Dissect the functions of genes in Arabidopsis thaliana
using reverse genetics strategy
Lv Haihui, Department of Biochemistry & Molecular Biology, Grade 98
Abstract
The Arabidopsis thaliana Genome Project had been finished and the genome sequence was
published in Nature1 by the end of last year, which should give a boost to almost every area of
plant science. Taking advantage of the known Arabidopsis genome sequence, we are setting up the
first Arabidopsis mutant collection in China, to identify the genes and determine their functions
using a reverse genetics strategy. T-DNA insertional mutagenesis was adopted to generate
Arabidopsis mutants. TAIL-PCR strategy was performed to amplify the unknown flanking
sequences of the T-DNA insertions. Then we compare the obtained sequences with the genome
sequence available on-line and specify the mutated genes.
Keywords Arabidopsis thaliana, T-DNA, Thermal asymmetric interlaced PCR (TAIL-PCR),
Mutant, Reverse genetics
Arabidopsis thaliana, a small annual weed of central Eurasian origin, has become a model plant
for the genetic analysis. As described in several excellent reviews2, 3, Arabidopsis is ideally suited
to laboratory studies. It is small, self-fertilizing and productive, with over 10,000 seeds per
individual. As plants go, its five weeks’ life cycle in the laboratory is extremely short, making
mutant identification and genetic analysis very rapid. From the prospective of molecular genetics,
its small genome size (about 120Mb) and paucity of repeated sequences are very convenient for
analysis. In recent years a substantial database of partially sequenced cDNAs or expressed
sequence tags (ESTs) has been setting up due to the efforts of a number of laboratories worldwide.
In 2000 came the successful completion of the Arabidopsis Genome Project. Arabidopsis is
among the few biological model systems that possess such an assemblage of features and
resources.
Reverse genetics, fundamentally different from forward genetics, begins with a mutant gene
sequence and asks the question “What is the resulting change in phenotype?” While forward
genetics is just the opposite. The recent completion of total genome sequences has only now
created the opportunity for pursuing reverse genetics in an exhaustive and complete manner. We
have now used this reverse genetics strategy in Arabidopsis to approach the genes with unknown
function (figure.1).
T-DNA insertion mutagenesis
T-DNA is a segment of the tumor inducing (Ti) plasmid of Agrobacterium tumefaciens and is
delimited by short imperfect-repeat border sequences. The T-DNA including any sequences
inserted between the borders, can be transferred by Agrobacterium to the plant cell. Engineered Ti
plasmids used in experiment have been disarmed of the tumor promoting and biosynthetic genes,
which are replaced with antibiotic resistance markers to allow the selection of transformants.
1
Arabidopsis Plants
Preparation of Agrobacterium containing engineered Ti
plasmid
________________________________________________
↓
Agrobacterium mediated transformation
↓
Seeds harvesting
↓
Screening by PPT resistance
↓
Planting transformed plants
↓
DNA isolation
↓
Amplify the flanking sequences of each insertion
(using TAIL-PCR strategy)
↓
DNA sequencing
↓
BLAST search to determine the inserted gene
↓
Genetic and biochemical analysis of the tagged gene
Figure 1. Using reverse genetics to dissect the gene function in Arabidopsis
Aside from the advantage of tagging the disrupted locus, T-DNA has other advantages over
traditional mutagenesis systems: low copy number and random insertion. After segregation
analysis of a large number of transformants, Feldmann4 concluded that the average number of
independent inserts is 1.5 per diploid genome. Also, within the context of the target gene, insertion
appears to be random. Because they are largely found in genes previously identified by physical
and chemical mutagenesis, it also appears that at the individual chromosome level T-DNA has no
significant target-site preference.
Saturation of the Arabidopsis Genome
Three variables determine the probability that a T-DNA insert will be found within a given gene:
the size of the gene, the size of the genome, and the number of T-DNA inserts distributed among
the population. According to analysis by Feldmann et al., the estimated average Arabidopsis gene
is 4kb in length. With an average of 15 inserts per line they predicted that 100,000 transformed
lines would be sufficient to give a probability of 95% of founding an insert in any average gene.
