J Integr Plant Biol. ›› 2004, Vol. 46 ›› Issue (5): 588-594.

• Research Articles • Previous Articles     Next Articles

Cloning and Expression of Two Chalcone Synthase and a Flavonoid 3''5''-Hydroxylase 3''-end cDNAs from Developing Seeds of Blue-grained Wheat Involved in Anthocyanin Biosynthetic Pathway

YANG Guo-Hua, LI Bin, GAO Jian-Wei, LIU Jian-Zhong, ZHAO Xue-Qiang, ZHENG Qi, TONG Yi-Ping, LI Zhen-Sheng   


Using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) strategies, two chalcone synthase (CHS) cDNAs were cloned from developing seeds of blue-grained wheat, both of the deduced peptides contain 394 amino acids, and share 98.9% of amino acid sequence identity and the nucleotide sequences have the identity of 96%, and one flavonoid 3''5''-hydroxylase (F3 ''5 ''H) 3''-end cDNA was isolated. Four CHS genomic DNAs were cloned from Thinopyrum ponticum (ThpCHS.tg), blue-grained wheat (TaCHS.bg), white-grained offspring of light blue-grained wheat (TaCHS.wg) and Chinese Spring (2n=42)(TaCHS.csg), respectively. Although these four genomic DNAs were isolated from different materials, they are very highly homologous and each has one intron. The difference of the four CHS genomic DNAs mainly exists in intron. Through DNA alignment we found that one CHS cDNA (TaCHS.t1) came from one of the parents, Th. ponticum, the other one (TaCHS.w1) had the identity of 100% with white-grained parent. This indicated that CHS genes from two parents expressed at the same developing stage in blue-grained wheat. Southern blotting analysis showed that they have at least four copies in wheat, the copy numbers in different color grains are not significantly different, but they are different from that of Th. ponticum. CHS in blue-grained wheat belongs to a CHS multifamily. Reverse Northern analysis indicated that the CHS expressed strongly in the developing blue-grained seeds at early stage (15 d after flowering, DAF), but F3 ''5 ''H and dihydroflavonol 4-reductase (DFR) transcripts accumulated less than that of CHS at early stage. However, at the later developing stage (21 DAF), F3 ''5''H and DFR transcripts accumulated more than that of CHS, the transcripts of CHS could hardly be detected. The expression order of the three genes is the same as the order of the biosynthetic steps in anthocyanin biosynthesis. At the same time, CHS genes cloned from seeds have not been detected in leaves of blue-grained wheat, but F3 ''5 ''H and DFR expressed strongly in leaves. This showed that the expression of CHS genes cloned by us had tissue specificity. RT-PCR indicated that the transcripts of F3 ''5''H accumulated a lot in the developing seeds of blue- and white-grained wheats at 21 DAF, but the transcripts of CHS and DFR accumulated in the blue-grained wheat more than those of white-grained wheat and Chinese Spring at the same developing stage. Therefore, we proposed that anthocyanin biosynthetic pathway existed in blue-grained wheat and the expression of the secondary structure genes in anthocyanin biosynthetic pathway was coordinately regulated by regulatory gene(s) during the period of blue pigment formation.

Key words: blue-grained wheat, anthocyanin biosynthetic pathway, chalcone synthase (CHS), flavonoid 3'5'-hydroxylase (F3'5'H), reverse Northern analysis

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