One ultimate goal of many plant
scientists is to gain in-depth knowledge on the function of each gene in
a given plant. Traditional approaches involve studying each plant gene,
one at a time. Since there are at least 20,000 genes in any given plant
species, it is believed that the functions of all these genes can be more
readily determined by using plant genomics, in which the functions of many
genes can be studied in parallel.
For convenience of discussion, a plant genomics project can
be arbitrarily divided into three phases. Phase I involves mapping the
genome by genetic and physical methods. Phase II includes cloning and sequencing
all, or at least most, of the genes. Phase III entails determining the
function of each gene. Phase III is not dependent on the completion of
either Phase I or II work.
Phase III work can be further divided
into three steps. Step one is to construct a complete insertional-mutagenesis
library for a large number of plants of a given species, with the hope
of disrupting each gene separately. Step two is to determine the DNA sequence
of one or both flanking regions of the inserted DNA and, in some cases,
the chromosomal location of the disrupted gene as well. Step three is to
identify the function of each disrupted gene by correlating it with a mutant
phenotype, followed by detailed analyses, including attempts to obtain
revertants or using complementation tests. Several additional approaches
are needed to study the function of a gene, especially if there is no obvious
phenotypic change. In this review article, I will cover mainly step one
of Phase III research.
For Arabidopsis and rice, the work
on Phase I was completed by mapping with a large number of RFLP, RAPD and/or
SSLP markers (see Pejic et al. 1998 for a review), as well as constructing
large contigs of YAC or BAC clones that span most of the genomes. Phase
II work is expected to be completed in mid-2000 for Arabidopsis and around
2003 for rice, when most of the DNA sequence information is expected to
have been obtained. Thus, the major tasks for genomics research over at
least the next 25-50 years will be to correlate data on the DNA sequence
of each gene with biological functions.
Work related to step one of Phase
III, functional genomics of Arabidopsis, was started ten years ago, when
a partial T-DNA insertional-mutagenesis library with approximately 8,000
insertional mutants was made (Feldmann et a! 1989, and subsequent work
by the same group). Some work on steps 2 and 3 was also initiated in 1989.
However, progress has been slow. Thus, new methods and strategies are required
to expedite the progress.
In work related to step two of Phase ifi, flanking sequences of T-DNA inserts can be determined by using TAIL PCR (Liu eta!. 1995) or suppression PCR (Schupp eta!. 1998), followed by sequence analysis. The chromosomal location of a gene-disrupted mutant can be determined by RFLP, RAPD, AFLP (Pejic et al. 1998), or SSLP (see Ponce et a!. 1999 for a review). In work related to step three of
Phase III, several methods involving cell biology and molecular biology
techniques are being used to determine the functions of genes. However,
additional methods need to be established. To assist with functional analysis,
the expression levels of all cDNAs in a library can be determined by using
the microarray method. In this method, small amounts of DNA samples from
thousands of cDNAs are applied onto a microchip. Four to ten DNA microchips
would therefore include all of the cDNAs in a library. Then, mRNAs are
isolated from different tissues of a given plant and separately hybridized
to several identical copies of DNA chips to determine tissue-specific expression
(see Pennisi 1998 for a review). Alternatively, a plant is subjected to
different abiotic stresses, or challenged with pathogens. Total mRNAs are
isolated after each treatment and separately hybridized to identical copies
of DNA chips to determine stress-induced gene expression, or pathogen-induced
gene expression, in different plant tissues.
In all three phases of a plant genome
project, large sets of data are generated. Therefore, extensive computational
analyses are required to handle these large data sets.
For step one of Phase III work,
to date, over 100,000 insertional mutant plant lines (T-DNA-tagged mutants)
have been produced by different groups of investigators. Since a T-DNA
insertional library represents a random library, for the estimated 25,000
genes in Arabidopsis, approximately 100,000 insertional mutants (with a
4-fold redundancy) are needed to cover most of the genome, such as 98%,
on a statistical basis. It is very time- consuming to produce a large number
of insertional mutants, since each one comes from a separate successful
transformation event.
In principle, the number of insertional
mutants should be proportional to the genome size, not the number of genes.
If the process of generating insertional mutants is random and, if on average,
there is a gene every 5 kb in the plant genome, one needs 24,000 mutants
for Arabidopsis, which has a genome size of 1.2 x 108 bp. By allowing a
margin of safety, most scientists would try to obtain a 10-fold redundancy,
which means 240,000 mutants are needed. By using the same calculations
and allowing a 10-fold redundancy, one needs 800,000 mutants for rice (genome
size 4 x i08 bp), and 4,800,000 for maize (genome size 2.4 x 109 bp).
