Cotton leaf curl virus (CLCuV) promoter and its use

Details Cotton leaf curl virus (CLCuV) promoter and its use Cotton leaf curl virus (CLCuV) promoter and its use

2001-09-14

2003-08-26

Mehta, Ashwin (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C435S468000, C536S024100

Reexamination Certificate

active

06610907

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to isolated cotton leaf curl virus promoters and method for expressing heterologous genes of interest in plant cells by using the promoter.
BACKGROUND OF THE INVENTION
Plant genetic engineering is aimed to breed transgenic plants that steadily express genes of interest at high level. Gene expression is controlled by the nucleotide sequence within the initiation site of transcription, called promoter elements, and include signals for promotion of transcription by RNA polymerase. Transcription results in the production of messenger RNA, which in turn results in the synthesis of protein.
So far, a variety of promoters derived from various resources have been applied in plant transformation. A main category of which was isolated from Agrobacterium. Another category was derived from plants. The expression levels of genes controlled by these two category of promoters are usually too low or the expression is species/varieties specific, or tissue/organ specific. The most extensively used promoters come from plant viruses. The 35S promoter of cauliflower mosaic virus is one of the most widely used promoters in dicotyledonous plant transformation as it can lead high level expression of heterologous genes in most tissues. Furthermore, in order to enhance the expression of heterologous genes, promoters are usually used in combination with enhancers, cis-elements, non-transcription sequences, intron, exon and 3′ control sequences and the like. For example, the expression level of GUS reporter gene could be enhanced up to 100-fold by using the intron 1 of maize shrunken-1 gene in tobacco cells, and could be enhanced up to 10-fold by using the first exon of maize shrunken-1 gene (C. Mass et al., 1991, “The Combination of Novel Stimulatory Element in the First Exon of the Maize Shrunken-1 Gene with the following Intron-1 Enhances Reporter Gene Expression up to 1000-fold”, Plant Mol. Biol., 16: 199-207).
Promoters from plant geminiviruses could be potentially used in heterologous gene expression. Geminivirus is a bipartite virus with twin particles. There are numerous members of geminivirus and they infect many crops of different kinds. The geminiviruses are classified into three subgroups based on their genome structure and the insect on which they rely for transmission. In subgroup I and II, the geminivirus genomes are mono partite, namely single genome. These two subgroups of geminiviruses are mainly transmitted by leafhopper. The differences between these two subgroups consist in that the former mainly infects monocotyledonous plants while the latter mainly infects dicotyledonous plants. The subgroup III of geminivirues mainly infect dicotyledonous plants and are transmitted by whitefly. Their genome is mostly bipartite, in which the larger one is called DNA A and the smaller one is called DNA B. The genome of some of the subgroup is mono partite.
A prominent characteristic of geminiviruses is that the genomic DNA is circular and single stranded, with a length of about 2400-3000 nucleotides. Another prominent characteristic of geminivirus genome is the bidirectional transcription controlled by the same promoter. The promoter can initiate transcription in two directions, therefore, the promoter is called a bidirectional promoter. One of the two directions controlsexpression of the viral replication protein gene, the other controls the expression of the coat protein gene. Thus, the two directions of the promoter are named the replication protein gene promoter and the coat protein gene promoter, respectively. The geminivirus promoter shares some features of a typical eukaryotic promoter which comprises a TATA box upstream of transcription initiation sites (P. A. Eagle et al., 1997, “Cis-Elements Tthat Contribute to Geminivirus Transcriptional Regulation and the Efficiency of DNA Replication”, J. Virol., 71: 6947-6955).
