Cloning,Structure and Expression Profile Analysis of CtANR2 and CtANR3 Genes from Carthamus tinctorius L.

doi:10.7668/hbnxb.20193450

Abstract

Abstract:

As part of flavone compounds,proanthocyanidins (PAs)play important roles in defense against pests and diseases,and they are also a kind of nutritionally valuable component of human diet.Anthocyanidin reductases (ANRs)are involved in the biosynthesis of PAs.We cloned two CtANR genes from Carthamus tinctorius L..Bioinformatic analysis showed that the full-length CDS sequences of CtANR2 and CtANR3 were 1 020 and 1 023 bp,respectively.Both of the genomic sequences contained five exons and four introns,the length of the first exon was different while the other four exons were the same,and the lengths of introns varied greatly.CtANR2 and CtANR3 genes encoded proteins with amino acid number of 339 and 340,respectively,and their secondary structures were mainly composed of α-helix and random curl.Both of them were hydrophilic proteins,CtANR2 protein was unstable,while CtANR3 was a stable hydrophilic protein.In addition,the two proteins both had no signal peptide sequence and transmembrane structure,and might be located outside the cells.Sequence alignment and phylogenetic analysis showed that CtANR2 and CtANR1 had the highest homology and the closest genetic relationship.The three CtANR proteins had the closest evolutionary relationship with ANR of Asteraceae,and also belonged to the same large branch with ANR of Solanaceae,Malvacede and Moraceae.Expression analysis showed that the tissue expression patterns of CtANR2 and CtANR1 were similar with the highest expression level in flowers and the lowest expression level in early fruit balls,while CtANR3 was highly expressed in bracts and almost not expressed in roots and early fruit balls.They displayed different expression patterns under different hormone treatments.The expressions of CtANR2 and CtANR1 were decreased after different hormone treatments,while the expression levels of CtANR3 were increased to different degrees after hormone treatments.These results suggest that CtANR genes might play different roles in different development stages and resistance to abiotic stresses of Carthamus tinctorius L..

Key words: Carthamus tinctorius L., Anthocyanidin reductase, Gene cloning, Gene structure, Expression profile

LU Dandan, TAN Zhengwei, YU Yongliang, LI Lei, XU Lanjie, YANG Hongqi, YANG Qing, DONG Wei, AN Sufang, LIANG Huizhen. Cloning,Structure and Expression Profile Analysis of CtANR2 and CtANR3 Genes from Carthamus tinctorius L.[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(1): 84-93. doi: 10.7668/hbnxb.20193450.

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Figures/Tables 11

References 36

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引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	用途 Usage
CtANR2-F	ATGGAAGAGAGTAAAAGCAAGTGCA	基因扩增
CtANR2-R	CTAGATATTGCCCGATCTCTTAGCAC
CtANR3-F	ATGGAAGAGAGTAAAAGCAGCAGGT	基因扩增
CtANR3-R	CTAGATATTACCAAATCTCTTTGCATATTC
qCtANR1-F	CGACAGGATTGAGGCAACAATCAA	实时荧光定量PCR
qCtANR1-R	AGCTGCAACAACAGAGGCGGTATA
qCtANR2-F	AACGACAGGATTGAGGCA	实时荧光定量PCR
qCtANR2-R	AACAACGGAGGCGGTATA
qCtANR3-F	AGTGAGGCGGCTCGTCTAT	实时荧光定量PCR
qCtANR3-R	CAAGGGTGTCCAGCAAGTTT
Ct60S-F	TGGAGCTCATCAAGAAGGG	内参基因
Ct60S-R	GGTAAGGACCACAAGACCGTA

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	用途 Usage
CtANR2-F	ATGGAAGAGAGTAAAAGCAAGTGCA	基因扩增
CtANR2-R	CTAGATATTGCCCGATCTCTTAGCAC
CtANR3-F	ATGGAAGAGAGTAAAAGCAGCAGGT	基因扩增
CtANR3-R	CTAGATATTACCAAATCTCTTTGCATATTC
qCtANR1-F	CGACAGGATTGAGGCAACAATCAA	实时荧光定量PCR
qCtANR1-R	AGCTGCAACAACAGAGGCGGTATA
qCtANR2-F	AACGACAGGATTGAGGCA	实时荧光定量PCR
qCtANR2-R	AACAACGGAGGCGGTATA
qCtANR3-F	AGTGAGGCGGCTCGTCTAT	实时荧光定量PCR
qCtANR3-R	CAAGGGTGTCCAGCAAGTTT
Ct60S-F	TGGAGCTCATCAAGAAGGG	内参基因
Ct60S-R	GGTAAGGACCACAAGACCGTA

在线网站 Online website	网址 Website	分析内容 Application
GSDS 2.0	http://gsds.gao-lab.org/	基因结构
CD-Search	https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi	保守结构域
ORF Finder	https://www.ncbi.nlm.nih.gov/orffinder/	最大开放阅读框
ProtParam	https://web.expasy.org/protparam/	理化性质
ProtScale	https://web.expasy.org/protscale/	亲疏水性
SOPMA	https://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl	二级结构预测
TMHMM Server v.2.0	http://www.cbs.dtu.dk/services/TMHMM/	跨膜结构
SignalP 4.0 Server	http://www.cbs.dtu.dk/services/SignalP-4.0/	信号肽
ProtComp 9.0	http://linux1.softberry.com/berry.phtml	亚细胞定位

在线网站 Online website	网址 Website	分析内容 Application
GSDS 2.0	http://gsds.gao-lab.org/	基因结构
CD-Search	https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi	保守结构域
ORF Finder	https://www.ncbi.nlm.nih.gov/orffinder/	最大开放阅读框
ProtParam	https://web.expasy.org/protparam/	理化性质
ProtScale	https://web.expasy.org/protscale/	亲疏水性
SOPMA	https://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl	二级结构预测
TMHMM Server v.2.0	http://www.cbs.dtu.dk/services/TMHMM/	跨膜结构
SignalP 4.0 Server	http://www.cbs.dtu.dk/services/SignalP-4.0/	信号肽
ProtComp 9.0	http://linux1.softberry.com/berry.phtml	亚细胞定位

蛋白质 Ptotein	理化性质 Physical and chemical properties					二级结构/% Secondary structure
蛋白质 Ptotein	氨基酸数目/个 Amino acid number	等电点 pI	分子质量/ku Molecular weight	亲水性指数 GRAVY	不稳定指数 Instability index	α-螺旋 Alpha helix	无规则卷曲 Random coil	延伸链 Extended strand	β-折叠 Beta turn
CtANR2	339	5.87	37.988 36	-0.231	42.92	40.71	37.76	14.45	7.08
CtANR3	340	6.32	38.188 59	-0.268	36.16	44.41	35.59	14.12	5.88

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