The Analysis and Identify of Hybridizing Pea DNA Segment with
Corn Chromosome and its Protein Expression Result
Peikun Zhu1 *, Xinjie Xia2, Yuan Gao3, Yong Xie4, Miaokang Lu5, Yang Xiang6 ,Zhongchen Guo7
(1 Bioroad Biotechnology CO., Ltd. at Shenzhen, 518172; 2 Institute of Science and Technology ,
Sichuan Agricultural University, Chengdu 611134; 3 China-UK HUST-Rees Crop Genetic
Engineering and Genomic Joint Laboratory, Huazhong University of Science and Technology,
Wuhan 430074; 4 Department of Biology, The Hong Kong University of Science and Technology,
Clear Water Bay, Hong Kong; 5 Life Science Department of Fudan University, Shanghai, 200433;
6 Changsha University of Science and Technology, Changsha 410076; 7 Institute of Botany,
Chinese Academy of Sciences, Beijing 100093; * Corresponding author, E-mail:
pkzhu2003@163.net)
Abstract: With the analysis and identification by chromosome fluorescence in situ
hybridization technology and Western blot, it is abundantly clear that there are many
pea gene signals in the chromosome of Pea-Corn (F3, Bioroad Code 216). The
Pea-Corn is peculiar to Bioroad Biotechnology CO., Ltd. at Shenzhen, and it
expresses the protein (whose molecular weight is 22.9kD, 150.5kD) which can react
with the pea antibody specifically. These proteins are owned by pea. It also expresses
the protein (whose molecular weight is 18.8kD, 31.0kD, 46.0kD, 136.6kD), which
can react with pea antibody specifically, these proteins are neither owned by pea nor
corn. Through the analysis and identification, it indicates that after the pea DNA
segment hybridized with the receptor corn chromosome, new types of chromosome is
formed and it expresses the new types of protein, which have immune response with
the pea antibody.
Key words: Pea-Corn, FISH, Western blot
With chromosome fluorescence in situ hybridization technology to carry out the
homochromy fluorescence in situ hybridization on the chromosome of the Pea-Corn
(No.216, F2) seedling root tip cells, it definitely shows several red signals (the
homologous sequence has been blocked), which means the signal of the pea gene is
emerged on the Pea-Corn chromosome. The red fluorescence signal just shows the
pea DNA sequence that heterogeneous with corn exists in the chromosome of
Pea-Corn (F2), while even the pea DNA that homologous with corn, of which has
hybridized on the Pea-Corn(No.216, F2) chromosome, also cannot show the red
fluorescence signal. In January 2004, the lead author put forward the view of
“segment hybridization” on DNA segment [1]. With the Western blot analysis on the
No.216 Chinese Grain F3, the protein that expresses itself just as the pea-corn protein
molecular weight of 22.9kD, 26.2kD, 39.3kD, 78.1kD, 79kD, 150.5kD, at the same
time we could also see many Chinese Grain proteins which are different from the pea
protein molecular weight, while could undivided responded with the pea antibody but
not exists in the control corn, of which the molecular weight is 18.8kD, 31.0kD,
46.0kD, 136.6kD. This result not only shows the No.216 Chinese Grain F3 could
inherit the F2 heredity hybrid character, but also shows that the new kind of Chinese
Grain chromosome expresses both the pea protein and many new kinds of protein
which can undivided responded with the pea antibody.
1. Materials and Methods
1.1 Plant materials and probes.
Plant materials for cytological research include maize (M), pea (P) and the 2nd
generation of inbred No.216 Pea-Corn. Extract the total pea DNA as the probe, the
blocking probe is GB57. Probes were labeled with biotin of cutting translation
method.
1.2 Chromosome preparation.
Collect root tips of plants with strong growth in the greenhouse. Immediately
fixed in fixative (3 ethanol: 1 acetic acid), leave overnight at 4℃. Wash the root tips
with dd H2O until the tips are clean. Place the root tips at 28℃ for 2h with the mixture
of 2% pectase and 2% cellulase. After the enzymolysis, squash the root tips on slide
glass, and then dry it over the flame.