Floral Dip
Several improvements in Agrobacterium-mediated transformation techniques have made T-DNA a
viable method for approaching genome wide mutagenesis. However, hundreds of thousands of
transformed plants necessary for saturation of the genome were beyond reach, until Clough and
2
Bent5 elaborated a new transformation method-floral dip. Arabidopsis transformants could be
obtained at a high rate (0.5-3% of all progeny seed) simply by dipping flowering plants in
Agrobacterium that are suspended in a solution containing sucrose and the surfactant SilwetL-77.
The three main requirements for successful transformation were: (1) Correct plant
developmental stage (maximum number of unopened floral bud clusters), (2) suger, (3) surfactant.
Covering of inoculated plants with a plastic dome to maintain high humidity during the first 12-24
hours after inoculation was beneficial, givng about twice as many transformants.
Isolation of T-DNA insert junctions by TAIL-PCR
Thermal asymmetric interlaced (TAIL)-PCR (Liu, 1995) is an efficient technique for isolation of
target DNA segments adjacent to known sequences. Requiring neither special DNA manipulations
before PCR nor laborious screening afterwards, it nevertheless yields specific products of high
purity even as sequencing templates. TAIL-PCR utilizes a set of nested sequence-specific primers
together with a shorter arbitrary primer of lower melting temperature. Annealing steps can be
carried out at different temperatures so that both primers – or alternatively, only the specific
primer – function well. Interspersing high and reduced stringency cycles enables target sequences
to be amplified preferentially over nonspecific products. Principle of this PCR strategy is shown in
Figure 2.
Material and Procedure
Plant Growth
Arabidopsis plants (ecotype Columbia) were planted 1-2 per pot in moistened soil. They were
grown to flowering stage in a shaded green house, at 19℃-24℃, with 15 hours’ lighting per day.
To obtain more floral buds per plant, inflorescences were cliped after most plants had formed
primary bolts, relieving apical dominance and encouraging synchronized emergence of multiple
secondary bolts. Plants were dipped when most secondary inflorescences were about 20cm tall.
Culture of Agrobacterium tumefaciens and Inoculation of Plants
1. Agrobacterium tumefaciens strain GV3101 carrying the binary plasmid pKSI015 were grown
for more than 24h at 28℃ in sterilized LB containing Kanamycin and Ampicillin (both of a
concentration of 50μg/mL)
2. PCR screening. First, bacteria culture was diluted in 10 folds and genome DNA was melted
at 100℃ for 10min.
Primer: T7 and K749
Table 1. Cycle settings used for PCR screening of transformed bacteria.
Thermal settings
Cycle No.
30
94℃, 60s; 60℃, 0s; 72℃, 120s
1
72℃, 10min
3.
4.
Through gel analysis, the band of 1.4kb indicated the transformed Agrobacterium.
The corresponding bacteria were grown to log phase in 500mL sterilized LB at 28℃, 250rpm
for 24h. Cells were harvested by centrifugation for 8min at 4℃, 5500g and then resuspended
in 1L 1/2 strength MS (Sigma Chemicals) Medium, with BAP and SilwetL-77.
The inoculum was added to a beaker, and plants were inverted into this suspension for about
15min. Then plants were moved to a plastic tray and covered with a plastic dome to maintain
humidity. After 24h plants were taken out and returned to the greenhouse for a further 3-5
3
weeks until siliques were brown and dry. Seeds were harvested, desiccated and stored at 4℃.
