Research Overview
Research Group: Regulation of Gene Expression and Plant Biotechnology
I. Introduction
Our group mainly focuses on
1. Regulation of plant gene expression and its application in plant biotechnology;
2. Expression and regulation of pathogenesis-related genes of plant bacterial pathogens;
3. Functional analysis of pathogenicity determinants of plant viruses and their interaction with plant host defense systems.
Group Members
- Principle investigator: Rong-Xiang Fang, Professor, Member of Chinese Academy of Sciences (CAS).
1967 Graduated from Dept. of Chemistry, Fudan University.
1968- Institute of Microbiology, CAS (IMCAS).
1979-1981 Laboratory of Molecular Biology, Gent University, Belgium.
1985-1988 Laboratory of Plant Molecular Biology. The Rockefeller University, USA.
1992-1999 Deputy Director of IMCAS.
2000-2004 Director of IMCAS.
- Other members: 2 Associate Research Fellows, 2 Assistant Research Fellows,
5 Ph.D. students, 2 Master students and 3 guest investigators.
II. Background, Significance and Progress
1. Regulation of plant gene expression and its application in plant biotechnology
Gene expression can be regulated at many of the steps in the pathway from DNA to RNA to protein. Transcriptional control is paramount in regulation of gene expression. In plant biotechnology, high-level transcription of transgenes is needed in most of cases, but sometimes inducible expression or expression at specific plant tissues is desired. Three plant promoters which are potentially applicable in plant biotechnology have been studied in our group. (1) The promoter of the rice HSP70 gene. This is a constitutively expressed promoter. By analysis of a series of 5'deletions, the fragment from the translation start codon to the upstream 493 bp was found to have the highest promoter activity which is higher than that of the most-used strong CaMV 35S promoter. And the 76-bp 5'-UTR is essential for the promoter activity. (2) The promoter of the rice cell wall glycine-rich protein gene. Our study revealed that it preferentially expressed in plant vascular tissues. We have further shown that a 99-bp sequence in the promoter is responsible for vascular-specific expression. This promoter could be used to develop disease-resistant plants against pathogens which are accumulated in vascular tissues. (3) The carrot invertase II gene promoter. It was known that the mRNA of carrot invertase II accumulates only in developing tap roots, implying the tap root-specificity of the gene promoter. We have conducted the 5'deletion analysis of a 2.7-kb region upstream of translation start codon and found that the fragment from -257 possesses the highest promoter activity which is 50% higher than that of the CaMV 35S promoter in carrot tap roots. Duplication of the -257 — -136 sequence increased the promoter activity by 48%. This promoter might be useful for expressing antigen proteins in carrot to develop oral vaccines. Vaccines against Rotaviruses and EPEC are currently being studied in our group.
In most of plant biotechnological applications, production of proteins in high amount is desirable. In this context, enhancement of translation efficiency or protein stability is of more direct effect on genetic manipulations. We found that fusion of a ubiquitin monomer (UBQ) to the N-terminus of GUS increased the GUS accumulation by 4-fold, and an N-terminal peptide (14 amino acids) of the Cucumber mosaic virus coat protein (NP14) was more potent as a fusion partner than UBQ, resulting a 7-fold increase in the GUS level. However, the NP14-GUS fusion protein can not be auto-cleaved within plant cells. We thus further designed an NP14-UBQ-GUS double fusion strategy to take advantage of both the higher enhancing activity of NP14 and the cleavability of the UBQ fusion protein by the plant endogenous ubiquitin C-terminal hydrolases.
2. Expression and regulation of pathogenesis-related genes of plant bacterial pathogens
Many bacteria use quorum sensing (QS) to regulate a diverse array of biological functions including pathogenicity on their hosts. Xanthomonas campestris pv. campestris (Xcc) is the causative agent of crucifer black rot disease. Unlike in other Gram-negative bacteria, no N-acylhomoserine lactones, a group of signal molecules of QS, have been identified in Xcc so far. However, sequence analysis of the Xcc genome revealed the presence of a single copy of luxR-like gene, named xccR, followed by a proline iminopeptidase gene (pip). A 20-bp lux box-like palindromic sequence was found at 60 bp upstream of the translation start site of the PIP gene. Our study focuses on the expression regulation of the locus xccR-pip box-pip and the relatedness to the Xcc pathogenicity. By mutagenesis we found PIP is crucial for the pathogenicity. Expression of pip was positively regulated by XccR via pip box. Furthermore, we found that the pip promoter was induced by the extract from the host cabbage, and XccR and pip box are essential for the induction. We will attempt to elucidate the mechanism of how Xcc utilize host factors to help it establish infection of the host.
3. Functional analysis of pathogenicity determinants of plant viruses and their interaction with plant host defense systems.
Our research is aiming at the functional analysis of virus-encoded proteins, especially the pathogenicity determinants, and virus-plant host interactions. One of the viruses currently studied in our group is Rice yellow stunt rhabdovirus (RYSV). In addition to N, P, M, G and L proteins which are common to other rhabdoviruses, RYSV encodes P3 which has been found in most of the plant rhabdoviruses but not in animal rhabdoviruses, and P6, the analog of which was encoded by some animal viruses but functionally unknown. We have shown that RYSV P3 is a movement protein responsible for the virus cell to cell transport. We are currently studying the function of RYSV P6. The other viral protein we are studying is the protein 2b of Cucumber mosaic virus (CMV). CMV 2b is a suppressor of the host RNA silencing system and one of the pathogenicity determinants of CMV. We attempt to (1) determine the protein motifs responsible for the suppressor activity and pathogenicity; (2) gain insights into the interactions between 2b and host factors involved in siRNA- and miRNA-mediated silencing pathways and in other plant defense systems; (3) explore a novel approach to anti-CMV infection by targeting 2b using RNA interference.
