Scientific report: "Application of PCR to detect the presence of two genes in the genome of rice AtAOS has been genetically modified" pdf

Application of PCR to identify the transgene Arabidopsis thaliana allene oxide synthase 2 in transgenic rice plants Ứng dụng PCR để phát hiện sự có mặt của gen AtAOS 2 trong genome củ

Application of PCR to identify the transgene

(Arabidopsis thaliana allene oxide synthase 2) in

transgenic rice plants

Ứng dụng PCR để phát hiện sự có mặt của gen AtAOS 2

trong genome của lúa đã được chuyển gen Nguyen Thi Thuy Hanh 1 , Han Oksoo 2

1

Faculty of Agronomy, Hanoi University for Agriculture

2

College of Agriculture and Life sciences, Chonnam National University, South Korea

TÓM TẮT

Allene oxide synthase (AOS) là một enzyme quan trọng trong số các enzyme tham gia vào quá trình tổng hợp axit jasmonic thông qua con đường sinh hoá octadecanoid Ở thực vật, axit jasmonic đóng vai trò then chốt trong việc hình thành các phản ứng đáp lại các tác động của môi trường trong

và ngoài tế bào Để làm tăng hàm lượng enzyme allene oxide synthase nội sinh ở lúa, gen allene oxide synthase 2 của Arabidopsis thaliana (AtAOS 2) đã được chuyển vào genome của lúa (Oryza

sativa L.Japonica cv Nakdong) Phản ứng PCR đã được sử dụng để phát hiện sự có mặt của gen AtAOS2 trong genome của lúa đã được chuyển gen Cụ thể là phản ứng PCR đã được sử dụng để phát hiện gen tổng hợp chất kháng sinh hygromycin, promoter CaMV35S (promoter này nằm ngay phía trước gen AtAOS2 trong cấu trúc của vector pCAMBIA1201/AtAOS) và gen AtAOS 2 trong genome của lúa

Từ khoá: Allene oxide synthase, axit jasmonic, con đường sinh hoá otadecanoid, thực vật chuyển gen.

SUMMARY

Allene oxide synthase (AOS) is an important regulator that steers the octadecanoid pathway toward jasmonic acid (JA) synthesis Plant jasmonic acid (JA) plays pivotal roles regulating cellular

responses against environmental cues, including the innate immune response(s) The Arabidopsis thaliana allene oxide synthase 2 (AtAOS 2) gene was introduced into rice (Oryza sativa L.Japonica

cv.Nakdong) genome The polymeraza chain reactions (PCRs) were applied to indentify the transgene

- AtAOS 2 in transgenic rice plants A set of PCRs was carried out to detect the hygromycin resistance gene, CaMV35S promoter (the pomoter located infront of the AtAOS 2 gene in pCAMBIA1201/AtAOS binary vector) and the AtAOS 2 gene in the transgenic rice genome

Key words: Allene oxide synthase, jasmonic acid, octadecanoid pathway, transgenic plants

1 INTRODUCTION

Plants have evolved defense strategies to

protect themselves from various abiotic and biotic

stresses One of the most prominent defense

mechanisms is the octadecanoid pathway, which

leads to jasmonic acid (JA) biosynthesis JA is a

genuine plant hormone that has various

physiological functions, including growth control,

senescence promotion, responses to wounding and

drought, and defenses against insects and pathogens

(Schaller et al., 2005) Furthermore, JA signaling

interacts with signaling of other plant hormones such

as ethylene and salicylic acid to form a complex

signaling network (Devoto et al., 2005) JA is

synthesized through the octadecanoid pathway (Figure 1) In the octadecanoid pathway, 13 (S) - HPOT is the pivotal intermediate, from which several metabolic pathways branch out For the JA synthesis, 13 (S) - HPOT is converted to an unstable allene oxide, 12,13 epoxy 9Z, 11E, 15Z -octadecatrienoic acid (12,13 - EOT), by an allene oxide synthase (AOS) The allene oxide is readily

converted to cis-(+)-OPDA by allene oxide cyclase

(AOC); cis-(+)-OPDA is further metabolized

through reduction and β-oxidation to JA On the

other hand, 13(S)-HPOT can also be metabolized by

other enzymes, including hydroperoxide lyase,

peroxygenase, hydroperoxide reductase, and divinyl

ether synthase A typical function of these branch

pathways is defence against insect and pathogens

(BlÐe, 2002) Therefore, AOS is an important

regulator that steers the octadecanoid pathway to JA

synthesis, thus affecting the synthesis of all

JA-related compounds (Kongrit et al., 2007)