Left border primers
short arbitrary degenerate
DL1 DL2 DL3
(AD)primers
Genome sequence flanking T-DNA insertion
nontarget sequence
Primary PCR with DL1 and AD primer
5 high stringency cycle
1 low stringency cycle
TAIL-Cycling (14 super cycles)
1 reduced stringency cycle
specific product
product yield
moderate (22n)
2 high stringency cycles
nonspecific product I
nonspecific product II
high (23n)
low (2n)
50-fold dilution
Secondary PCR with DL2 and the same AD primer
product yield
high (detectable)
low (undetectable)
10-fold dilution
Tertiary PCR with DL3 and the same AD primer
Agarose gel analysis
Figure 1. TAIL-PCR procedure for specific amplification of genomic sequence flanking a T-DNA insertion.
Three PCR reactions are carried out sequentially to amplify target sequences using nested T-DNA-specific
primers on one side and an AD primer on the other. One or more sites within the flanking sequence are adapted for
annealing to the AD primer through a special low-stringency cycle. Even after creation of sites adapted for the AD
primer, however, high-temperature annealing still favors the specific primer, resulting in a linear amplification of
target molecules. To achieve adequate thermal asymmetry, the specific and AD primers were designed to have Tms
of 57℃-62℃ and 44℃-46℃ respectively. By interspersing reduced-stringency cycles to allow AD priming,
double-stranded molecules can be formed, and the preferential linear amplification of target molecules becomes
logarithmic. In the secondary and tertiary reactions non-specific product I fail to be reamplified and thus is not
shown.
Selection of Putative Transformants
4
As Ti plasmid contains PPT resistant gene, transformed plants can grow in culture with PPT.
Seeds were treated in 30% bleach for 10min, followed by thrice rinses with sterile water. Then
they were planted in solid MS Medium with 2.5% PPT. Plates were first kept in the greenhouse
(as mentioned in Plant Growth before) with all-day lighting for 1-2 days, then at 4℃ for about 3
days, and last returned to the greenhouse. Plates were sealed with surgical tape for the first week
of growth. Transformants were identified as PPT resistant seedlings that produced green leaves
and well established roots within the selective medium.
DNA Isolation
Preparation of genomic DNA using CTAB from the T-DNA insertion lines was done as follows:
1. 3-4 leaves were taken from each plant, stored frozen in a 1.5mL Eppendorf tube, and
grounded to powder. Add 600μL isolation buffer (2% CTAB, 0.2-0.5% mercapto-ethanol,
incubated in 60℃ water bath for 1 hour).
2. The Eppendorf tube was heated in 65℃ water bath for more than 30min (shaken once every
2-3 minutes). 600μL chloroform was added to the tube, mixed, and centrifuged at 8,000rpm
for 10min. Equal volume of isoamyl alcohol was added to the supernatant and DNA was
recovered by centrifugation at 12,000rpm for 20 min. The pellets were washed with 70%
ethanol and then 100% ethanol, desiccated in vacuum, and then dissolved in 40μL TE buffer.
Finally, 0.6μL RNase was added and the tube was kept for 0.5h at 37℃ to eliminate RNA.
PCR Screening
Before TAIL-PCR, a PCR to test the bar gene in T-DNA and cop1 gene in Arabidopsis genome
was taken. For transformants, amplified products after electrophoresis should be of 300bps and
700bps respectively for a sequence of cop1 gene and bar gene.
Primers:
COP1-5: 5’ > TGACTATGCTCTGTTTCAGCT < 3’
COP1-3: 5’ > TTAGTAAACCAAGGAACACCA < 3’
BAR5-S: 5’ > TCGACTCTAGCGAATTCCTC < 3’
BAR3-S: 5’ > ATAGGCGTCTCGCATATCTC < 3’
A reaction mixture (25μL) was prepared with 2.5μmol dNTPs, 12.5μmol BAR5-D and BAR3-D
primer, 3.1μmol COP1-5 and COP1-3 primer, and 0.5μTaq enzyme. 10-20ng DNA templates were
added.
Table 2. Cycle settings used for PCR screening of bar and cop gene
Thermal settings
Cycle No.
1
95℃, 2min
35
94℃, 1min; 60℃, 1min; 72℃, 1min
1
72℃, 10min
Flanking Sequence Amplification Using TAIL-PCR
Three successive reactions were taken to segregate the disrupted gene sequences for the tested
transformants.