Publications (2001-2005) (* the corresponding author)
1. W.L. Lei, R.X. Fang*, G.H. Zhang, X.Y. Chen and X.Q. Zhang
Recombination with coat protein transgene in a complementation system based on Cucumber mosaic virus (CMV).
Science in China (Series C) (2001) 44: 263-273.
2. C.Q. Cai, R.X. Fang*
Technical improvements in genetic manipulation of Pichia pastoris and their application in hirudin expression
Chinese Journal of Biotechnology（2001）17：155-160。
3. X.G. Wang, G.H. Zhang, C.X. Liu, Y.H. Zhang, C.Z. Xiao, R.X. Fang*
Purified cholera toxin B subunit from transgenic tobacco plants possesses authentic antigenicity.
Biotechnology and Bioengineering (2001) 72: 490-494.
4. T.X. Guo, R.X. Fang*, G.H. Li, Y. Qian
A fusion protein of rotavirus VP6 and cholera toxin B subunit: expression in Escherichia coli and analysis of biological activities.
Chinese Journal of Biotechnology（2001）17：621-625。
5. Z.M Liu, Z.Z. Liu, Q.W. Bai, R.X. Fang*
Agroinfiltration, a useful technique in plant molecular biology research.
Chinese Journal of Biotechnology（2002）18：411-414。
6. W.Q. Cai, R.X. Fang*, H.S. Shang, X. Wang, F.L. Zhang, Y.R. Li, J.C. Zhang, X.Y. Cheng, G.L. Wang, K.Q. Mang
Development of CMV-and TMV-resistant chili pepper: field performance and biosafety assessment.
Molecular Breeding (2003) 11: 25-35.
7. Z.Z. Liu, J.L. Wang, X. Huang, W.H. Xu, Z.M. Liu, R.X. Fang*
The promoter of a rice glycine-rich protein gene, Osgrp-2, confers vascular-specific expression in transgenic plants.
Planta (2003) 216: 824-833.
8. Z.Z. Liu, J.L. Wang, Q. Wang, X. Huang, W.H. Xu, L.H. Zhu, P. He and R.X. Fang*
Structure, expression pattern and chromosomal localization of the rice Osgrp-2 gene.
Science in China (2003) 46: 584-594.
9. K.X. Mi, J. Li, Z.S. Zhang, R.X. Fang*
GM1-binding ability and immunogenicity of CTB/CS3 fusion protein expressed in E. coli.
Prog. Biochem. Biophys.（2003）30: 278-254。
10. H.G. Jia, Y.Q. Pang, R.X. Fang*
Agroinoculation as a simple way to deliver a tobacco mosaic virus-based expression vector.
Acta Botanica Sinica (2003) 45: 770-773.
11. Y.W. Huang, H. Zhao, Z.L. Luo, X.Y. Chen and R.X. Fang*
Novel structure of the Rice yellow stunt nucleorhabdovirus genome: identification of the gene 6-encoded virion protein.
Journal of General Virology (2003) 84: 2259-2264.
12. Q.H. Sun, W. Wu, W. Qian, J. Hu, R.X. Fang, C.Z. He*
High-quality mutant libraries of Xanthomonas oryzae pv. oryzae and X. campestris pv. campestris generated by an efficient transposon mutagenesis system.
FEMS Microbiology Letters (2003) 226: 145-150.
13. L.H. Wang, Y.W. He, Y.F. Gao, J.E. Wu, Y.H. Dong, C.Z. He, S.X. Wang, L.X. Weng, J.L. Xu, L. Tay, R.X. Fang and L.H. Zhang*
A bacterial cell-cell communication signal with cross-kingdom structural analogues.
Molecular Microbiology (2004) 51: 903-912.
14. H.G. Jia, L.F. Lv, Y.Q. Pang, X.Y. Chen, R.X. Fang*
Using green fluorescent protein as a reporter to monitor elimination of selectable marker genes from transgenic plants.
Chinese Journal of Biotechnology (2004) 20: 10-15.
15. H.N.Guo, X.Y.Chen, H.L.Zhang, R.X.Fang, Z.Q.Yuan, Z.S.Zhang, Y.C.Tian*
Characterization and activity enhancement of the phloem- specific pumpkin PP2 gene promoter
Transgenic Research (2004) 13: 559-566.
16. Y.W. Huang, Y.F. Geng, X.B. Ying, X.Y. Chen and R.X. Fang*
Identification of a movement protein of rice yellow stunt rhabdovirus
Journal of Virology (2005) 79: 2108-2114.
III. Future Research Plan
1. Manipulation and optimization of transgene expression, mainly for the purpose of using plants as bioreactors to produce oral vaccines and pharmaceutical proteins.
2. Elucidation of expression regulation of the xccR-pip locus of Xcc, with emphases on identification of plant host factors and the Xcc genes downstream of pip in the pathogenesis pathway.
3. Study on the mechanisms of the counter-host defense function of the silencing suppressor CMV 2b. To seek if the host equips with a counter-counter-defense mechanism targeting on the suppressor.