ANTIB IOTIC MULTI PLE CLONING
RESISTANCE S ITES FOR CHIMERIC
GENE
GENE INSERTION
(for selection in plants)
"
,
75
BORDER / /
, , ,
RIGHT
� "
(
TRANSFERRED REG ION(T-DNA) \ \ BORDER r� - - - -
----
-,
----
- - - -J
�
- - - I r, , ,
\ \ \, ,
, , , , ... ... ...
VECTOR BINARY....
ANTIBIOTIC RESISTANCE GENE(for selection in bacteria)
,
" " I I I I I , r I IBinary vectors:
(1) Replicate in Agrobacterium tumefacians.
(2) Contain a plant selectable marker gene within the T-DNA, which usually confers resistance to an antibiotic and is used to indicate successful transfonnation.
(3) Contains the Ti-plasmid sequences required for integration of the T-DNA into the plants genomic DNA.
76
(4) Contains a polylinker within the T-DNA, which is a short sequence of DNA into which
chimeric genes can be inserted.
1.3.5.1.4 Regeneration of Plants Transformed with a Binary Vector.
As wounding is required for crown gall tumour growth in nature, plant cell transfonnation is perfonned by innoculation of wounded plant tissue with Agrobacterium tumefacians containing a binary vector.
The presence, within the binary vector of sequences required for transfer of T-DNA, results in the incorporation of the chimeric and plant selectable marker genes into the plant's genomic DNA. Subsequent shoots which grow from infected plant tissue and are able to survive and proliferate on the selectable marker usually contain the chimeric gene.
1.3.5.2 FOREIGN GENE EXPRESSION IN PLANTS.
Table 12 summarises a number of examples of foreign genes that have been transferred into plants. Stable genetic transfonnation is now routinely obtained with the following
economically important plants: tomato, potato, cotton, white clover, lucerne, flax, rape, tobacco, Lotus sp., Douglas fir and poplar. However, despite this progress, regeneration of soybean, cereals and temperate grasses is not yet possible as these species are not able to be transfonned by Agrobacterium tumefacians and are difficult to regenerate by presently available tissue culture techniques (Weising et al., 1988).
1.3.5.3 THE LEVEL OF EXPRESSION OF FOREIGN GENES IN PLANTS.
The level of expression of a foreign gene in plants would appear to depend upon the source of the gene and the plant tissue in which the gene is to be expressed. The levels of
77
Table 1 2: Examples of genes from animals, yeast,
plant viruses, bacteria and plants which have been transferred to either tobacco, tomato or petunia plants
by tumefacians mediated transformation.
AUTHOR ORIGIN OF TRANSFORMED
GENE SPECIES
ANIMAL Ow et a/. (1986) Firefly
Luciferase Gene Tobacco ' Hunt et al. (1 987) Human
Growth Hormone Gene Tobacco '
YEAST
Barton et al. (1983) Aldehyde
Dehydrogenase Gene Tobacco # PLANT VIRUS
Bevan et al. (1985) Tobacco Mosiac Virus
Coat Protein Gene Tobacco Turner et al. (1987) Lucerne Mosiac Virus
Coat Protein Gene Tobacco and Tomato BACTERIA
Della Cioppa et al. EPSP Synthase
(1987) Gene (1 ) Tobacco
PLANT
Lawton et al. (1987) Soybean Petunia 8-conglycinin Gene
Keith and Chua Wheat Tobacco •
(1986) rbcS Gene (SSU) (2) Hilder et al. (1987) Cowpea
Gene Tobacco
("') Transient expression only
(#) No expression (1 ) Enolpyruvylshikimate-3-phosphate (2) Ribulose-1 .5-bisphosphate carboxylase
78
expression of foreign seed storage genes in the seeds of transformed plants, although generally lower than in the native seed, can be as high as 2-8% of total seed protein (Sengupta-Gopalan et a/., 1985; Altenbach et at., 1989; Vandekerckhove et a/., 1989), whereas the level of expression of foreign genes in the leaves of transformed plants is very low and generally in the range of 0.001 -0. 1 % of total leaf protein (Wei sing et aI., 1988).
Low levels of expression of herbicide and insect resistance genes in the leaves of
transformed plants gives acceptable levels of plant protection (Weising et al., 1 988; Schulz
et aI. , 1990). However, it has been estimated that the level of expression of genes coding
J
or proteins containing a high proportion of SAA, in the leaves of lucerne would need to be :'at least 5% of total leaf protein in order to have any positive benefits on animal production(T.1. Higgins, personal communication).
In contrast to the generally low level of expression of foreign genes in the leaves of their
new hosts, there are four examples of foreign genes showing relatively high levels of expression in the leaves of transgenic plant hosts.
(1) Glutamine synthatase (GS) is an enzyme which plays a key role in the detoxification of
ammonia in plants, and therefore its overproduction is of potential use where herbicides are
being used which inhibit GS activity (Eckes et at. , 1 989). A leaf-specific GS from lucerne has been engineered for expression in the leaves of tobacco. The foreign GS gene was expressed at a level of 5% of total tobacco leaf protein, whilst tobacco plants expressing the
'�S gene at that level.;:were 20-fold more tolerant of the GS inhibiting herbicide, L
phosphinothricin (Eckes et aI., 1989).
(2) The gene coding for vicilin, a pea seed protein, when engineered for expression in the leaves of tobacco, expressed vicilin at a level of 0.01 % of total leaf protein (T.1. Higgins, personal communication). Munro and Pelham (1 987) identified an amino acid sequence (lys-asp-glu-Ieu), which occurred at the carboxy terminus of some proteins and appeared to be responsible for targeting those proteins for retention in the endoplasmic reticulum. When the before mentioned vicilin gene was modified to include the endoplasmic reticulum targeting sequence, vicilin was expressed in the leaves of tobacco at a level of
1 % of total leaf protein (T.J. Higgins, personal communication).
(3) When the seed-specific cowpea trypsin inhibitor gene (CpT!), was engineered for
expression in the leaves of tobacco, the trypsin inhibitor was expressed at a level of ! % of total leaf protein. Expression of the CpT! gene enhanced plant resistance to insect pests (Hilder et a/., 1987).
79 (4) When mouse antibody cDNA's derived from hybridoma mRNA, were engineered for leaf expression, functional antibodies were expressed at a level of 1 .3% of total leaf protein in tobacco (Hiatt et aL., 1989).
These examples of relatively high levels of foreign gene expression in the leaves of a transgenic host suggest it is worthwhile pursuing high levels of expression of genes coding for proteins containing a proportion of SAA, in the leaves of agriculturally important legumes, such as lucerne and white clover.
However, there is no experimental data available to indicate whether 5% of total leaf protein is the level of expression required to achieve significant responses in animal production, particularly wool growth. There is a need for experiments to determine the relationship between the level of expression of high sulphur protein genes in lucerne and the animal production response to grazing that lucerne, in an effort to establish exactly what level of expression is required. In the case of PA 1 , SF8 and thionin, a suitable source of large quantities of pure protein for orally or intraruminally supplementing grazing animals is not available. However, ovalbumin is more readily obtainable and thus a more likely test protein for use in experiments to determine the ideal level of transgenic protein expression.
2.
80