[논문]Metabolic engineering of aliphatic glucosinolates in Chinese cabbage plants expressing Arabidopsis MAM1, CYP79F1, and CYP83A1

Zang, Yun-Xiang; Kim, Jong-Hoon; Park, Young-Doo; Kim, Doo-Hwan; Hong, Seung-Beom

doi:10.5483/bmbrep.2008.41.6.472

[국내논문] Metabolic engineering of aliphatic glucosinolates in Chinese cabbage plants expressing Arabidopsis MAM1, CYP79F1, and CYP83A1 원문보기

BMB reports, v.41 no.6, 2008년, pp.472 - 478

Zang, Yun-Xiang (Department of Molecular Biotechnology, Konkuk University) , Kim, Jong-Hoon (Department of Molecular Biotechnology, Konkuk University) , Park, Young-Doo (Department of Horticultural Biotechnology, Kyunghee University) , Kim, Doo-Hwan (Department of Molecular Biotechnology, Konkuk University) , Hong, Seung-Beom (Department of Molecular Biotechnology, Konkuk University)

Abstract ▼ AI-Helper

Three Arabidopsis cDNAs, MAM1, CYP79F1, and CYP83A1, required for aliphatic glucosinolate biosynthesis were introduced into Chinese cabbage by Agrobacterium tumefaciens-mediated transformation. The transgenic lines overexpressing MAM1 or CYP83A1 showed wild-type phenotypes. However, all the lines overexpressing CYP79F1 displayed phenotypes different from wild type with respect to the stem thickness as well as leaf width and shape. Glucosinolate contents of the transgenic plants were compared with those of wild type. In the MAM1 line M1-1, accumulation of aliphatic glucosinolates gluconapin and glucobrassicanapin significantly increased. In the CYP83A1 line A1-1, all the aliphatic glucosinolate levels were increased, and the levels of gluconapin and glucobrassicanapin were elevated by 4.5 and 2 fold, respectively. The three CYP79F1 transgenic lines exhibited dissimilar glucosinolate profiles. The F1-1 line accumulated higher levels of gluconapoleiferin, glucobrassicin, and 4-methoxy glucobrassicin. However, F1-2 and F1-3 lines demonstrated a decrease in the levels of gluconapin and glucobrassicanapin and an increased level of 4-hydroxy glucobrassicin.

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가설 설정

(A) wild-type control; (B) M1-1; (C) F1-1; (D) F1-2; (E) F1-3; (F) A1-1; (G) A representative GUS assay for control (left) and transgenic plants (T₀ of M1-1, right). (H) A representative fourth rosette leaf from control (left), F1-1 (middle), and F1-2 (right) plants. (I) Wild-type control leaf with thorn (left) and F1-1 leaf without thorn (right).

제안 방법

PCR analysis was carried out with genomic DNA to verify the presence of transgenes using Ex Taq DNA polymerase (Takara, Japan) and a GUS gene primer set: 5'-GTC ACT CAT TAC GGC AAA GTG TGG GTC AAT-3' (forward) and 5'-GTT CGT TGG CAA TAC TCC ACA TCA CCA CGC-3' (reverse).
RT-PCR was performed in a total volume of 20 μl containing 0.5 μl of each of the above primers (100 μM), 1 μl of total RNA, 8 μl of DEPC water, and 10 μl of 2X ReddyMix RT-PCR Master Mix with re-verse transcriptase (ABgene, Surrey, UK), using primer sets: 5'-CTC CTA CGT ACT CCA TAG TG A TGG CTT CAT-3' (forward) and 5'-ATA CAA GAG ATA CAA CGT GT T TAC ACA TTC-3' (reverse) for MAM1; 5'- ACT ACA CGC ATA CAT ACA TC A TGA TGA GCT-3' (forward) and 5'- ACC ATC TTG TTT TCT TCT TC T TAA GGA CGG-3' (reverse) for CYP79F1; 5'-AAA AGT AGT AAC AAC TAA GAA TGG AAG ATA-3' (forward) and 5'-ATT TAT TTG ATA ATT AGC TAA TAC TTG-3' (reverse) for CYP83A1.
The reaction was initiated with first cDNA strand synthesis at 47℃ for 30 min. PCR was then performed at 94℃ for 2 min followed by 35 cycles of denaturation (94℃; 30 sec), annealing (58, 60, and 49℃ for MAM1, CYP79F1 and CYP83A1, respectively; 30 sec) and extension (7℃; 2 min) and was completed with a final extension of 5 min at 72℃. The amplification products were analyzed by 1% agarose gel electrophoresis.

이론/모형

These amplified sequences were then inserted into the pC11 multiple cloning site in the sense direction, yielding the individual constructs named pCM1, pCF1, pCA1, respectively. All of the constructs were transformed into the Agrobacterium tumefaciens strain GV3101 using the freeze-thaw method (31). The transformants that grew on kanamycin (50 mg/L) LB plates were analyzed for the presence of the plasmids by restriction digest analysis
The assay was conducted according to the histochemical staining method (33). Leaf fragments were dipped into the GUS assay mix containing 50 mM NaH2PO4, 50 mM Na2HPO4, 0.

성능/효과

Hence, it has been suggested that the CYP79F1 gene may modulate cytokinin level at the site of bud initiation and the changes in cytokinin levels at these sites are responsible for the altered shoot branching pattern (15). Taken together, these results indicate that glucosinolate biosynthesis may be intricately linked with hormone homeostasis, and the modulation of cytokinin levels by the CYP79F1 gene may differ between the Arabidopsis and Chinese cabbage.
In conclusion, ectopic overexpression of the Arabidopsis cDNAs, MAM1, CYP79F1, and CYP83A1 in Chinese cabbage changes the endogenous glucosinolates content. In the Chinese cabbage plant, alkenyl glucosinolates are the most abundant form of aliphatic glucosinolate.

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