Construction and Identification of Bait Vector Containing BBTV nsp Gene in Yeast Two-hybrid System

doi:10.7525/j.issn.1673-5102.2015.05.017

Abstract

Abstract: The nuclear shuttle protein(NSP) which encoded by banana bunchy top virus(BBTV) DNA6 plays an important role in infection, replication and transportation of virus. In order to research the interaction of BBTV DNA6 and host banana protein, the yeast two-hybrid system were used for the assay. pGBKT7-nsp genes was amplified using two pairs of specific primers, and their PCR products were mixed and amplified to obtain the 1/4 DNA products which contained EcoRⅠ and BamHⅠ enzyme site sequence. And then the DNA was cloned to pGBKT7 of yeast two-hybrid system. After confirmation with sequence analysis, the plasmid was transformed into the yeast cell Y2H, and its toxicity and transcriptional activation were tested by color assay. The pGBKT7-nsp had no selfactivation, and non-toxic to yeast cells, and it was consistent with the requirements of yeast two hybrid bait plasmids for the next step in the experiment.

Key words: banana bunchy top virus(BBTV), nsp genes, yeast two-hybrid, bait vector, self-activation

CLC Number:

WANG Xiang；ZHANG Xiu-Chun；YU Nai-Tong；LIANG Jie；ZHOU Peng；LIU Zhi-Xin*. Construction and Identification of Bait Vector Containing BBTV nsp Gene in Yeast Two-hybrid System[J]. Bulletin of Botanical Research, 2015, 35(5): 741-745.

References

1.华有群.也谈香蕉束顶病的发生和防止［J］.植物保护,1989,5(2):39-40.

2.余乃通,冯团诚,王建华,等.香蕉束顶病毒海口分离物Rep基因的克隆、原核表达、抗血清制备及检测［J］.植物保护,2011,37(4):38-43.

3.Harding R M,Burns T M,Dale J L.Virus-like particles associated with Banana bunchy top disease contain small single-stranded DNA［J］.Journal of General Virology,1991,72:225-230.

4.Beetham P R,Harding R M,Dale J L.Banana bunchy top virus DNA-2 to -6 are monocistronic［J］.Archives of virology,1999,144:89-105.

5.Burns T M,Harding R M,Dale J L.The genome organization of Banana bunchy top virus:analysis of six ssDNA components［J］.Journal of General Virologyl,1995,76:1471-1482.

6.Wanitchakorn,R,Harding R M,Dale J L.Sequence variability in the coat protein gene of two groups of banana bunchy top isolates［J］.Archives of virology,2000,145:593-602.

7.Dala J L.Banana bunchy top:an economically important tropical plant virus disease［J］.Advances in virus research,1987,33:301-325.

8.Niu S N,Wang B S,Guo X F.Identification of two RNA silencing suppressors from Banana bunchy top virus［J］.Archives of virology,2009,154(11):1775-1783.

9.Ding S W.RNA-based antiviral immunity［J］.Nat Rev Immunol,2010,10(9):632-644.

10.Auer C,Frederick R.Crop improvement using small RNAs:applications and predictive ecological risk assessments［J］.Trends Biotechnol,2009,27(11):644-651.

11.Zhang X,Sato S,Ye X,et al.Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene［J］.Phytopathology,2011,101(11):1264-1269.

12.Xie W S,Hu J S.Molecular-cloning,sequence-analysis,and detection of Banana bunchy top virus in Hawaii［J］.Phytopathology,1995(85):339-347.

13.Sanderfoot,Lazarowitz S G.Cooperation in viral movement:the geminivirus BL1 movement protein interacts with BR1 and redirectsit from the nucleus to the cell periphery［J］.Plant Cell ,1995,7:1185-1194.

14.Karan M,Harding R M,Dale J L.Evidence for two groups of Banana bunchy top virus isolates［J］.Journal of General Virology,1994,75:3541-3546.

15.Yu N T,Zhang Y L,Feng T C,et al.Cloning and sequence analysis of two Banana bunchy top virus genomes in Hainan［J］.Virus Gene,2012,44(3):488-494.

16.Zhang X,Yuan Y R,Pei Y,et al.Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonautel cleavage activity to counter plant defense［J］.Genes Dev,2006,20:3255-3268.

17.Lu L M,Du Z G,Qin M L.Pc4,a putative movement protein of Rice stripe virus,interacts with a type I DnaJ protein and a small H_sp of rice［J］.Virus Genes,2009,38:320-327.

18.Bagewadi B,Chen S,Lal S K,et al.PCNA interacts with Indian mung bean yellow mosaic virus Rep and down regulates Rep activity［J］.Journal of Virology,2004,78(21):11890-11903.

[1]	Anying HUANG, Dean XIA, Yang ZHANG, Dongchen NA, Qing YAN, Zhigang WEI. Cloning and Drought Tolerance Expression Analysis of PtrWRKY51 Gene in Populus trichocarpa [J]. Bulletin of Botanical Research, 2022, 42(6): 1005-1013.
[2]	ZANG Li-Na；ZHENG Tang-Chun；DAI Li-Juan；LIU Cai-Xia；QU Guan-Zheng. Identification of Protein Interactions between Flowering Repressors BpFLC and BpSVP from Betula platyphylla* [J]. Bulletin of Botanical Research, 2015, 35(6): 866-872.