1.3 Fluorescence in situ hybridization.
Extract the total pea DNA [4] with CTAB method[4]. Three step amplification to
detect biotin-labeled probes: streptavidin-Cy3, biotinylated anti-streptavidin and
streptavidin-Cy3. Antibody should be added after each step, then place in humidified
37℃ for 1h. Wash with PBS for 3×5 min. Add 40μl of 1μg/ml DAPI containing 20%
antifade Vectashield to each slide, and counterstained the slide.
1.4 Cytological measurements and analysis.
Observe chromosomes with fluorescence microscope. Capture gray scale images
in different florescence channel, and then merge the imaes with software V++
Precision Digital Imaging System. Measure the digital images with Meta Imaging
Series 4.6 software, and integrate the final images with Adobe Photoshop v5.1.
2. Production of rabbit antibody to total pea protein
Take pea seedling (after three weeks of budding) as experimental material.
Produce total pea protein with saturated ammonium sulphate method. Take the total
pea protein as antigen. Add Freund’s adjuvant, after emulsifications inject rabbit.
3. Western blot[5][6]
2. Experimental results
2.1 The identification of 3rd generation of inbred Pea-Corn (No.216)
From analysis of the monochromatic fluorescence in situ hybridization, it is
observed there is the chromosome with pea gene signal in Pea-Corn. Shown from
(Fig.) 1(a), after blocking, the original sequence on control corn chromosome that
homologous with pea DNA cannot reveal special red signal, but blue signals. Red
signals are clear in (Fig.) 1(b) because in the Pea-Corn chromosome there is
non-blocking DNA sequence from pea that heterogeneous with corn, so these
heterogeneous sequence must combine with the heterogeneous sequence of the pea
DNA probe. By comparison with the two picture, it is found that pea (donor)
chromatin has transferred into corn cell (receptor) and has hybridized with corn
chromatin.
2.2 The result and analysis on Western blot
(Fig.) 2 shows the Western blot of Pea-Corn (No.216-1-4, No.216-1-66 and
No.216-1-73). In the molecular weight of 19.0kD, 24.0kD, 53.4kD and 82.7kD, the
Control corn has no protein band, while at the same place the pea and Pea-Corn (No.
216) hold clear protein band. This phenomenon indicates the Pea-Corn (No. 216)
express the proteins whose molecular weight are 19.0kD, 24.0kD, 53.4kD, 82.7kD
and have immune response with the pea antibody.
In addition, from the Western blot of the No.216-1-4, No.216-1-61, No.216-1-66
and No.216-1-73, it indicates that the Pea-Corn expresses some protein bands
respectively which are absent in the corn and their molecular weight are different
from the ones of pea. Certainly, the express of these protein bands is attributed to their
immune response with pea antibody. This result also indicates that after segment
hybridization, many genes in the new types of chromosome of Pea-Corn have
expressed many new types of proteins which have immune response with pea
antibody. Furthermore, the No.216-1-4, No.216-1-61, No.216-1-66 and No.216-1-73
Pea-Corn have expressed many proteins with the same molecular weight. Also they
express some proteins with different molecular weight. The difference between the
protein expressions of Pea-Corn (No.216, F3) indicates the Pea-Corn has not yet
completely stabilized.
3. Discussion
The hybrid plant is very short with the height of 21cm, and its ears are so small
with the length of 3.6cm. The plump seeds are light yellow with ball shape like pea
(Fig. 3(a). And one seed has grown up to a plant with the height of 159.4 centimeters
and bear bigger ears. The seeds have distinctive shapes, such as like pea, flat,
umbilicate，and some are intermediate shapes [Fig. 3(b)] mentioned above. Select
different types of seeds to plant, the height and leave shape of Pea-Corn show distinct
differences [Fig. 3(c)].
From the Western blot, it is found the protein band map of Pea-Corn is more
similar to pea rather than corn. The protein band with molecular weight of 22.9kD and
150.5kD Pea-Corn express is peculiar to pea, while the protein band with molecular
weight of 51.7kD is just possessed by corn. In addition, the protein band with
molecular weight of 18.8kD, 31.0kD, 46.0kD, 136.6kD exists neither in corn nor in
pea, it is only owned by Pea-Corn (No.216). These proteins only owned by Pea-Corn
are classified into two types, one is the protein with molecular weight of 46.0kD
which is close to the corn protein with molecular weight of 51.7kD, and another one
is the proteins with molecular weight of 31.1kD, 136.6kD close to the pea protein
with molecular weight of 32.4kD, 119.0kD. It shows there are three different types of
proteins with different molecular weight, one is identical with the molecular weight of
pea, and the other is the same as the molecular weight of corn. The majority proteins
are the one which have immune response with the pea antibody and are absent in pea
or in corn. From the comparison between chromosomes of Pea-Corn and corn, it is
found the chromosomes and DNA segments of Pea-Corn have great change. These
great changes are attributed to the new types of chromosomes which express new
kinds of proteins.
