马缨杜鹃查尔酮异构酶基因RdCHI1的克隆与功能解析

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

摘要/Abstract

摘要：

为了研究马缨杜鹃（Rhododendron delavayi）查尔酮异构酶（CHI）的功能，以其花朵为材料，克隆获得了1个RdCHI1基因。通过多序列比对、系统进化分析、qRT-PCR、拟南芥的遗传转化和抗逆试验，探究RdCHI1参与花色苷合成与抗逆的作用。结果表明，RdCHI1的CDS序列长663 bp，编码 220个氨基酸，预测其蛋白分子量为23.728 kD。多序列比对分析显示，RdCHI1与拟南芥、苜蓿CHI的相似性分别为56.52%和56.07%。开花过程中RdCHI1的表达水平先上升后下降，在雌蕊中表达量最高，花瓣和根中相对较低。拟南芥转化试验表明，RdCHI1可以恢复CHI突变体（tt5）缺失的花色苷与黄酮醇，使其幼苗子叶与下胚轴颜色由绿色恢复为紫色。逆境表明，RdCHI1过表达拟南芥植株在氯化钠和甘露醇胁迫下比野生型具有更好的适应力与存活率，证明RdCHI1在植物中具有抵抗逆境的潜在作用。

关键词: 马缨杜鹃, 查尔酮异构酶, 花色, 花色苷, 功能解析

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

中图分类号:

S685.21

孙威, 孙世宇, 陈一然, 王聿晗, 张艳, 鞠志刚, 乙引. 马缨杜鹃查尔酮异构酶基因RdCHI1的克隆与功能解析[J]. 园艺学报, 2022, 49(11): 2407-2418.

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.

图/表 10

表1 本研究所用引物序列

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

图1 马缨杜鹃CHI1基因的PCR扩增产物检测

Fig. 1 Gel electrophoresis of the PCR product of RdCHI1

图2 RdCHI1（马缨杜鹃）、AtCHI（拟南芥）和MsCHI（苜蓿）多序列比对粉色框氨基酸参与（2S）-柚皮素的结合，红色星标记氨基酸参与氢键的互作，绿色框氨基酸决定CHI对柚皮素查尔酮和异甘草素的偏好性。

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.

图3 马缨杜鹃CHI1的系统进化分析

Fig. 3 Phylogenetic analysis of RdCHI1

图4 马缨杜鹃CHI1的表达量

Fig. 4 Expression levels of RdCHI1

图5 马缨杜鹃CHI1在拟南芥tt5突变体中的功能互补分析

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

图6 马缨杜鹃CHI1转基因拟南芥花色苷与黄酮醇的分析 A ~ D：花色苷分析;E ~ H：黄酮醇分析。

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

图7 拟南芥野生型（WT）及其RdCHI1过表达株系（OE1、OE2）和tt5突变体在氯化钠与甘露醇处理下的发芽率不同字母表示差异显著（P < 0.05）。下同。

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.

图8 拟南芥野生型（WT）及其RdCHI1过表达株系（OE1、OE2）和tt5突变体经氯化钠处理时的生长情况

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

图9 拟南芥野生型（WT）及其RdCHI1过表达株系（OE1、OE2）和tt5突变体经甘露醇处理时的生长情况

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

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