Rice AOS (OsAOS) and Arabidopsis

thaliana AOS belong to the CYP74A subfamily

A database search indicates that, besides the

characterized OsAOS1 (Agrawal et al., 2004),

there are at least four additional OsAOS

(OsAOS2-5) genes in the rice genome (Figure 2) Most of the rice AOS genes are located on chromosome 3, except for the OsAOS3 gene, that

is located on chromosome 2 The OsAOS proteins show putative localization sites such as mitochondria (MC), endoplasmic reticulum (ER), plasma membrane (PM), microbody (MB), and

cytosol (CS) (Agrawal et al., 2004) Three Arabidopsis thaliana AOS genes (AtAOS1-3)

have been identified All AtAOS genes are located

on chromosome 5 Arabidopsis thaliana AOS

proteins localize on chloroplast (CP), microbody (MB), mitochondria (MC) In order to increase the level of endogenous JA in rice, we try to over express AOS genes by transforming AtAOS2 gene

to the rice genome

Fig 1 The octadecanoid pathway leading to jasmonic acid (JA) biosynthesis in plants

AOC: allene oxide cyclase; OPR: 12-oxo-phytodienoic acid reductase; 13 (S)-HPOT: (9Z, 11E, 15Z, 13S)-13 - hydroperoxy - 9, 11, 15-octadecatrienoic acid; 12, 13 - EOT: (9Z, 11E, 15Z, 13S, 12R) - 13 -

epoxy - 9, 11, 15 - octadecatrienoic acid; OPDA: 12 - oxo- 10, 15 (Z) - octadecatrienoic acid; OPC - 8:0: 3-oxo-2 (2' (Z) -pentenyl)-cyclopentane-1-octanoic acid (Schaller, 2001)

Fig 2 Phylogenetic tree of AOS homologues and related proteins from other species

CP: chloroplast; CPS: chloroplast stroma; CS: cytosol; ER: endoplasmic reticulum; MB: microbody;

MC: mitochondria; PM: plasma membrane (Agrawal et al., 2004)

Fig 3 Structure of the pCAMBIA1201/AtAOS2 binary vector

(pCAMBIA1201 from CAMBIA,USA; pCAMBIA1201/AtAOS2 was contructed by Dr.Eunsun Kim)

In this study, a set of PCRs was carried out to

detect the hygromycin resistance gene, CaMV35S

promoter (the promoter located in front of the

AtAOS2 gene), and the AtAOS2 gene in the

transgenic rice genome The results of PCRs would

confirm the appearance of the AtAOS2 gene in the

transgenic rice genome

2 MATERIALS AND METHODS

2.1 Extraction of rice DNA

The leaves of one-month-old wild-type (Oryza

sativa L Japonica cv Nakdong) and 5 lines of T1

transgenic rice plants (T1 transgenic seeds were

obtained from Dr.Eunsun Kim) were harvested,

frozen in liquid nitrogen, and ground to fine

powder with a mortar and pestle under liquid

nitrogen Total genomic DNA from non-transgenic

and transgenic rice plants were extracted by using

the DNeasy Plant Mini Kit (QIAGEN) according to

the instructions of the manufacturer

2.2 PCR analysis

2.2.1 Design of Primers

The Primer 3 program was used to design all

primers for PCRs analysis

2.2.2 PCR analysis to detect hygromycin

resistance gene

The primer pair employed to detect the

phosphotransferase) was: foward primer

5'-AGTCAATGACCGCTGTTATGCG-3' PCR was

carried out according to the PCR Amplification

Kit by using HiPi PCR Premix (ELPis Biotech,

Korea) in a 20 µl reaction volume, with thermal

cycling parameters as follows: initial

denaturation at 940C for 5 min; following by 35

cycles of 940C for 1 min, 610C for 1 min, 720C

for 2 min; and 720C for 5 min for final extension

2.2 3 PCR analysis to detect the CaMV 35S promoter

The primer pair, forward primer 5'-

designed from the sequence of CaMV35S promoter which located infront of AtAOS2 gene

