight of 26.1 kD with an isoelectric point of 5.86. Its molecular formula is C1153HI822N322O356S7. ScCYS had a predicted molecular weight of 26.2 kD and an isoelectric point of 5.91. Its molecular formula is C1152H1825N323O356S7.2. Two aspartic proteinase inhibitor genes SpAPI and ScAPI were isolated from wild potato, which cDNAs all contained an open reading frame of 663 bp coding a same-size protein (220 amino acid residues) named SpAPI and ScAPI. SpAPI protein had a predicted molecular weight of 24.2kD with an isoelectric point of 6.39. Its molecular formula was C1082H1697N285O319S12. ScAPI had a calculated molecular weight of 24.2 kD with an isoelectric point of 6.79. Its molecular formula was C1082H1702N286O319S12.3. Semi-quantitive RT-PCR analysis indicated that SpCYS, ScCYS, SpAPI and ScAPI were expressed in leaves, stem, roots and tubers. But mRNA level was the highest in tubers. Mechanical damage treatment induced increased expression in leaves. But there was difference in expression pattern between CYS and API. The expression of SpCYS and ScCYS was induced increasing at 3 h, reached the top at 9 h, then decreased to control level. The expression of SpAPI and ScAPI was induced inhancing similarly at 3 h but higher expression level lasted to 24 h after treatment.4. Two over-expreession vectors 1304-SpCYS were constructed using the vector pCAMVBIA1304, then 1304-SpCYS was used for transforming potato and tobacoo. Two transgenic potato plants and two transgenic tobacoo plants were generated respectively using Agrobacterium-mediated transformation.

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CLC: > Agricultural Sciences > Crop > Tuber Crops > Potatoes ( potatoes )
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