The specific primers and their annealing positions (Figure 2) within the T-DNA region of the
transfer vector pKSI015.
DL1:
5’ > GACAACATGTCGAGGCTCAGCAGGA < 3’
5
5’ > TGGACGTGAATGTAGACACGTCGA < 3’
5’ > GCTTTCGCCTATAAATACGACGG < 3’
DL2:
DL3:
5’>GGATCGACTGCTTCTCTCGCAACGCCATCCGATGGATGATGTTTAAAAGTCCCATGT
GGATCACTCCGTTGCCCCGTCGCTCACCGTGTTGGGGGGAAGGGCACATGGCTCAGTT
CTCAATGGAAATTATCTGCCTAACCGGCTCAGTTCTGCGTAGAAACCAACATGCAAGC
TCCACCGGGTGCAAAGCGGCAGCGGCGGCACCATATATTCAATTGTAAATGGCTTCAT
GTCCGGGACCAATTTTTTTTCAATTCAAAAATGTAGATGTCCGCAGCGTTATTATAAAAT
GAAAGTACAATCTACATGGATCGTAATGAGTATGATGGTCAATATGGAGGAAAAGAAA
GAGTAATTAATTTTGATAAAACGACAAATTACGAT CCGTCGTATTTATAGGCGAAAGC
AATAAACAAATTATTCTAATTCGGAAATCTT TATTTCGACGTGTCTACATTCACGTCCA
AATGGGGGCTTAGATGAGAAACTTCACGATCGATATCTAGATCTCGAGCTCGAGATCTA
GATATCGATAAGCTTGCATGCCTGCAGGTCCTGCTGAGCCTCGACATGTTGTCGCAAA
ATTCG CCCTGGACCC <3’
Figure 3.
Annealing positions of the three specific primers within the T-DNA region of the transfer vector
pKSI015. Single frame box: DL1; double frame box: DL2; triple frame box: DL3; Black box: left border
And the Arbitrary Degenerate Primer:
AD2:
AD2-2:
5’ > NGTCGAG/CA/TGANAA/TGAA < 3’
5’ > NGTGCAG/CA/TGTNTA/TGAA < 3’
Table 3. Cycle settings used for TAIL-PCR
Reaction
Cycle No.
Primary
1
5
1
15
Secondary
Tertiary
1
12
20
Thermal settings
95℃ (2min)
94℃ (1min), 62℃ (1min), 72℃ (2min30s)
94℃ (1min), 25℃ (3min), 72℃ (2min30s)
94℃ (30s), 68℃ (1min), 72℃ (2min30s),
94℃ (30s), 68℃ (1min), 72℃ (2min30s),
94℃ (30s), 44℃ (1min), 72℃ (2min30s),
72℃ (5min)
94℃ (30s), 64℃ (1min), 72℃ (2min30s),
94℃ (30s), 64℃ (1min), 72℃ (2min30s),
94℃ (30s), 44℃ (1min), 72℃ (2min30s),
72℃ (5min)
94℃ (30s), 44℃ (1min), 72℃ (2min30s)
6
1
72℃ (5min)
Different reaction mixture was prepared for each reaction (The same AD primer is used in
successive reactions), and 10-20ng DNA templates were added.
Primary reaction: 5μmol dNTPs, 5μmol DL1 primer, 25μmol AD2/AD2-2 primer, and 0.5μ Taq
enzyme.
Secondary reaction: 5μmol dNTPs, 5μmol DL2 primer, 25μmol AD2/AD2-2 primer, and 0.5μ Taq
enzyme.
Tertiary reaction: 5μmol dNTPs, 5μmol DL3 primer, 25μmol AD2/AD2-2 primer, and 0.5μ Taq
enzyme.
PCR procedure (table 3):
The products of the primary reaction were diluted 50 folds to provide the template for the
secondary reaction, while the secondary products were diluted 10 fold for the tertiary reaction.