in pCAMBIA1201/AtAOS binary vector (Fig 3) PCR was carried out according to the PCR Amplification Kit by using HiPi PCR Premix (ELPis Biotech, Korea) in a 20 µl reaction volume Template DNA were initially denatured

at 940C for 5 min, followed by 35 cycles with thermal cycling parameters of 940C for 50 second,

580C for 50 seconds, 720C for 1 min.30 seconds

A final 5 min incubation at 720C was allowed for completion of primer extension

2.2.4 PCR analysis to detect the introduced gene- AtAOS2

The primer pair employed to detect the introduced genes was: forward primer 5'-ACGACCAAGGAGCTGAAGAG-3' and reverse primer 5'-CGCCGGTGGTAGACTAAATG-3' PCR was carried out according to the PCR Amplification Kit by using HiPi PCR Premix (ELPis Biotech, Korea) in a 20 µl reaction volume with thermal cycling parameters as follows: 940C for 5 min; following by 35 cycles of 940C for 40 seconds, 530C for 40 seconds, 720C for 1 min.40 seconds; and 720C for 5 min

3 RESULTS AND DISCUSSION The agarose gel banding patterns of the PCR products were shown in figure 4 For the hygromycin resistance gene, a 590 base pair amplified segment was detected in pCAMBIA1201/AtAOS plasmid and transgenic rice genome with the specific designed primers, but was not detected in non-transgenic (Fig 4a) For the CaMV35S promoter, which was located infront of the AtAOS2 gene, the amplified band size was 399 base pairs, and it was detected in the plasmid, transgenic rice genome (Fig 4b) but was not detected in the non-transgenic genome

A 599 base pair segment that was located in the

Arabidopsis thaliana cDNA region was detected

in the plasmid genome and transgenic rice

genome, but was not detected in the non-transgenic rice genome (Fig 4c)

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Fig 4 Representative PCR analysis for the presence of the hygromycin resistance gene (a);

CaMV35S promoter (b) and AtAOS2 gene (c) in transgenic rice plants

Lane 1: molecular size marker; Lane 2:

pCAMBIA1201/AtAOS plasmid (positive control);

Lane 3: untransformed plant (negative control); Lanes 4 - 8: transformed plants

The result in fig.4c shows that beside expected

band (599 bp) in transgenic rice plants, a strong

band (about 200 bp) was also detected in both non -

transgenic and transgenic rice plants This is

because there are AOS genes in the rice genome

As shown in fig 2, five different rice AOS genes

have been identified The alignment of the rice AOS genes with a primer pair, which was used to

detect the Arabidopsis thaliana AOS2 gene in the

rice genome, shows that rice AOS5 (AK066137) may have a 208 base pair segment with this primer pair (Fig 5)

Fig 5 Alignment of rice AOS5 (AK066137) with the primer pair that was used to detect

the Arabidopsis thaliana AOS2 gene in the rice genome

4 CONCLUSION

From the above results, the hygromycin

resistance gene, CaMV35S promoter (the promoter

located in front of the AtAOS2 gene), and the

AtAOS2 gene were clearly detected in the transgenic

rice genomes

These results strongly confirmed the appearance

of the transgene - Arabidopsis thaliana AOS2- in

transgenic rice plant genomes

REFERENCES

Agrawal, G.K., Tamogami, S., Han, O., Iwahashi,

H., Rakwal, R (2004) Rice octadecanoid

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Blée, E (2002) Impact of phyto-oxilipins in plant

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Devoto, A., Turner, J.G (2005) Jasmonate-regulated Arabidopsis stress signaling network,

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Kongrit, D., Jisaka, M., Iwanaga, C., Yokomichi, H., Katsube, T., Nishimura, K., Nagaya, T., Yokota, K (2007) Molecular cloning and functional expression of soybean allene oxide

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