Cloning and Expression Analysis of SRPP2 Gene in Taraxacum kok-saghyz Rodin

doi:10.13560/j.cnki.biotech.bull.1985.2019-0684

Abstract

Abstract: TkSRPP2 is a member of SRPP family in Taraxacum kok-saghyz Rodin,and plays an important role in the biosynthetics of natural rubber and stability of rubber particles. PCR was applied to clone the full length of TkSRPP2 gene from T. kok-saghyz Rodin,then the bioinformatics analysis of TkSRPP2 gene,subcellular localization and quantitative expression analysis of tobacco were conducted. The results demonstrated that the full length of TkSRPP2 gene was 633 bp,encoding 210 amino acids,the relative molecular weight of the protein was 23.15 kD,and the theoretical isoelectric point（pI）was 8.51. The phylogenetic analysis demonstrated that it was most closely related to SRPP3.The subcellular localization of tobacco revealed that TkSRPP2 protein was in cytoplasm. TkSRPP2 gene was expressed in many tissues of T. kok-saghyz Rodin,with the highest expression in 6 months roots.

Key words: Taraxacum kok-saghyz Rodin, Small rubber particle protein（SRPP）, Bioinformatics analysis, Real-time quantitative PCR, Protein Subcellular Localization

ZHANG Rui-zhu, JIANG Yu-chen, HUANG Jun, YAN Jie. Cloning and Expression Analysis of SRPP2 Gene in Taraxacum kok-saghyz Rodin[J]. Biotechnology Bulletin, 2020, 36(1): 9-14.

References

[1] Beilen JBV, Poirier Y.Establishment of new crops for the production of natural rubber[J]. Trends in Biotechnology, 2007, 25(11):522-529.
[2] Lin T, Xu X, Ruan J, et al.Genome analysis of Taraxacum kok-saghyz Rodin provides new insights into rubber biosynthesis[J]. National Science Review, 2018, 5:78-87.
[3] Dennis MS, Light DR.Rubber elongation factor from Hevea brasiliensis. Identification, characterization, and role in rubber biosynthesis[J]. Journal of Biological Chemistry, 1989, 264(31):18608.
[4] Berthelot K, Lecomte S, Estevez Y, et al.Rubber particle proteins, HbREF and HbSRPP, show different interactions with model membranes[J]. Biochimica et Biophysica Acta(BBA)- Biomembranes, 2014, 1838(1):287-299.
[5] Berthelot K, Lecomte S, Estevez Y, et al.Homologous Hevea brasiliensis REF(Hevb1)and SRPP(Hevb3)present different auto-assembling[J]. Biochimica et Biophysica Acta(BBA)- Proteins and Proteomics, 2014, 1844(2):473-485.
[6] Laibach N, Hillebrand A, Twyman RM, et al.Identification of a, Taraxacum brevicorniculatum, rubber elongation factor protein that is localized on rubber particles and promotes rubber biosynthesis[J]. The Plant Journal, 2015, 82(4):609-620.
[7] Amerik AY, Martirosyan YT, Gachok IV.Regulation of natural ru-bber biosynthesis by proteins associated with rubber particles[J]. Russian Journal of Bioorganic Chemistry, 2018, 44(2):140-149.
[8] Men X, Wang F, Chen G, et al.Biosynthesis of natural rubber:Current state and perspectives[J]. Int J Mol Sci, 2019, 20. doi:10.3390/ijms20010050.
[9] Oh SK, Kang H, Shin DH, et al.Isolation, characterization, and functional analysis of a novel cDNA clone encoding a small rubber particle protein from Hevea brasiliensis[J]. Journal of Biological Chemistry, 1999, 274(24):17132-17138.
[10] 于一帆, 朱小彬, 葛会敏, 等. 基于绿色荧光蛋白瞬时表达的植物亚细胞定位方法[J]. 江苏农业科学, 2014, 42(12):58-61.
[11] 覃碧, 潘敏, 余海洋, 等. 橡胶草实时荧光定量PCR内参基因评价[J]. 植物生理学报, 2016(7):1059-1065.
[12] Bushman BS, Scholte AA, Cornish K, et al.Identification and comparison of natural rubber from two Lactuca species[J]. Phytochemistry, 2006, 67(23):2590-2596.
[13] Cornish K.Similarities and differences in rubber biochemistry among plant species[J]. Phytochemistry(Oxford), 2001, 57(7):1123-1134.
[14] Wang X, Shi M, Lu X, et al.A method for protein extraction from different subcellular fractions of laticifer latex in Hevea brasiliensis compatible with 2-DE and MS[J]. Proteome Science, 2010, 8(1):35.
[15] Ko JH, Chow KS, Han KH.Transcriptome analysis reveals novel features of the molecular events occurring in the laticifers of Hevea brasiliensis(para rubber tree)[J]. Plant Molecular Biology, 2003, 53(4):479-492.
[16] Collins JE.The role of small rubber particle proteins in rubber biosynthesis[D]. Reno:University of Nevada, 2009.
[17] Fricke J, Hillebrand A, Twyman RM, et al.Abscisic acid-dependent regulation of small rubber particle protein gene expression in Taraxacum brevicorniculatum is mediated by TbbZIP1[J]. Plant & Cell Physiology, 2013, 54(4):448-464.
[18] 李先昆, 段翠芳, 聂智毅, 等. 巴西橡胶树小橡胶粒子蛋白(SRPP)基因启动子的克隆及其功能分析[J]. 中国农学通报, 2009(1):241-247.
[19] Li JF, Norville JE, Aach J, et al.Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9[J]. Nature Biotechnology, 2013, 31(8):688-691.
[20] 冯伟强, 仝征, 靳翔, 等. 巴西橡胶树REF/SRPP家族基因的克隆及表达分析[J]. 分子植物育种, 2016, 15(11):3024-3032.