Direct Sequencing of TAIL-PCR Products
The products showing bright fluorescence can be deposited and sequenced directly. The sequenced
fragments contained junctions of T-DNA with other sequences, confirming their origin as flanking
sequences. Other specific products are sequenced through the following procedures.
Purification of DNA
1. 3 identic amplification product were poured to one 1.5mL Eppendorf tube, added 200μL
equilibrated phenol and mixed. Another 200μL chloroform (with 1/25 volume isoamyl
alcohol) was added, mixed and centrifuged at 12,000rpm for 5min. The aqueous phase was
transferred to a new tube, added 300μL chloroform, mixed, and then centrifuged at
10,000rpm for 4min. The supernatant was transferred to a fresh tube and added 1/10 volume
NaAc (3mol/L) and 2.5 volume 100% ethanol. The mixture was stored at –20℃ for more
than 30min.
2. DNA was recovered by centrifugation at 12,000rpm for 15min. The DNA pellet was washed
with 70% ethanol and then 100% ethanol, followed by centrifugation at 15,000rpm for 2min.
Desiccate the pellet in vacuum.
Recovery of DNA from Low-melting temperature Agarose Gel
1. A gel was prepared containing 1% concentration of Low-melting-temperature Agarose in 1×
TAE buffer and cooled at 4℃ to ensure complete setting.
2. Sample DNA was dissolved in 20μL ddH2O, mixed with 5μL loading buffer (10×), and
loaded into the slots of gel. Electrophoresis was taken at 75-80V for about 3h.
3. Photos were taken using a nucleo vision. A slice of agarose gel was cut out for each band of
interest and transferred to a clean Eppendorf tube. TE buffer were added to the agarose gel to
a total volume of 500μL. Then the gel was melted in 65℃ water bath for 5 minute.
4. The solution was cooled to room temperature, and then added equal volume of equilibrated
phenol and vortexed. The aqueous phase was recovered by centrifugation at 12,000rpm for
10min, transferred, and extracted once with phenol : chloroform. Then 1/10 volume NaAc
and equal volume ethanol was added to the supernatant. Store the mixture at -20℃ over
7
5.
6.
night.
DNA pellets were recovered by centrifugation at 12,000rpm for 15min, and then washed with
70% ethanol and 100% ethanol, succeeded by centrifugation at 15,000 for 2min. The DNA
pellets were desiccated and dissolved in 20μL ddH2O.
DNA concentrations were determined by a semi-quantitative analysis strategy. 1/5 volume
(0.2μM) EB was added to sample DNA and standard DNA of different concentration
respectively. The concentrations were determined by comparison of the fluorescence.
DNA sequencing
Recovered DNA was sequenced by Ms Zhang Li using dideoxy-mediated sequencing strategy.
Acquired sequences were analyzed by BLAST search to determine the insertion position in
Arabidopsis Genome. Sometimes homologous genes from different plant species have been
investigated and useful information of the gene function is available. Then our research of the
specific gene are proceeded with these resources.
Results
Using the strategy and concrete methods discussed before, a system for tagging, segregating and
analyzing the disrupted genes was established. The segregation and analysis of 16 T-DNA
insertions were finished in my research.
1. Results of PCR screening for transformants (figure 4.)
Table 4. Statistics of T-DNA transformation
DNA sample
LY
98
BAR(+)
Transformation
rate
80
82％
M
.
BAR
(700bp)
Cop1
Figure 4. PCR screening for bar gene and cop1 gene. The two bands of 300bps and 700bps are a sequence of cop1
gene and bar gene respectively. M: DNA Marker λDNA/EcoRⅠ+ Hind Ⅲ
2. Results of TAIL-PCR (figure 5)
Table 5. Statistics of TAIL-PCR results.
LY
BAR (+)
TAIL (+)
T/B
11
5
45％
8
M Ⅱ Ⅲ
Ⅱ Ⅲ Ⅱ Ⅲ Ⅱ Ⅲ Ⅱ
Ⅲ Ⅱ Ⅲ
.