A second type of insertional library
makes use of a transposable element, such as AciDs of maize, to produce
a relatively small number (such as 1,000) of primary anchor mutants. After
crossing a Ds-containing plant with an Ac-containing plant, a large number
of secondary insertional mutants can be generated from each primary mutant
after transposition. One major advantage of this method is that from 1,000
anchor plant lines, over 200,000 secondary insertional-mutant plant lines
can be generated without the need of additional time-consuming transformation
steps (Hehi and Baker, 1989; Bancroft and Dean, 1993). The AciDs system
was improved by using enhancer-trap and gene-trap plasmids to transform
Arabidopsis. This allows disrupted genes, which are non-phenotypic, to
be detected by the expression of a reporter gene (such as Gus). Eventually,
results from enhancer-trap and gene-trap experiments can be used to infer
gene function. So far, this type of insertional-mutant library includes
less than 10,000 AciDs- tagged plant lines (Sundaresan et a!. 1995; Martienssen
1998). Therefore, many additional plant lines are needed to cover approximately
98% of the genome. Another advantage of Ac/Ds-tagged plants over the T-DNA-tagged
plants is that revertants can be obtained more easily.
K. Shimamoto’s group has published
several papers on using the AciDs system to produce insertional mutant
rice plants (Izawa eta!. 1997; Enoki eta!. 1999). These plants are simple
insertional mutant lines without gene- or enhancer-trap features. So far,
approximately 6,000 mutant plant lines have been produced, demonstrating
that Ac can be used efficiently for functional analysis of the rice genome.
However, a much larger population is needed to cover the entire rice genome.
C.-d. Han’s group produced several hundred enhancer/gene trap Ds-vector
or Ac/Ds-vector transformed mutant rice plant lines. This group plans to
generate over 10,000 mutant plant lines over the next several years (Chin
et a!. 1999). The advantage of using the AciDs system in rice, especially
when enhancer-trap or gene-trap features are included, is that one can
start with a mutant plant line which shows a phenotypic change and try
to identify the gene that is responsible for it by using the flanking sequence
adjacent to the Ds element, following the classical forward genetics approach.
Recently, two abstracts reported
production of AciDs insertional mutants, which include enhancer-trap features.
Greco et a!. (1999) constructed plasmids carrying the maize transposon
systemsAc-Ds and En-I (spm) as enhancer and activation traps and transformed
Japonica rice varieties with them. Having demonstrated transposition activity
using these plasmids, this group is now developing transposon-tagged rice
plant lines for functional genomics. Another group reported preliminary
results on the genetic transformation of Basmati rice with AciDs transposons
for isolation of important genes (Dhaliwal eta!. 1999).
A third type of insertional-mutant
library is produced by making use of an endogenous transposon, such as
the Ac transposon or the mutator in maize (see references quoted in Hehl
and Baker 1989; Walbot 1992; Bensen eta!. 1995; Martienssen 1998), or the
rice transposon, tosl7 (Hirochika et a!. 1996). tosl7 transposes only during
the tissue-culture stage; thus, transposition events can be controlled.
So far, the tosl 7-based insertional mutant library has less than 9,000
rice lines (Sato et a!. 1999). In this approach, there are usually multiple
copies of the transposable element, often as many as 5 to 20 copies per
plant. The major use of this type of mutant library is to determine the
function of a gene by the reverse genetics approach. This is done by identifying
a mutant plant line with PCR-based screening of the entire mutant library
using two primers: one primer sequence derived from a specific gene of
interest and the other derived from a portion of the endogenous transposon
sequence (Ballinger and Benzer 1989; Bensen et a!. 1995; Sato et al. 1999).
Even though it is difficult to obtain revertants to confirm the specific
assignment of a mutation with a given phenotype, one can use complementation
tests to confirm an assigned gene function. As discussed earlier, by including
a margin of safety of 10-fold redundancy, perhaps 800,000 mutant plant
lines are needed. Of course, if a mutant plant line includes an average
of five copies of the endogenous transposon, the number of mutant plant
lines can be reduced fivefold.
The advantage to the above approach
is that a large number of plant lines can be screened relatively quickly
using PCR. The disadvantage is that by using a gene-specific primer with
the hope of learning more about the function of this gene, the identified
insertional mutant plant line may not show any phenotype. Since in Arabidopsis
and rice, so far, only a low percent of insertional mutant plant lines
give identifiable phenotypic changes, a considerable amount of effort may
be consumed without learning the function of the gene of interest.
All of the insertional mutant libraries
described above have been constructed based on random insertions of a DNA
(a T-DNA, an endogenous transposon, or a plasmid that contains a transposable
element that can be introduced and result in transposition) into the plant
genome. In other words, the insertional mutagenesis libraries are produced
by a “shotgun”-type approach because the site of insertion is presumed
to be random. In shotgun libraries, one usually needs to include a 4-fold
redundancy in order to cover most of the genome on a statistical basis.
Since, in fact, insertions are not random, one needs to include perhaps
a 10-fold redundancy. It is very labor-intensive to produce and analyze
approximately 800,000 insertional mutant plant lines in rice. Thus, a new
method needs to be discovered to produce an insertional mutant library
using a systematic approach. In this case, only a slight redundancy (perhaps
50%) would need to be included to cover most of the genome. Thus, in rice,
only 120,000 insertional mutants would be needed. If a systematic approach
can be devised to produce insertional mutant libraries, it would save a
great deal of time and labor in both the construction and subsequent analyses
of plant lines.
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