Cloning of chalcone synthase genes from potato and antisense inhibition of anthocyanin biosynthesis = 감자의 chalcone synthase 유전자의 클로닝과 안토시아닌 색소의antisense저해

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The chalcone synthase(CHS), the key enzyme of the anthocyanin synthesis pathway, was studied to establish the system of gene screening as a model system. A potato cDNA was synthesized from mRNA extracted from in vitro microtuber in early tuberization stage. Degenerate oligonucleotides encoding conserved sequences of CHS gene in other plant were used for PCR and two independent partial length CHS cDNA clones were isolated. Finally, we have isolated a chalcone synthase gene 2(ST-CHS2) from potato by rapid amplification of cDNA ends PCR. CHS2 cDNA had high homology to tomato LET-CHS2(98%), petunia PHCHSJ(94%), potato ST-CHS1B(92%), petunia PHCHSA (92%), and LET-CHS1(90%) at the overall 389-amino acid level. Genomic hybridization analysis indicated that CHS genes of potato comprise a family of at least six individual members. To inhibit the synthesis of anthocyanin, we constructed vectors of pBI/CH1-Asand pBI/CH2-S containing antisense and sense cDNA encoding potato CHS under the control of CaMV 35S promoter, and a nos-nptII marker gene. Potato leaf disc cultured in vitro were transformed by the Agrobacterium strainLBA4404 containing pBI/CH1-AS and pBI/CH2-S from three varieties of potatoes (cv.Red Pontiac, Desiree, Superior). At stage of selection for transformant, 50 mg/L kanamycin medium was effectively used without reducing regeneration capability of transformed potato. The polymerase chain reaction (PCR), amplifying the 700bp neomycin phospho transferase (NPTII) sequences with NPTII primer, proved to be a routine analytical tool for successfully analyzing the potato transformants for the presence of foreign gene. Red Pontiac exhibit a characteristic pattern of red pigmentation on the tuber skin. The extraction and quantitative analysis of anthocyanin from transgenic microtubers showed that red skin color of microtuber was decreased. The relative absorbance at 513nm decreased in the all transgenic lines by 55%-25%. Transgenic line No.8(CS1RP8) is the most decre...
Byun, Si-MyungJoung, Hyouk변시명정혁
한국과학기술원 : 생물과학과,
Issue Date
108900/325007 / 000835630

학위논문(박사) - 한국과학기술원 : 생물과학과, 1996.2, [ xiii, 102 p. ]


Antisense inhibition; Potato; 감자; Chalcone synthase; Chalcone synthase; 색소저해

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