Expression Analysis of Anthocyanin Biosynthesis Pathway Genes in

Indosasa hispida MeClure cv. ‘Rainbow’

Chunsheng Zhang

, Guisha Peng

and Rui Shi

3,* c

Office of Academic Affairs, Yunnan University of Finance and Economics, 650221, Kunming, Yunnan, China

Key Laboratory for Forest Disaster Warning and Control in Yunnan Province, Southwest Forestry University, 650224,

Kunming, Yunnan, China

Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China, Ministry of

Education, Southwest Landscape Architecture Engineering Research Center of National Forestry and Grassland

Administration, Southwest Forestry University, 650224, Kunming, Yunnan, China

Keywords: Indosasa Hispida MeClure cv. ‘Rainbow’, Anthocyanin Biosynthesis, Gene Expression.

Abstract: Anthocyanins existed in diverse plants have many health beneficial functions such as anti-inflammatory, anti-

oxidation and antihypertensive. Studies have demonstrated that anthocyanin accumulation in plant organs is

related to gene expression in anthocyanin biosynthesis pathway. In order to probe which gene or genes have

correlation with the anthocyanin biosynthesis in bamboo species, the expression level of some genes involved

in anthocyanin biosynthesis pathway was determined by real time PCR semi-quantitatively at seven different

growth stages of Indosasa hispida cv. ‘Rainbow’. Results showed that all of the genes studied were expressed

with similar patterns in most tissues, and which also showed a positive correlation between the level of CHS,

F3H, DFR, UFGT gene expression and anthocyanin accumulation.

1 INTRODUCTION

Anthocyanins are accumulated in cell vacuoles and

produce a diverse pigmentation from orange to red,

purple and blue in flowers, fruits and vegetables

(Oancea, Oprean, 2011, Horbowicz et al, 2008). Anti-

oxidant effects, protecting DNA and the

photosynthetic machinery from high radiation fluxes,

resistance to cold and drought stress, anti-aging and

anti-cancer properties and recruitment of pollinators

are some well-known roles of anthocyanins (Oancea,

Oprean, 2011, Horbowicz et al, 2008). Studies have

demonstrated that anthocyanin biosynthesis is

mediated by the anthocyanin pathway some

genes/transcription factors (Li et al, 2021).

Indosasa hispida MeClure cv. ‘Rainbow’ (Y. M.

Yang et J. Wang) with high ornamental value, a new

variety of Indosasa hispida, exhibits different degree

of purplish red at different stages of its growth, and

the purplish red material was ascertained as

anthocyanin after isolation and analysis (Wang et al,

https://orcid.org/0000-0002-8542-8676

https://orcid.org/0000-0002-7018-1965

https://orcid.org/0000-0003-2593-7302

2012, Miao et al, 2014, Wang et al, 2014). Although

the anthocyanin pathway has been thoroughly

characterized and the correlation between gene

expression level and anthocyanin accumulation in

many plants having been identified (Yonekura-

Sakakibara et al, 2019, Boss et al, 1996, Jenog et al,

2004), the question about how their biosynthesis is

regulated in bamboo species is rarely known at

present. In this study, ten structural genes related to

anthocyanin biosynthesis of I. hispida MeClure cv.

‘Rainbow’ were determined by real time PCR semi-

quantitatively at seven different growth stages to

interpret a correlation between anthocyanin

biosynthesis and gene expression level.

184

Zhang, C., Peng, G. and Shi, R.

Expression Analysis of Anthocyanin Biosynthesis Pathway Genes in Indosasa hispida MeClure cv. ‘Rainbow’.

DOI: 10.5220/0011194200003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 184-188

ISBN: 978-989-758-595-1

2 MATERIALS AND METHODS

2.1 Plant Materials

I. hispida MeClure cv. ‘Rainbow’ was obtained from

greenhouse of Yunnan Academy of Forestry, and

which was divided into seven growth stages

according to colour differences (Figure 1). The culm

tissues for RNA isolation were collected directly into

liquid nitrogen and were stored at -80℃ until used.

Description of the growth stages: 1, 2: Tender apex without visible anthocyanin pigments; 3: Intermediate stem with purplish

red tissues and colourless skin; 4: Intermediate stem with purplish red tissues and skin; 5: Intermediate stem with little purplish

red tissues and purplish red skin; 6: Mature stem with colourless tissues and purplish red skin; 7: Mature stem without visible

anthocyanin pigments.

Figure 1: The cross profile of I. hispida MeClure cv. ‘Rainbow’ at different growth stages.

2.2 Main Reagents

DEPC-treated Water; UltraPower Nucleic Acid Stain;

75% ethanol; isopropyl alcohol; chloroform; Takara

RT-PCR Kit; PCR mix Kit; TRNzol® Reagent.

2.3 RNA Extraction and cDNA

Synthesis

Total RNA was isolated from culm tissues using

TRNzol® Reagent (TIANGEN Biotech (Beijing)

Co., Ltd.). Purity and concentration of isolated RNA

was determined by protein nucleic acid analyzer, and

integrity of which was verified by electrophoresis on

1.2% agarose gel, respectively. RNA was reverse

transcribed to cDNA with the first strand cDNA

synthesis kit (TaKaRa Biotechnology (Dalian) Co.,

Ltd.) according to the manufacture’s protocols. The

reverse transcription system and conditions are as

follows: Oligo (dT) Primer 1 Μl, dNTP Mixture 1 μL,

Total RNA 3 μL, RNase-free dH2O up to 10 μL; 65℃,

5 min annealing response, ice chill; 5×PrimeScript®

Buffer 4 μL, RNase Inhibitor 0.5 μL, PrimeScript®

RTase 1μL, RNase Free dH2O 4.5 μL; 30℃ 10 min,

42℃ 60 min, 95℃ 5min.

