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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (7): 1021-1031.doi: 10.3724/SP.J.1006.2018.01021

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Functional Analysis of R2R3 MYB Genes Involved in Anthocyanin Biosynthesis in Potato Tuber

Huan TAN1,Yu-Hui LIU1,*(),Li-Xia LI1,Li WANG2,Yuan-Ming LI1,Jun-Lian ZHANG1,*()   

  1. 1 Gansu Key Laboratory of Crop Genetic Improvement and Germplasm Enhancement / College of Horticulture / Gansu Provincial Key Laboratory of Aridland Crop Science, Gansu Agricultural University, Lanzhou 730070, Gansu, China
    2 College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, Gansu, China
  • Received:2017-10-27 Accepted:2018-03-26 Online:2018-07-10 Published:2018-04-09
  • Contact: Yu-Hui LIU,Jun-Lian ZHANG E-mail:lyhui@aliyun.com;zhangjunlian77@163.com
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (31601356), Gansu Science Foundation for Distinguished Young Scholars (17JR5RA138), the Agricultural Biotechnology Research and Application Development Foundation of Gansu Province (GNSW-2015-15), Gansu Scientific Research Foundation for the Universities (2016A-030), and Fuxi Talent Project of Gansu Agricultural University (Gaufx-02Y04).

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Abstract:

The anthocyanins biosynthesis is regulated by transcription factors, and R2R3 MYB is the most important transcriptional regulator in plant. In this study, three homologous genes were isolated from tetraploid potato, belonging to R2R3 MYB gene family. The structure and function analysis of the three homologous genes were characterized by bioinformatics analysis, stable tobacco genetic transformation and qPCR assays. The three homologous genes contained R2 and R3 conserved domains, differed in the number of repeats (R) consisting of 10 amino acid sequences, and named as StAN1-R0, StAN1-R1, and StAN1-R3 according to the number of R. The coding proteins are hydrophilic with molecular weight of 28 047.91, 29 458.35, 31 527.60 Da, and isoelectric points (pI) of 6.14, 6.90, and 8.39, respectively. The accumulation of anthocyanin was significantly increased in StAN1-R0, StAN1-R1 and StAN1-R3 overexpressed plants. The leaf color of StAN1-R1-overexpressed plants was dark red with the highest anthocyanin content among the three transgenic events. The qPCR assays showed that exogenous StAN1 genes enhanced the expression of endogenous NtbHLH transcription factor as well as NtCHS, NtCHI, NtF3H, NtF3’H, NtDFR, NtANS, and NtUFGT, involved in anthocyanin biosynthesis in transgenic tobacco leaves. The overexpression of StAN1-R1 resulted in higher expression of NtDFR, NtANS, and endogenous NtbHLH in transgenic tobacco. The results showed that three homologous genes of StAN1 can regulate anthocyanin biosynthesis, among them StAN1-R1 containing one R has the strongest regulatory capacity.

Key words: potato, anthocyanin, R2R3 MYB, gene cloning, bioinformatics analysis, stable tobacco transformation, gene expression analysis

Table 1

Primer sequences for qRT-PCR in this study"

基因
Gene		 登录号
Accession number		 上游引物
Forward primer (5'-3')		 下游引物
Reverse primer (5'-3')
StAN1 AY841129.1 GGCCACATATCAAGAGAGGTGACTTTG TCACATCGTTAGCTGTCCTTCCTGG
NtCHS AF311783.1 TTGTTCGAGCTTGTCTCTGC AGCCCAGGAACATCTTTGAG
NtCHI AB213651.1 GTCAGGCCATTGAAAAGCTC CTAATCGTCAATGCCCCAAC
NtF3H AB289450.1 CAAGGCATGTGTGGATATGG TGTGTCGTTTCAGTCCAAGG
NtF3'H AB289449.1 AGGCTCAACACTTCTCGT CATCAACTTTGGGCTTCT
NtDFR EF421429.1 AACCAACAGTCAGGGGAATG TTGGACATCGACAGTTCCAG
NtANS AB289447.1 TGGCGTTGAAGCTCATACTG GGAATTAGGCACACACTTTGC
NtUFGT FG627024.1 GAGTGCATTGGATGCCTTTT CCAGCTCCATTAGGTCCTTG
NtbHLH HQ589208.1/HQ589209.1 ATGKGCGCAAACGAGGTTGATAGC TRGCTGAGGTTGTTGTTGCTCA
NtEF-1α D63396.1 TGAACCATCCAKGACAGATTGG TGGGCTCCTTCTCAATCTCCTT