X. Zhan et al. (X. Zhan et al., 1991, “Analysis of the Potential Promoter Sequence of African Cassava Mosaic Virus by Transient Expression of the &bgr;-Glucuronidase Gene”, J. Gen. Virol., 72: 2849-2852) isolated a bidirectional promoter from African cassava mosaic virus(ACMV) genome which belongs to subgroup III. Transient expression in tobacco protoplast showed that GUS reporter gene expression activity controlled by replication protein gene promoter was 40-fold lower than that of CaMV 35S promoter. The activity of the coat protein gene promoter was very low when used alone. However, the activity can be increased up to 3-fold by the ACMV coded AC2 protein under the control of CaMV 35S promoter (Haley, A. et al., 1992, “Regulation of African Cassava Mosaic Virus Promoters by the AC1, AC2, AC3 Gene Products”, Virology, 188: 905-909).
By transforming a plant using TGMV introduced into Agrobacterium as a gene vector, R. J. Hayes et al. found that the reporter gene activity of neomycin phosphotransferase (NPTII) controlled by coat protein gene promoter from tomato golden mosaic virus(TGMV) activated by AC2 was not as strong as the CaMV 35S promoter, and was 2-fold lower than that of CaMV 35S promoter. (R. J. Hayes et al., 1989, “Replication of Tomato Golden Mosaic Virus DNA B in Transgenic Plants Expressing Open Reading Frames (ORFs) of DNA A: Requirement of ORF AL2 for Production of Single-Stranded DNA”, Nucl. Acids Res., 17: 10213-10222).
C. Brough et al. (C. Brough et al., 1992, “Kinetics of Tomato Golden Mosaic Virus DNA Replication and Coat Protein Promoter Activity in Nicotiana Tabacum Protoplast”, Virology, 187: 1-9) found that transient expression activity of GUS gene controlled by the coat protein gene promoter was up to 2-fold higher than that by the CaMV 35S promoter, which was demonstrated by using non-replicative TGMV vectors for plant transformation. However, the activity of replicative TGMV coat protein gene promoter was about 60-90-fold of that of CaMV 35S promoter.
Xu Yuquan et al. (Y. Xu et al., 1998, “Activity and Transcriptional Regulation of Bidirectional Promoter from Tobacco Yellow Dwarf Geminivirus”, Chinese Journal of Virology, 14: 68-74) isolated a subgroup III bidirectional promoter from tobacco yellow dwarf geminivirus, and found that the replication protein gene promoter was stronger and has an activity of 15-20% of the CaMV 35S promoter as demonstrated by transient expression of GUS as a reporter gene in tobacco protoplast and maize cell suspension. The activity of the coat protein gene promoter was weaker but could be increased by C1: C2 protein(corresponding to AC2 protein) by 3-fold.
Most of the previous studies on bidirectional promoters have been carried out by using transient expression. X. Zhan et al. (X. Zhan et al., 1991, “Analysis of the Potential Promoter Sequence of African Cassava Mosaic Virus by Transient Expression of the &bgr;-Glucuronidase Gene”, J. Gen. Virol., 72: 2849-2852) found that the activity of ACMV replication protein gene promoter was lower than that of the CaMV 35S promoter as demonstrated by Agrobacterium mediated transformation. Briefly, the activities of the presently isolated geminivirus promoters are lower than that of the CaMV 35S promoter. According to this invention, a promoter was isolated from cotton leaf curl virus which belongs to subgroup III of geminivirus and the activity of the promoter was studied by using GUS as the reporter gene.
Cotton Leaf curl virus (CLCuV) is a geminivirus that was found to have infested various crops in Pakistan, Sudan, and India in recent years. According to the statistics for 1993-1994 published by the Pakistan Government, the area of cotton field infested by CLCuV was up to 900,000 hectares and the loss of yield was about 80% (M. Ali et al., 1995, “Cotton Leaf Curl Virus in the Punjab: Current Situation and Review of Work”, Multan: Central Cotton Research Institute/Ministry of food, Agriculture and Livestock, Government of Pakistan/Asian Development Bank). It has been demonstrated that CLCuV was transmitted by whitefly. The virus affects a wide spectrum of crops, including French bean, okra, tobacco, tomato, cotton and t

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