Figure 5. Agarose gel analysis of TAIL-PCR products amplified from T-DNA insertion lines. The size difference
of 60bp between each II and III bands indicated the targeted sequences. M: DNA Marker λDNA/EcoRⅠ+-Hind
Ⅲ; Ⅱ secondary reaction; Ⅲ tertiary reaction. The arbitrary primer used is AD2。
3. Recovery of DNA from Low-melting temperature Agarose Gel (figure 6).
Table 6. Statistics of successfully sequenced transformants.
LY
M
突变体数目
已得序列的突变体数目
测序成功率
41
20
49％
1
2
3
4
5
.
Figure 6. Recovery electrophoresis of T-DNA flanking sequences. Multiple bands of each sample indicate
different annealing position of AD primer. M: DNA MarkerλDNA/EcoRⅠ+-Hind Ⅲ
4. Sequences were analyzed using BLAST search provided at National Center for Biotechnology
Informatics - http://www.ncbi.nlm.nih.gov.
9
Table 7. BLAST search of sequences
Ly040102-3
II
GenBank
ID
AC007019
Ly040109-3
II
AC004411
Sample No.
Chr
T16B14
Insertion
position
23573
F19D11
3160
product = "putative inorganic
pyrophosphatase"
Clone ID
Protein & note
Ly040110
Ⅳ
ATT22B4
T22B4
91375
strong similarity to disease
resistance response protein
206-d - Pisum sativum,
PID:g508844
Ly040402-1
Ly040402-2
Ⅴ
ATF1N13
F1N13
60468
similarity to UVB-resistance
protein UVR8-rabidopsis
thaliana
Ly040205-3
I
AC011000
F16P17
71531
Ly040206
I
AC025417
T12C24
92934
Ly040207-2
Ⅲ
ATT18N14
T18N14
73872
Ly040208-1
I
AC023279
F12K21
46926
Ly040209-2
Ⅲ
AP000603
MRP15
49176
Ly040306
Ⅴ
AB025606
F6N7
73523
Ly040307-1
Ⅲ
AB028609
K7P8
3246
Ly040307-3
Ⅲ
AB028609
K7P8
3246
Ly040308-1
Ly040308-2
Ⅲ
ATT5P19
T5P19
12707
Ly040309-3
Ly040310-2
Ly040403
Ⅱ
Ⅳ
Ⅲ
AC004411
ATT4F9
AC018907
F19D11
T4F9
F28L1
45606
52582
21359
Ly040502-2
I
AC007259
T28P6
82022
Contains weak similarity to
5-epimerase from
Saccharopolyspora erythraea
similar to cytochrome P450
emb|CAA16713.1
similarity to
brassinosteroid-insensitiv
e protein
BRI1 - Arabidopsis thaliana,
PIR:T09356
unknown protein; similar to
ESTs gb|AW560705.1, and
gb|AW017741.1
product=”lysyl-tRNA
synthetase”
similar to unknown protein
contains similarity to
spliceosomal protein
contains similarity to
spliceosomal protein
similarity to leucine-rich
receptor-like protein
kinase - Malus domestica
hypothetical protein
putative protein
hypothetical protein
Hypothetical protein;
N-terminal end of protein is
similar to katanin p80
Table 8. Different types of insertion location
Type
Intron
Exon
Non-coding region
Promotor
Repeat_region
Number
2
6
5
5
1
Discussion
The construction of a mutant database large enough is the prerequisite for the research of gene
function using reverse genetics strategy. To obtain such a mutant collection, we carried out wide
reading and a long time’s groping, and finally established a molecular genetics analysis system.
10
Through one-year’s research work, the mutant collection was enlarged and the methods for gene
isolation and molecular genetic analysis of putative tagged lines were optimized. As discussed
previously, these methods have advantages over other methods with respect to simplicity,
specificity, efficiency, speed and sensitivity.