Fig. 1

Electrophoresis of the total RNARS1: GN, red skin; WS: XD, white skin; PS: HM, purple skin; RS2: QS, red skin; WF1: GN, white flesh; WF: XD, white flesh; PF: HM, purple flesh; WF2: QS, white flesh; RFV: QS, red vascular bundle; M: DNA marker."

Fig. 2

PCR map of gene cloningRS1: GN, red skin; WS: XD, white skin; PS: HM, purple skin; RS2: QS, red skin; WF1: GN, white flesh; WF: XD, white flesh; PF: HM, purple flesh; WF2: QS, white flesh; RFV: QS, red vascular bundle; M: DNA marker."

Fig. 3

Sequence comparison chart R indicates the repeat structure for TIAPQPQEGI."

Table 2

Primary structure prediction of potato StAN1 gene encoding proteins"

基因
Gene		 蛋白质分子式
Formula		 分子量
Molecular weight (Da)		 等电点
pI		 不稳定系数
Instability index		 平均疏水性
Average of hydropathicity		 脂肪系数
Aliphatic index
StAN1-R0 C1227H1936N362O365S14 28047.91 8.39 46.97 -0.693 79.14
StAN1-R1 C1284H2019N377O390S15 29458.35 6.90 48.64 -0.720 74.88
StAN1-R3 C1371H2164N402O423S15 31527.60 6.14 50.76 -0.712 76.15

Table 3

Secondary structure analysis of StAN1 gene encoding protein (%)"

基因
Gene		 α-螺旋
Alpha helix		 β-螺旋
Beta turn		 无规则卷曲
Random coil		 延伸链
Extended strand
StAN1-R0 34.84 9.84 40.57 14.75
StAN1-R1 35.27 8.91 42.25 13.57
StAN1-R3 28.06 7.55 47.84 16.55

Fig. 4

Multiple alignment of amino acid sequence of R2R3 MYB in some solanaceae plants and other subfamilyStAN1-R0_PSAK277, StAN1-R1_PSAK277, StAN1-R3_PSAK277: Solanum tuberosum; AAF66727.1: Petunia×hybrida; AAA33482.1: Zea mays; AAG42001.1-AAG42002.1: Arabidopsis thaliana; ABB84753.1: Malus pumila; ABD72956.1: Vitis vinifera; ABO26065.1: Solanum chilense; ABX71488.1: Pyrus pyrifolia; ACI195261.1: Solanum habrochites; ACN79541.1: Medicago truncatula; ACO52470.1: Nicotiana tabacum; ADW94950.1: Petunia×hybrida; AFD31843.1: Solanum tuberosum; AIP93873.1: Solanum melongena; AMK01804.1: Solanum melongena; BAF45114.1: Ipomoes batats; NP_001311547.1: Capsicum annuum; ABX71493.1: Cerasus avium; sp_O23892.1: Oryza sativa."

Fig. 5

Phylogenetic tree analysis of the amino acid sequence of StAN1 genes and some R2R3 MYB transcription factor families in solanaceae and other plants"

Fig. 6

Phenotypic analysis of StAN1 transgenic tobacco a: whole plant of StAN1 transgenic tobacco; b: leaves of StAN1 transgenic tobacco."

Fig. 7

PCR identification of StAN1 transgenic tobaccoM: DNA marker; CK: non-genetically modified tobacco; R0, R1, R3: the transgenic tobacco of StAN1-R0, StAN1-R1, StAN1-R3, respectively."

Fig. 8

Average content of anthocyanin in StAN1 transgenic tobacco and controlDuncan’s multiple comparisons were used to analyze (P < 0.05,n = 3)."

Fig. 9

Gene expression relative to anthocyanin biosynthesis in transgenic tobaccoDuncan’s multiple comparisons were used to analyze (P < 0.05, n = 3)."

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