Cloning of Mango Gene MinMYB10 and Vector Construction for Expression Analysis

doi:10.3969/j.issn.1000-2561.2019.07.008

Abstract

Abstract:

The MYB (myeloblastosis) transcription factors are mainly involved in the regulation of flavonoid metabolism, growth and development in plants. In order to study how the flavonoid metabolic pathways are regulated in mango fruit, a MYB gene, designated as MinMYB10, was cloned from the flesh of Jinhuang mango fruit. The full-length cDNA sequence of MinMYB10 was 1021 bp long, containing an open reading frame of 837 bp which would encode a peptide of 278 amino acid residues with a molecular weight of 31.60 ku and an isoelectric point of 5.89. Phylogenetic analysis showed that the encoded protein sequence was closely related to its homolog from Arabidopsis thaliana. We have also constructed a silencing vector, pTRV2-MinMYB10, and an over expression vector, pGreenII 62-SK-MinMYB10, aiming to decipher the role of MinMYB10 in the metabolism of anthocyanin in mango fruit in our upcoming researches.

Key words: mango, MinMYB10 gene, vector construction

CLC Number:

S667.7

BAI Beibei,JING Yonglin,LAN Li,WANG Jia,ZHAO Zhichang. Cloning of Mango Gene MinMYB10 and Vector Construction for Expression Analysis[J]. Chinese Journal of Tropical Crops, 2019, 40(7): 1297-1303.

Figures/Tables 10

Fig. 1 Electrophoresis of the total RNA extracted from the fruit of ‘Jinhuang’ mango 1: Mature green; 2: Semi mature; 3: Ripening.

Fig. 2 Electrophoresis of the full length cDNA of MinMYB10 M: DL 2000 Marker; 1: PCR product of the full length MinMYB10 gene.

Fig. 3 Alignment of MinMYB10 with other MYB10 homologous proteins MinMYB10: MYB10 of Mangifera indica L.; TcaMYB10: MYB10 of Theobroma cacao; HumMYB10: MYB10 of Herrania umbratica; DziMYB10: MYB10 of Durio zibethinus; VviMYB10: MYB10 of Vitis vinifera.

Fig. 4 Protein domain prediction analysis for MinMYB10

Fig. 5 The hydrophobic region of MinMYB10 protein

Fig. 6 The secondary structure of the MinMYB10 protein

Fig. 7 The predicted tertiary structure of MinMYB10 protein

Fig. 8 Phylogenetic tree analysis of MinMYB10 and other MYB proteins

Fig. 9 Construction of the pTRV2-MinMYB10 silencing vector M: DL 2000 marker; A: pTRV2-MinMYB10 double enzyme digestion, 1-2: doubleenzyme digestion; B: PCR detection for the cloned targets in Agrobacterium, 1-8: Target band.

Fig. 10 Construction for pTRV2-MinMYB10 over-expression vector M: DL 2000 Marker; A: pTRV2-MinMYB10 double enzyme digestion; 1-2: doubleenzyme digestion; B: PCR detection for the cloned targets in Agrobacterium, 1-7: Target band.