T-DNA nutagenesis has prominent advantages over traditional mutagenesis systems: low copy
number and random insertion. An average 1.5 copies insertions in a transformant is especially
suited for our research for individual genes, facilitating genetic analysis of the T2 generations and
the confirmation of the phenotype for specific gene mutation. The new floral dip method for
transformation of Arabidopsis thaliana make the T-DNA saturation of Arabidopsis genome
practical. This strategy requires no expensive apparatus, and no time-consuming regeneration
procedures. Nevertheless, it provided satisfactory result: a high transformation rate of 0.5-3% of
all progeny seed.
The task of transformation and screening for mutant plants is easier compared to the succeeded
work of isolation and analysis of insert junctions. Thermal asymmetric interlaced (TAIL-) PCR
is an efficient technique for amplifying genomic sequences flanking T-DNA insertions from
transgenic lines. Highly specific amplification is achieved without resort to complex
manipulations before or after PCR. Different from specific PCR, this technique require special
conditions to obtain desired results. The two series of primers are designed deliberately. The long
specific primers have nested locations in T-DNA to facilitate gel screening for products. In
addition, the loci of these primers should be stable through the insertion process. The DL primers
we chose are located near the left border. Through static work from large scale mutants, we
discovered this segment is rarely lost in the process of T-DNA insertion. The AD primers were
designed with Tms of 44-46℃, require to be at least 10℃ lower than that of the specific primers.
(In our PCR procedure, the specific primers have Tms of 57-63℃, to obtain a higher specificity.)
The specific products from TAIL-PCR are usually 300-700bp, and sometimes longer products of
more than 1kb are obtained. Excluding the length of the T-DNA part, a well-sequenced segment of
more than 100bp is enough for sequence analysis.
Despite so many advantages of this PCR strategy, we discovered that it is not very stable for
the Arabidopsis system. The same pair of primer and a fixed reaction program may function quite
differently for different mutant DNA. While the isolation of the flanking sequence is the key
procedure for molecular analysis of mutant DNA, we have to adapt the primers, thermal and cycle
settings used in PCR. One AD primer may combine well with a specific region in genomic DNA
through annealing, but may hardly combine with the wanted flanking region. A single AD primer
usually only works well for less than half of the mutants DNA. So we use a second AD primer for
the PCR screening of the others, and then we change for a third one. Finally, a high rate of specific
amplification is obtained among all the mutants DNA. Thermal settings are also adjusted referring
to the results of gel analysis. Annealing temperature are raised to reduce nonspecific bands and
eliminate primer dimers, while decreased in cases that specific products are little. Due to such
restriction, we have to preserve the products of the primary reaction in case of mistakes made in
the following reactions.
At first, we use low-melting-temperature Agarose gels to recover DNA from PCR products.
This method provide pure DNA sample for efficient sequencing, however, it is laborious and
time-consumming. As the Arabidopsis genome sequencing is already completed, a sequenced
DNA fragment of more than 100bp is enough for analyzing. So well amplified DNA segments are
11
selected for direct sequencing. After several amplification reactions, nearly all the products have
the concentration high enough for direct sequencing. Thus we established a series of efficient
methods for isolation and analysis of insert junctions.
Some of the mutated genes obtained now have shown their value in further research. Mutant
Ly040110 shows strong similarity to disease resistance response protein 206-d - Pisum sativum.
It suggests the same resistance in Arabidopsis. In the succeeded research, resistance will be tested
on the F2 progeny of the mutant and the cosegregation phenomena will be tested.
Future Prospects
After the modification made in the research technique, we greatly improved our rate of progress.
In the last three months we isolated hundreds of new transformants. However, to achieve a higher
efficiency, the gel analysis and recovery strategy have to be farther optimized.
When thousands of isolated transformants are available, we will choose the genes with
important properties for further research. Using the references available we will study the
expected phenotype for gene knock-out, through anatomic, physiological and biochemical analysis.
