Transgenic aloe plants for production of proteins and related methods

Abstract

The present inventions provide transgenic aloe plants and recombinant constructs for transforming aloe plants, aspects of which, may be applied to other monocots. The recombinant constructs may include one or more DNA sequences encoding mammalian proteins and at least one promoter capable of directing the expression of recombinant proteins in an aloe plant. The present inventions also provide methods for constructing and reproducing a transgenic aloe plant. The present inventions include methods for transfection of an aloe plant with several genes of interest simultaneously. The aloe plant production methods of the inventions may provide the potential to inexpensively and more safely mass-produce some biologically active compounds including biopharmaceuticals for disease therapy, diagnosis and prevention, and is more accessible to the less affluent countries. The aloe plant production methods may also produce proteins for cosmetics.

What is claimed is: 
     
         1 . A transgenic aloe plant comprising a recombinant DNA construct, said construct comprising a promoter, a sequence encoding an exogenous protein, a termination sequence, and a translocation sequence encoding a secretion signal peptide,
 wherein the promoter is an ubiquitin promoter from maize having SEQ ID NO. 44; and   wherein the exogenous protein is expressed from the DNA construct.   
     
     
         2 . The transgenic aloe plant of  claim 1 , wherein at least a portion of the exogenous protein is translocated from an aloe cell into the gel of an aloe leaf of the transgenic aloe plant 
     
     
         3 . The transgenic aloe plant of  claim 1 , wherein the exogenous protein is a mammalian protein selected from interferons, immunoglobulins, mammalian growth factors, mammalian hormones, blood factors, and histocompatibility antigens. 
     
     
         4 . The transgenic aloe plant of  claim 1 , wherein the exogenous protein is a mammalian protein selected from a-interferon, y-interferon, prothrombin, dermicidin and human growth hormone. 
     
     
         5 . The transgenic aloe plant of  claim 1 , wherein the translocation sequence is the alpha amylase secretory sequence from rice. 
     
     
         6 . The transgenic aloe plant of  claim 5 , wherein the translocation sequence is the alpha amylase secretory sequence from  Oryza sativa  rice having SEQ ID No. 29. 
     
     
         7 . The transgenic aloe plant of  claim 1 , wherein the recombinant DNA construct has at least one enhancer element, at least one selectable marker gene, or a combination thereof. 
     
     
         8 . The transgenic aloe plant of  claim 1 , wherein the recombinant DNA construct has at least one selectable marker gene conferring resistance to a compound chosen from the group consisting of kanamycin, chloramphenicol, G418, hygromycin B, paromomycin, glufosinate, glyphosate and gentamycin. 
     
     
         9 . The transgenic aloe plant of  claim 1 , wherein the recombinant DNA construct further comprises an expression cassette encoding eGFP, the expression cassette encoding eGFP having an internal ribosomal entry site and a termination sequence, wherein both the sequence encoding the exogenous protein and eGFP sequences are expressed as a single mRNA transcript. 
     
     
         10 . The transgenic aloe plant of  claim 1 , wherein the recombinant DNA construct further comprises an expression cassette encoding a protein conferring resistance to kanamycin, the sequence encoding the protein conferring resistance to kanamycin having an internal ribosomal entry site and a termination sequence, wherein both the sequence encoding the exogenous protein and the kanamycin-resistant sequences are expressed as a single mRNA transcript. 
     
     
         11 . A method for producing an exogenous protein in an Aloe plant, the method comprising:
 providing a transgenic Aloe plant comprising a recombinant DNA construct comprising a promoter, a sequence encoding the exogenous protein, a termination sequence and a translocation sequence encoding a secretion signal peptide;   wherein the promoter is a ubiquitin promoter from maize having SEQ ID No. 44; and   cultivating the plant so that the exogenous protein from the DNA construct is expressed, wherein at least a portion of the exogenous protein is translocated from an aloe cell into the gel of a leaf of the transgenic plant.   
     
     
         12 . The method of  claim 11 , wherein the exogenous protein is extracted from the gel of the leaf of the transgenic plant. 
     
     
         13 . The method of  claim 11 , wherein the exogenous protein is a mammalian protein selected from α-interferon, γ-interferon, prothrombin, dermicidin and human growth hormone. 
     
     
         14 . The method of  claim 11 , wherein the translocation sequence is the alpha amylase secretory sequence from rice ( Oryza sativa ). 
     
     
         15 . The method of  claim 11 , wherein the transgenic Aloe plant is generated by a process comprising:
 isolating aloe cells from an aloe seed, meristem or plant;   growing the aloe cells in culture in a nutrient medium containing an auxin, and a cytokinin to form a callus;   transforming the callus with the recombinant DNA construct;   selecting for transformed aloe cells containing the recombinant DNA construct while growing the callus in a shooting and selection medium comprising 0.2 mg/L of 1-naphthaleneacetic acid (NAA) or indole 3-acetic acid (IAA), and 2 mg/L of 6-benzylaminopurine (BAP);   growing a shoot regenerated in the shooting and selection medium in a rooting medium comprising 0.2 mg/L NAA; and   when roots have begun to form, transferring the plantlet to soil.   
     
     
         16 . The method of  claim 15 , wherein the aloe cells are embryonic aloe cells from an aloe seed, or meristematic cells from an aloe plant, an aloe shoot meristem, or an aloe root meristem. 
     
     
         17 . A method of producing a transgenic Aloe plant that expresses a protein exogenous to the plant, the method comprising:
 culturing undifferentiated callus of aloe cells in a nutrient medium containing an auxin, and a cytokinin;   introducing into the callus a recombinant DNA construct comprising a promoter, a sequence encoding the exogenous protein, a termination sequence and a translocation sequence encoding a secretion signal peptide;   wherein said promoter comprises SEQ ID No. 44;   wherein the exogenous protein is expressed from the DNA construct;   growing the callus in a shooting and selection medium comprising 0.2 mg/L of 1-naphthaleneacetic acid (NAA) or indole 3-acetic acid (IAA), and 2 mg/L of 6-benzylaminopurine (BAP);   growing a shoot regenerated in the shooting and selection medium; and   once roots have begun to form, transferring the plantlet to soil.   
     
     
         18 . The method of  claim 17 , wherein at least a portion of the exogenous protein is translocated from an aloe cell into the gel of an aloe leaf of the transgenic aloe plant. 
     
     
         19 . The method of  claim 17 , wherein the exogenous protein is a mammalian protein selected from α-interferon, γ-interferon, prothrombin, dermicidin and human growth hormone. 
     
     
         20 . The method of  claim 17 , wherein the callus is from aloe seed.