2.4 PCR Cloning and Analysis of the

Products

Primers were designed on the basis of transcriptome

data acquired by our group earlier, and the designed

primers for amplifying gene are as follows (Tab. 1).

Conditions for PCR of genes involved are as follows:

95℃ for 5 min followed by 35 cycles at 95℃ for 45s,

at annealing temperature listed below for 30 s, and at

72℃ for 5 min, with a final extension at 72℃ for 8

min. The PCR products were assessed by agarose gel

electrophoresis, taking actin primer as a reference

gene.

Expression Analysis of Anthocyanin Biosynthesis Pathway Genes in Indosasa hispida MeClure cv. ‘Rainbow’

185

Table 1: PCR primer and reaction condition for PCR.

Abbreviations: PAL, phenylalanine ammonia lyase; 4CL, coenzyme A ligase; CHS, chalcone synthase; CHI, chalcone

isomerase; F3H, flavanone-3-hydroxylase; F3'5'H, flavonoid-3’,5’-hydroxylase; LDOX, leucoanthocyan-idin dioxygenase;

DFR, dihydroflavonol 4-reductase; ANS, Anthocyanidin Synthase; UFGT, UDP glucose-flavonoid 3-O-glucosyl transferase.

3 RESULTS

3.1 Analytical Results of Isolated Total

RNA

Figure 2: Electrophoresis Image of total RNA at different

stages.

The agarose gel electrophoresis of total RNA at

different stages was shown in figure 2. The data of

A260/A280 ratio and concentration of total RNA

listed Tab. 2 shows that total RNA isolated for study

can be used for cDNA synthesis.

Table 2: Purity and concentration of total RNA from culm

tissues.

3.2 Expression of the Anthocyanin

Biosynthesis Pathway Genes

As depicted in figure 3, the target genes with the

exception of F3H, DFR and UFGT were all expressed

with different levels at the first two stages. No

anthocyanin pigments were seen in young tissues,

presumably because UFGT is missing. In the

pigmented tissues, the expression levels of several

genes increased dramatically, but the expression

levels were gradually decreased toward the end of

ripening. Half of genes were not expressed in the

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

186

ripening tissues without visible anthocyanin pigments

compared with the first two stages, in which 4CL and

CHI still showed higher levels.

Figure 3: Electrophoresis Image of PCR products at

different growth stages.

4 DISCUSSION

It was evident that anthocyanin accumulation in plant

is associated with gene expression in anthocyanin

biosynthesis pathway (Rouholamin et al, 2015). Ten

structure genes related to anthocyanin biosynthesis

pathway were investigated at seven different stages of

a new variety of Indosasa hispida. Similar expression

patterns of PAL and 4CL were observed at the whole

growth stages, and the difference between them is that

there was no expression of PAL in the unpigmented

ripening tissue. The results indicated that they are not

only the precursor genes of anthocyanin biosynthesis

pathway, but also related to the accumulation of

anthocyanin.

Only weak expression of CHS and CHI was

detected at the first two stages while the expression of

the two genes increased dramatically to a very high

level subsequently. Notably, the high levels of

expression of CHI lasted to the unpigmented ripening

stage. Therefore, there is a significant positive

correlation between the accumulation of anthocyanin

and the expression of CHS and CHI, especially CHS

gene.

Previous study showed that the colour of grape

skins is blue or red was determined by the ratio of

F3’5’H/F3H (Simone, Gabriele, 2007). In the study,

both F3’5’H and F3H were expressed in culm tissues

contained visible anthocyanin pigments, where levels

of F3’5’H was higher. It can be deduced that F3’5H

and F3H might commonly regulate anthocyanin

biosynthesis of I. hispida MeClure cv. ‘Rainbow’ to

account for the fact of purplish red.

The expression of LDOX and ANS showed same

trends, both of them had no expression in the

unpigmented ripening tissue. DFR and UFGT were

only expressed obviously in the pigmented tissues,

which demonstrate that DFR and UFGT are two key

encoding enzyme genes to regulate anthocyanin

biosynthesis of I. hispida MeClure cv. ‘Rainbow’,

and DFR expression was consistent to the study on

other species (Hasegawa, 2001).

5 CONCLUSIONS

For preliminary study the expression of genes

involved in anthocyanin biosynthesis pathway of I.

hispida MeClure cv. ‘Rainbow’, ten genes was

determined at seven different growth stages. It can be

seen from the results that the appearance of

anthocyanin at the onset of ripening coincides with

increased expression of each of the genes encoding

biosynthetic enzymes in this pathway, and which

suggested that the induction of anthocyanin synthesis

is triggered by regulatory genes in I. hispida MeClure

cv. ‘Rainbow’. Further, the content analysis of

anthocyanin in various tissues is currently being

undertaken by our group to understand the deeper

correlation between anthocyanin biosynthesis and

gene expression level.

ACKNOWLEDGEMENTS

This work was supported by China Agriculture

Research System (CARS-21), Yunnan Provincial

Key Programs (202102AE090042, 2019ZG00901,

202002AA10007), High-end Foreign Experts

Program of Yunnan (202105AQ130011, 2019013),

and Yunnan Provincial Financial Forestry Science

and Technology Promotion Demonstration Special

Project (2020, No: TS09).

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