References 22

[1]	Latchman D S . Transcription factors: an overview[J]. International Journal of Experimental Pathology, 1997,29(12):1305-1312.
[2]	Liu L, White M J, Macrae T H . Transcription factors and their genes in higher plants[J]. European Journal of Biochemistry, 1999,262(2):247. DOI URL
[3]	左然, 徐美玲, 柴国华 , 等. 植物MYB转录因子功能及调控机制研究进展[J]. 生命科学, 2012,24(10):1133-1140.
[4]	Legay S, Lacombe E, Goicoechea M , et al. Molecular characterization of EgMYBl, a putative transcriptional repressor of the lignin biosynthetic pathway[J]. Plant Science, 2007,173(5):542-549. DOI URL
[5]	Fornalé S, Lopez E, Salazar-Henao J E , et al. AtMYB7: a new player in the regulation of UV-sunscreens in Arabidopsis thaliana[J]. Plant Cell Physiology, 2014,55(3):507-516. DOI URL
[6]	Li M, Dong X J, Peng J Q , et al. De novo transcriptome sequencing and gene expression analysis reveal potential mechanisms of seed abortion in dove tree (Davidia involucrata Baill.)[J]. BMC Plant Biology, 2016,16(82):1-21. DOI URL
[7]	Schwinn K E, Boase M R, Bradley J M , et al. MYB and bHLH transcription factor transgenes increase anthocyanin pigmentation in petunia and lisianthus plants, and the petunia phenotypes are strongly enhanced under field conditions[J]. Frontiers in Plant Science, 2014,5:603.
[8]	罗学兵 . 芒果的营养价值、保健功能及食用方法[J]. 中国食物与营养, 2011,17(7):77-79.
[9]	罗小杰, 韦虎 . 芒果酒的研制[J]. 中国酿造, 2007(5):89-90.
[10]	Dubos C, Stracke R, Grotewold E , et al. MYB transcription factors in Arabidopsis[J]. Trends in Plant Science, 2010,15(10):573-581. DOI URL
[11]	Ma L G, Sun N, Liu X G , et al. Organ-specific expression of Arabidopsis genome during development[J]. Plant Physiology, 2005,138(1):80-91. DOI URL
[12]	Song S, Qi T, Huang H , et al. The Jasmonate-ZIM domain proteins interact with the R2R3-MYB transcription factors MYB21 and MYB24 to affect Jasmonate-regulated stamen development in Arabidopsis[J]. The Plant Cell, 2011,23(3):1000-1013. DOI URL
[13]	Mccarthy R L, Zhong R, Fowler S , et al. The poplar MYB transcription factors, PtrMYB3 and PtrMYB20, are involved in the regulation of secondary wall biosynjournal[J]. Plant & Cell Physiology, 2010,51(6):1084-1090.
[14]	Ma Q H, Wang C, Zhu H H . TaMYB4 cloned from wheat regulates lignin biosynjournal through negatively controlling the transcripts of both cinnamyl alcohol dehydrogenase and cinnamoyl-CoA reductase genes[J]. Biochimie, 2011,93(7):1179-1186. DOI URL
[15]	Zhong R, Lee C, McCarthy R L , et al. Transcriptional activation of secondary wall biosynjournal by rice and maize NAC and MYB transcription factors[J]. Plant & Cell Physiology, 2011,52(10):1856-1871.
[16]	Stracke R, Werber M, Weisshaar B . The R2R3-MYB gene family in Arabidopsis thaliana[J]. Current Opinion in Plant Biology, 2001,4(5):447-456. DOI URL
[17]	Lin-Wang K, Micheletti D, Palmer J , et al. High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex[J]. Plant, Cell & Environment, 2011,34:1176-1190.
[18]	Shen X, Zhao K, Liu L , et al. A role for PacMYBA in ABA-regulated anthocyanin biosynjournal in red-colored sweet cherry cv. Hong Deng (Prunus avium L.)[J]. Plant & Cell Physiology, 2014,55(5):862-880.
[19]	Fischer T C, Mirbeth B, Rentsch J , et al. Premature and ectopic anthocyanin formation by silencing of anthocyanidin reductase in strawberry (Fragaria ananassa)[J]. New Phytologist Trust, 2014,201(2):440-451. DOI URL
[20]	Fournier-Level A, Lacombe T, Le Cunff L , et al. Evolution of the VvMybA gene family, the major determinant of berry colour in cultivated grapevine (Vitis vinifera L.)[J]. Heredity, 2010,104(4):351-362. DOI
[21]	Vimolmangkang S, Han Y, Wei G , et al. An apple MYB transcription factor Md MYB3 is involved in regulation of anthocyanin biosynjournal and flower development[J]. BMC Plant Biology, 2013,13:176. DOI URL
[22]	赵佳, 刘荣, 杨帆 , 等. 月季花青素苷相关R2R3-MYB蛋白基因的克隆和表达分析[J]. 中国农业科学, 2015,48(7):1392-1404.