Cloning and Function Analysis of Chalcone Isomerase Gene RdCHI1 in Rhododendron delavayi

doi:10.16420/j.issn.0513-353x.2021-0988

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (11): 2407-2418.doi: 10.16420/j.issn.0513-353x.2021-0988

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Cloning and Function Analysis of Chalcone Isomerase Gene RdCHI1 in Rhododendron delavayi

SUN Wei¹, SUN Shiyu¹, CHEN Yiran¹, WANG Yuhan¹, ZHANG Yan¹, JU Zhigang²^,^**(), YI Yin¹^,^**()

¹Key Laboratory of Plant Physiology and Development Regulation，Guizhou province School of Life Sciences，Guizhou Normal University，Guiyang 550025，China
²Pharmacy College，Guizhou University of Traditional Chinese Medicine，Guiyang 550025，China

Received:2022-08-05 Revised:2022-09-26 Online:2022-11-25 Published:2022-11-25
Contact: JU Zhigang,YI Yin E-mail:juzhigangz@163.com;yiyin@gznu.edu.cn

Abstract

Abstract:

In order to investigate the function of Chalcone Isomerase（CHI）from Rhododendron delavayi，the flower of R.delavayi was used as study material to clone RdCHI1 gene. Multiple sequence alignment，phylogenetic analysis，qRT-PCR，genetic transformation of Arabidopsis thaliana and stress assay were employed to study the function of RdCHI1 on anthocyanin synthesis and stress resistance. The results showed that CDS sequence length of RdCHI1 is 663 bp，encoding 220 amino acids，and the predicted molecular weight of the protein is 23.78 kD. Analysis of multiple sequence alignment revealed that RdCHI1 shares 56.52% and 56.07% similarity with CHI of Arabidopsis thaliana and Medicago. The expression levels of RdCHI1 showed a trend of rising first and then decreasing during flower development，and the transcripts were the highest in pistil，but relatively lower in petal and root. Transformation of Arabidopsis found RdCHI1 could restore the biosynthesis of anthocyanins and flavonols which missed in CHI mutant（tt5），and turn the color of cotyledons and hypocotyls of seedlings from green back to purple. Meanwhile，stress assay indicated that Arabidopsis overexpressed RdCHI1 showed better adaptability and survival rate than the wild type when under NaCl and mannitol stresses，suggesting RdCHI1 playing a potential role to stress resistance in plants.

Key words: Rhododendron delavayi, chalcone isomerase, flower color, anthocyanin, function analysis

CLC Number:

S685.21

SUN Wei, SUN Shiyu, CHEN Yiran, WANG Yuhan, ZHANG Yan, JU Zhigang, YI Yin. Cloning and Function Analysis of Chalcone Isomerase Gene RdCHI1 in Rhododendron delavayi[J]. Acta Horticulturae Sinica, 2022, 49(11): 2407-2418.

Figures/Tables 10

Table 1 Primer sequences used in this study

用途 Use	引物名称 Primer name	序列（5'-3'） Sequence
CDS 全长扩增Amlification of CDS	CHI1-CDS-F	GAGCAATGTCTTCACCACTG
	CHI1-CDS-R	GAAACAGAAGAATCAACAGGGA
qRT-PCR	CHI1-qPCR-F	CATCGTATCTGGTCCCTTTGAG
	CHI1-qPCR-R	CTATACTGATGCAGAGGCCAAA
定量内参引物Primers for internal control	β-actin-F	GTATGCCCTTCCTCATGCTATC
	β-actin-R	GGCCGTGGTAGTGAACATATAA
过表达载体引物Primers for overexpression	CHI1- pBI121-F	GCTCTAGAATGTCTTCACCACTGGCG
	CHI1- pBI121-R	CGGGATCCTTATGTCTCCTTGAATAA
拟南芥actin引物Primers for Arabidopsis actin	Atactin1-RT-F	GGCCGTGGTAGTGAACATATAA
	Atactin1-RT-R	GTATGCCCTTCCTCATGCTATC

Fig. 1 Gel electrophoresis of the PCR product of RdCHI1

Fig. 2 Sequence alignment of RdCHI1 with AtCHI（Arabidopsis thaliana，P41088）and MsCHI（Medicago sativa，P28012） Residues for binding（2S）-naringenin are shown with pink boxes，and those for hydrogen bond network in active site are shown with red stars. The green box identifies residues postulated to determine substrate preference for naringenin chalcone and isoliquiritigenin.

Fig. 3 Phylogenetic analysis of RdCHI1

Fig. 4 Expression levels of RdCHI1

Fig. 5 Complementation of RdCHI1 function in Arabidopsis tt5 mutant P < 0.05.

Fig. 6 Anthocyanin and flavonol analysis of RdCHI1transgenic Arabidopsis A-D：Anthocyanin analysis;E-H：Flavonol analysis.

Fig. 7 Germination of wild-type，tt5 and 35S::RdCHI1 OE1 and OE2 plants under NaCl and mannitol Different letters indicate significantly different values（P < 0.05）. The same below.

Fig. 8 Growth of wild-type，tt5 and 35S::RdCHI1 OE1 and OE2 plants under NaCl treatment

Fig. 9 Growth of wild-type，tt5 and 35S::RdCHI1 OE1 and OE2 plants under mannitol treatment

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Cloning and Function Analysis of Chalcone Isomerase Gene RdCHI1 in Rhododendron delavayi

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