Cloning and Function Identification of Anthocyanidin Synthase Gene TgANS in Tulipa gesneriana

doi:10.16420/j.issn.0513-353x.2021-0638

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (9): 1935-1944.doi: 10.16420/j.issn.0513-353x.2021-0638

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Cloning and Function Identification of Anthocyanidin Synthase Gene TgANS in Tulipa gesneriana

HUANG Ling, HU Xianmei, LIANG Zehui, WANG Yanping, CHAN Zhulong^*(), XIANG Lin^*()

Key Laboratory of Horticultural Plant Biology,Ministry of Education,College of Horticulture and Forestry Sciences,Huazhong Agricultural University,Wuhan 430070,China

Received:2021-08-16 Revised:2021-10-13 Online:2022-09-25 Published:2022-10-08
Contact: CHAN Zhulong,XIANG Lin E-mail:zlchan@mail.hzau.edu.cn;xianglin@mail.hzau.edu.cn

Abstract

Abstract:

In this study,the contents of anthocyanins in the tepals were determined from flower of red Tulipa gesneriana‘Strong Fire’at four developmental stages by high performance liquid chromatography（HPLC）. The results showed that the content of anthocyanins continued to increase along with the development of flowers,and the highest content was up to 3 002.75 μg · g^-1 FW in fully flowering stage flower. The sequence of TgANS was cloned,and the GenBank accession number was MZ442383. TgANS has 1 071 bp ORF region,encoding 356 amino acids. The expression level of TgANS differed significantly at different flower stages,and showed a trend of rising during the flower development. The expression pattern was consistent with the accumulation of anthocyanin in the four stages of flower development. Ectopic expression of TgANS in transgenic tobacco plants showed that the flowers of transgenic plants obviously turned red and TgANS was expressed at high levels in transgenic plants. The anthocyanin content in the tepals of the transgenic plants was slightly higher than that in empty vector control. Further analysis of the flower color related genes indicated that the expression of genes NtF3H,NtF3’H and NtDFR were increased significantly,and NtCHS,NtCHI were decreased in transgenic tobacco plants. These results indicated that tulip TgANS gene is involved in the biosynthesis of anthocyanin.

Key words: Tulipa gesneriana, anthocyanidin synthase（ANS）, anthocyanin, gene function

CLC Number:

S682.263

HUANG Ling, HU Xianmei, LIANG Zehui, WANG Yanping, CHAN Zhulong, XIANG Lin. Cloning and Function Identification of Anthocyanidin Synthase Gene TgANS in Tulipa gesneriana[J]. Acta Horticulturae Sinica, 2022, 49(9): 1935-1944.

Figures/Tables 6

Table 1 The primers used for qRT-PCR

引物 Primer	正向引物序列（5′-3′） Forward sequence	反向引物序列（5′-3′） Reverse sequence
TgANS	CGCTCGAGATCCTCAGCAAT	GTCCGGGGAGGAAACTTAGC
TgUBQ10	GTCGCACGTTGGCTGATTAC	GGGCTTAAAGACCACCACGA
NtCHS	AAGCAAGAGAAACTAAAGGCTACAAG	AAATCCAAAAAGCACACCCCAT
NtCHI	CGGGTGCCTCCATTCTTTTTACT	CCTGACACTCTTTCGGCGATACTAC
NtF3H	CCAGACAAACCAGATGGATGGATAG	CAAGGGTAAGGTCGGGCTGTG
NtF3’H	TGGCTATTTCATTCCAAAAGGCTCA	CTTCAAAGTCATTTCCTCGCACATC
NtDFR	GCAGTTGCTTCCCTTTTCTACC	TTCCCCATTGGTTGACTTTCC
NtFLS	CTTGAAGGGAAAAGGGGTTGG	CGCAACTTCTCGCAGCCTCT
NtEF1α	TGGTTGTGACTTTTGGTCCCA	ACAAACCCACGCTTGAGATCC

Fig. 1 Analysis of anthocyanins content and relative expression levels of TgANS in tulip flower development The values indicates averages of three independent replicates and SEs. P < 0.05,Tukey’s method.

Fig. 2 Alignment of the deduced amino acid sequences of ANS from other plant species TgANS sequence was aligned with ANS from Tulipa fosteriana（TfANS,AGJ50591）;Lilium cernuum（LcANS,AGC92011）;L. hybrida（LhANS,ALA55544）;L. speciosum（LsANS,BAS69334）;Iris sanguinea（IsANS,QCI56004）;Phoenix dactylifera（PdANS,XP_008783582）. The black underline shows the conserved domain 2OG-Fe II_Oxy. * and # indicate conserved residues which are required for ferrous-iron coordination,and 2-oxoglutarat binding,respectively.

Fig. 3 Phylogenetic analysis of ANS The numbers at node represent the bootstrap values,with 1 000 replicates.

Fig. 4 Analysis of flower color phenotype of transgenic tobacco EV：The controls that only transformed empty vetor. *** P < 0.001,student’s t test.

Fig. 5 qRT-PCR analysis of flavonoid-related genes expression in TgANS transgenic tobacco

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Cloning and Function Identification of Anthocyanidin Synthase Gene TgANS in Tulipa gesneriana

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