Homology analysis of gene and protein product can provide useful clues for our research. Once
such a phenotype has been observed, several steps must be taken to prove that the phenotypic
characteristic is indeed controlled by the gene of interest. The first step is to follow the
co-segregation of the T-DNA with the corresponding phenotypes. Considering the benefit of the
probability of finding more than one T-DNA insert in a given gene family from generating a large
collection of T-DNA-transformed lines, we can obtain double mutant through hybridization and
clarify the interactional relation of the two genes. Southern Blotting and Northern Blotting
analysis was carried out to clarify the insertion copy number and the specific expression of the
interest gene in spatial-temporal pattern.
Acknowledgements
The author thanks Professor Gu Hongya and Professor Qu Lijia for their enlightment and
directions on research theory and idea throughout the whole year’s research work. She also thanks
Dr. Qin Genji, Shen Yunping, and Dong Yiyu for their instructions about the techniques and other
difficulties met in the research. Thanks also to Dr. Kang Dingming and his research group for
growing plants, producing the T-DNA-tagged lines and extracting DNA.
Thanks to the “Tai Zhao Fundation” for providing this opportunity for me and funding for the
research.
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作者简介
吕海慧，生物化学及分子生物学系 98 级本科生。1998 年 8 月入学，以山东
省高考理科 900 分成绩进入生物化学系，并被选入理科实验班。本科三年成绩名
列前茅，年年获得理科实验班雏鹰奖一等奖。在生物化学系成绩排名第四，一年
级获得住友商事奖学金（1600 元），二年级获得汇凯奖学金（2000 元），三年级
获得住友商事奖学金，并被评为三好学生。
2000 年 11 月获得泰兆基金资助，进入“北京大学-耶鲁大学”植物分子遗传
学合作实验室开始实验工作。资助的实验课题为：拟南芥功能基因组研究。在导
师顾红雅教授和瞿礼嘉教授的指导下，经过一年的努力，在实验理论和技术方面
都有了很大的提高，并在实验的上游工作——拟南芥突变体库的建立中取得较好
的实验结果。在接下来的一年中，将继续深入上游的工作并逐步开展下游工作
——基因功能的研究。
感悟与寄语
要做好一个实验课题，首先需要有深厚的基础知识，要对本课题的相关知识
有透彻的了解。其次要有严谨的态度，科学工作不能有半点松懈，每一个步骤都
要专心致志，不能放过任何小小的例外。第三要有周密的计划。实验中既要有统
筹全局的计划，又要对每一点时间合理安排，最大限度的利用时间。第四是遇到
失败不能气馁，要坚持不懈，同时要认真分析错误原因，虚心请教他人，尽快解
决存在的问题。第五要勇于探索，实验流程不是一成不变的。不同的实验目的、
材料和条件决定了不同的实验方法。特别是我们的拟南芥突变 体库建立和
TAIL-PCR 的工作，没有太多的经验可以参考，许多东西是一点点摸索出来的，
仍有待完善。在实验中我们就尝试过改变一些条件如反应温度、时间以及原料用
量，使实验结果更加理想。最后是要关注科学新进展，借鉴他人的经验，少走弯
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路，提高实验效率多。
这一年的实验使我受益匪浅，最重要的就是培养了独立实验的能力。对实验
中遇到的问题，通过查阅文献，独立思考和大胆的尝试来解决。这样的经历，对
我将来的学习和工作都会有很大的帮助。
感谢学校和泰兆基金会为我提供这样一个宝贵的机会，希望将来有更多的优
秀学生能得到这样的锻炼机会。
指导教师简介：顾红雅，女，1960 年 5 月生于江苏省扬州市。现任北京大学生
命科学学院教授，副院长，生物技术系副系主任。1982 年毕业于南京大学生物
系，1987 年获美国华盛顿大学生物系博士学位。主要从事植物分子生物学及植
物系统与进化的研究，承担并主持了国际自然科学基金青年基金、面上基金、重
点基金的课题，国家 863 项目的课题，国家转基因专项课题，国家 973 重大基础
研究项目等多项课题。
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