From the homochromy chromosome fluorescence in situ hybridization [Fig. 1(b)],
it is clear that the pea DNA segment has hybridized with corn chromosome and form
new types of chromosomes. The lead author put forward his view of “segment
hybridization” according to the chromosome karyotype change of Pea-Corn hybrid
plant (No.031016-1). From the analysis of chromosome in situ hybridization of
Pea-Corn, it is found that the exogenous pea DNA segment has indeed hybridized
with corn chromosome. The “segment hybridization” is the recombination of
exogenous DNA segment (including hundreds or thousands genes) and chromosomes
of receptor cell. Because the DNA segment is huge, the structure of new types of
chromosomes is instable. If the recombination is at different arms but the same parts,
the structure of new types of chromosomes will be inclined to stability [Fig. 1(b)].
For this is a new rule of plant’s life phenomena, it remains to further research. The
“segment hybridization” between exogenous DNA segment and the chromosome of
the receptor cell could be a feasible approach for creating new plant species or
subspecies. According to this result, it’s easy to understand the great difference
between the proteins Pea-Corn express and the one of corn as well as the Pea-Corn
express proteins which has the same molecular weight of pea. The segment
hybridization of pea and corn not only form the new types of chromosomes (the
chromosome number of Pea-Corn is identical with the one of receptor corn 2n=2x=20,
but also express proteins with same (Mt=22.9kD, 150.5kD) or close to the molecular
weight (Mt=31.1kD, 136.6kD) of pea protein. By the experiments, it is disclosed that
“segment hybridization” is another rule of the plant’s life phenomena, also it indicates
with the discovery of “segment hybridization”, human being could reconstruct plants
with biology engineering more effectively and extensively.
Fig.1 Comparison of chromosome fluorescence in situ hybridization between Pea-Corn and corn
(Fig.) a is the chromosome fluorescence in situ hybridization of corn. (Fig.) b is
the chromosome fluorescence in situ hybridization of Pea-Corn (No.209-1-81). The
red signals stand for pea DNA segment, and the blue signals represent chromosome
(chromatin) of hybrid plant (Pea-Corn). This experiment uses pea DNA as probe, the
red signal is the DNA segment which pea transfers into corn. In (Fig.) a blue signals
stand for the chromosome of corn (the homologous sequence of pea and corn have
been blocked). From the comparison between the two figures, it is found the DNA
segment of pea has transferred into corn cell (receptor) and hybridized with corn
chromosome, and thus form chromosome (chromatin) of Pea-Corn.
Fig.2 Western Blot Hybridization electrophoresis of Pea-Corn
The Pea-Corn express protein bands with molecular weight of 22.9kD which is
peculiar in pea, and it also expresses protein band with molecular weight of 51.7kD
which is just possessed by pea but not corn. In addition, the protein bands with
molecular weight of 18.8kD, 31.0kD, 46.0kD, 136.6kD exist neither in corn nor in
pea which are only owned by Pea-Corn (No.216-1-67).
Fig.3 Ear of F1, F2 and the plant of F3 of Pea-Corn No.216
(Fig.) 3- a、b、c are the growth development of No.216 Pea-Corn (F1, F2 and F3)
in the field. The plump seeds (a) show light yellow, and its shapes are spherical like
pea, different from the dent corn which used in this experiment. The seeds [Fig.
b]show different shapes, some are like pea, some are flat, some are dent and some are
intermediate shapes. Select some different shapes of seeds (b) to plant. (Fig.) 3-c is
the different shapes of seeds in b which are grown in the field. The height and leave of
Pea-Corn show distinct differences. These characters show the truth that the Pea-Corn
is crossbreed.
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