中国农业科学Issue(14)：2784-2794,11.DOI:10.3864/j.issn.0578-1752.2014.14.009
利用酵母双杂交技术筛选介体灰飞虱中与水稻条纹病毒病害特异蛋白互作的蛋白质
Screening of Putative Proteins in VectorLaodelphax striatellus Which are Interacted with Disease-Specific Protein ofRice stripe virus by Yeast Two-Hybrid Based on the Split-Ubiquitin
摘要
Abstract
[Objective]The objective of this study is to screen the proteins in the small brown planthopper (Laodelphax striatellus, SBPH) interacted with the disease-specific protein (SP) ofRice stripe virusand to further clarify their functions for understanding the mechanism of interactions between virus and vector insect.[Method]The high-quality total RNA of the SBPH was isolated and purified by TaKaRa D9086 OligtexTM-dT30 mRNA Purification Kit and used as the template to synthesize the double-strand cDNAs by SMART technology. The ds cDNAs dealed withSfiⅠwere ligated with vector pPR3-N for construction of a cDNA library with high quality. cDNA library was amplified by electro transformation and tested the quality and titer. The target gene (SP) was got by the primers designed withSfiⅠsequence. The gene sequence,SP, was ligated with vector pDHB1 and the product was called bait vector, pDHB1-SP. Then pDHB1-SP was transformed into the NMY51 yeast, and total proteins were extracted. Western Blot was applied to check whetherSP was expressed or not and functional assay was used to identify whether the bait vector, pDHB1-SP, was suited to this yeast two-hybrid system. The library screening stringency was optimized by using a pilot screen. Proteins interacted with the bait pDHB1-SP were screened from the cDNA library using the yeast two-hybrid system based on the split-ubiquitin and analyzed using gene ontology (GO), then 20 proteins were screened for further interaction assay.[Result]The high quality RNA was extracted for synthetised ds cDNA by SMART technology and the compounded ds cDNA size was ranged from 0.1 to 4.5 kb. The unamplified cDNA library was consisted of 1.2×106 independed clones, the titer of library was 1.12×108 cfu/mL, and the average size of inserts was above 1.5 kb in the cDNA library. The result of restriction enzyme digestion suggested that the bait vector, pDHB1-SP, was constructed successfully. pDHB1-SP was expressed in NMY51 using Western Blot and suited to the yeast two-hybrid system by functional assay. A total of 534 clones from the library were got, and 76 proteins may be interacted with RSV SP after sequencing and blast. Twenty of them selected based on the subcellular localization of SP in SBPH and functional analysis in terms of GO were further confirmed by theβ-galactosidase assay and co-transformation assay and the results suggested that they were all interacted with SP.[Conclusion]Some proteins interacted with RSV-SP were screened from the cDNA library of SBPH and are important for understanding the mechanism of interaction between RSV and vector insect.关键词
水稻条纹病毒/SP/酵母双杂交技术/灰飞虱/cDNA文库Key words
Rice stripe virus (RSV)/SP/the yeast two-hybrid/Laodelphax striatellus/cDNA library引用本文复制引用
秦发亮,刘文文,李莉,王锡锋..利用酵母双杂交技术筛选介体灰飞虱中与水稻条纹病毒病害特异蛋白互作的蛋白质[J].中国农业科学,2014,(14):2784-2794,11.基金项目
国家“973”计划（2010CB126200）、国家自然科学基金 （）
