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

doi:10.3864/j.issn.0578-1752.2014.14.009

摘要/Abstract

摘要： 【目的】筛选介体灰飞虱（Laodelphax striatellus，SBPH）中与水稻条纹病毒（RSV）病害特异蛋白（disease-specific protein，SP）可能发生相互作用的蛋白质，为明确介体灰飞虱传播RSV的分子机制及SP在传毒过程中的作用打下基础。【方法】提取高质量灰飞虱总RNA并通过TaKaRa D9086 OligtexTM-dT30 mRNA Purification Kit对mRNA进行纯化。利用SMART技术合成双链cDNA，利用Axygen的PCR纯化回收试剂盒将ds cDNA回收纯化，纯化后的ds cDNA再经SfiⅠ限制性内切酶处理纯化后与文库载体pPR3-N相连接以构建高质量灰飞虱cDNA文库，通过电转化的方法对灰飞虱cDNA文库进行扩增并进行cDNA文库质量和滴度检测。设计带有SfiⅠ酶切位点的引物以扩增得到目的基因即RSV SP。将带有酶切位点的SP基因序列连接到载体pDHB1上构成诱饵载体pDHB1-SP。将诱饵载体pDHB1-SP转化酵母菌NMY51中并提取总蛋白，通过Western Blot方法检测诱饵载体上的目的基因SP是否表达，并通过功能验证试验验证诱饵载体是否适合本研究采用的分裂泛素酵母双杂交系统。确定构建的诱饵载体适合本系统后，用诱饵载体与文库质粒pPR3-N共转化到酵母菌NMY51中以优化cDNA文库筛选条件并明确3-AT浓度。对灰飞虱cDNA文库进行筛选并对筛选结果进行分析，对筛选出来的蛋白进行体内试验以进一步验证是否发生互作。【结果】提取到高质量的灰飞虱总RNA，通过SMART技术合成的ds cDNA大小介于0.1—4.5 kb。构建的灰飞虱cDNA文库的库容量超过1.2×106 cfu，文库滴度为1.12×108 cfu/mL，扩增文库插入片段平均长度大于1.5 kb。载体酶切验证表明诱饵载体pDHB1-SP成功构建；Western Blot结果表明诱饵载体pDHB1-SP能够在NMY51中正常表达；功能验证表明诱饵载体pDHB1-SP适合该系统。在3-AT浓度为20 mmol?L-1的筛选条件下从构建的高质量的灰飞虱cDNA文库中筛选得到534个克隆，经测序、Blast比对最终得到76个可能与RSV的SP发生互作的灰飞虱蛋白质。根据SP在灰飞虱体内的亚细胞定位结果以及通过gene ontology(GO)对蛋白质的功能分析，挑选出20个蛋白质进行共转和β-galactosidase检测，结果表明这20个蛋白质均与RSV SP互作。【结论】通过构建高质量灰飞虱cDNA文库及酵母双杂交技术，筛选出了与RSV SP互作的蛋白质，为明确介体灰飞虱传播RSV的分子机制，及RSV的SP在传毒过程中的功能打下了基础。

关键词: 水稻条纹病毒 , SP , 酵母双杂交技术 , 灰飞虱 , cDNA文库

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) of Rice stripe virus and 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 with SfiⅠ were 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 with SfiⅠsequence. 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 whether SP 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 the β-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.

Key words: Rice stripe virus (RSV) , SP , the yeast two-hybrid , Laodelphax striatellus , cDNA library

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, 47(14): 2784-2794.

QIN Fa-Liang, LIU Wen-Wen, LI Li, WANG Xi-Feng. Screening of Putative Proteins in Vector Laodelphax striatellus Which are Interacted with Disease-Specific Protein of Rice stripe virus by Yeast Two-Hybrid Based on the Split-Ubiquitin[J]. Scientia Agricultura Sinica, 2014, 47(14): 2784-2794.

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