核桃JrGA2ox基因的克隆、亚细胞定位及功能验证

doi:10.11707/j.1001-7488.20190206

Abstract

Abstract: [Objective] GA2-oxidase (GA2ox) is a key enzyme which plays a negative role in regulating the gibberellins.GA2ox could convert bioactive gibberellins to inactive gibberellins during gibberellin biosynthesis.Accordingly,it has a certain inhibitory effect on plant growth.This experiment was aimed at cloning and verifying function of GA2ox encoding gene (JrGA2ox) in walnut (Juglans regia).It helps to further explore the role of JrGA2ox gene in the process of plant growth and development,especially in regulating the plant height.This further assists in discovering and utilizing more high-qualified genes in walnut and cultivating more improved walnut varieties.[Method] The full length of JrGA2ox gene was cloned and verified based on PCR technique.Furthermore,the 35S::JrGA2ox::GFP overexpression vector with a strong promoter was constructed by In-Fusion cloning technology.The JrGA2ox homologous amino acid sequences were obtained by the online tool of BLAST,and their comparative analysis and phylogenetic tree analysis were carried out.Thereafter subcellular localization assay was carried out to reveal where it worked.The overexpressed vector was transformed into walnut somatic embryos using the Agrobacterium-mediated method and the positive transformation plants of JrGA2ox overexpression types were obtained.Furthermore,the biological properties of JrGA2ox gene were further analyzed.[Result] The 1 056 bp open reading frame (ORF) of JrGA2ox was gained by cloning method,encoding 351 amino acids and its molecular weight was 39.25 kDa.Blast analysis showed that JrGA2ox had a common structural feature of GA2-oxidase family which was a conserved protein domain named 20G-FeⅡ-Oxy.It indicated that JrGA2ox belongs to GA2-oxidase gene family.The results of phylogenetic tree analysis showed that JrGA2ox and Populous tomentosa GA2ox were clustered into the same branch.And the protein encoded by JrGA2ox gene had a high homology with those proteins of woody plants,Morus notabilis GA2ox1,Pyrus communis GA2ox,Prunus persica GA2ox1,and Malus domestica GA2ox1.The analysis of subcellular localization assay in epidermal cells of tobacco (Nicotiana benthamiana)-leaves showed that JrGA2ox was mainly localized in the nucleus and plasma membrane.The results of GFP fluorescence detection and PCR inspection in the transformed somatic embryos showed that 35S::JrGA2ox::GFP had been transformed into walnut somatic embryos successfully.Comparing with the regenerated wild type walnut plants,there was a significant difference in plant height.The average height of the transformed plants was 1/2 of that of the control and it was negatively correlated with the expression of JrGA2ox gene.[Conclusion] The results indicated that JrGA2ox was located in the nucleus and plasma membrane.In addition,JrGA2ox gene regulated the height of walnut plants negatively.When the relative expression level raised in the transgenic walnut plants with 35S::JrGA2ox::GFP,these plants showed obvious dwarfing characteristics.This study provides a technical basis for further analysis of the functions of JrGA2ox gene in growth and development and for the breeding of dwarf walnut varieties.

Key words: Juglans regia, GA2ox gene, over-expression, subcellular localization, transformation

CLC Number:

S718.46

Zhang Jiaqi, Hu Hengkang, Xu Chuanmei, Hu Yuanyuan, Huang Youjun, Xia Guohua, Huang Jianqin, Chang Yingying, Ye Lei, Lou Heqiang, Zhang Qixiang. Cloning, Subcellular Localization and Function Verification of Gibberellin 2-Oxidase Gene in Walnut (Juglans regia)[J]. Scientia Silvae Sinicae, 2019, 55(2): 50-60.

References

白牡丹, 王彩虹, 田义轲, 等. 2012. 苹果茎尖组织中GA2ox基因的结构特征及蛋白序列的生物信息学分析. 华北农学报,27 (5):55-59.
(Bai M D, Wang C H, Tian Y K, et al. 2012. Gene structure identification and protein sequence bioinformation analysis for GA2ox from apical tissue of apple shoots. Acta Agriculturae Boreali-Sinica, 27 (5):55-59.[in Chinese])
程飞飞. 2012. 矮生梨'中矮1号'GA2-oxidase基因的克隆与功能分析. 北京:中国农业科学院硕士学位论文.
(Cheng F F. 2012. Cloning and functional analysis of GA2-oxidase gene in the dwarf pears named 'Zhongai 1'. Beijing:MS thesis of Chinese Academy of Agricultural Sciences.[in Chinese])
姜志昂,彭建营,孙建设. 2014. 苹果MdKS、MdKOA1基因克隆与表达分析. 植物遗传资源学报,15 (2):362-368.
(Jiang Z A, Peng J Y, Sun J S. 2014. Isolation and expression of MdKS and MdKOA1 gene in apple. Journal of Plant Genetic Resources, 15 (2):362-368.[in Chinese])
屠煦童,张仕杰,陈小云,等. 2015. "南通小方柿"GA2ox基因的克隆、亚细胞定位及表达分析. 中国农业科学,48 (1):197-206.
(Tu X T, Zhang S J, Chen X Y, et al. 2015. Cloning, subcellular localization and expression analysis of Gibberellin 2-Oxidase gene in Diospyros kaki Linn. cv. Nantongxiaofangshi. Scientia Agricultura Sinica, 48 (1):197-206.[in Chinese])
王西成,任国慧,房经贵,等. 2012. 葡萄赤霉素合成相关基因克隆、亚细胞定位和表达分析. 中国农业科学,45 (11):2224-2231.
(Wang X C, Ren G H, Fang J G, et al. 2012. Cloning, subcellular localization and expression analysis of genes related to the synthesis of gibberellin from grapevine. Scientia Agricultura Sinica, 45 (11):2224-2231.[in Chinese])
吴国良,刘群龙,郑先波,等. 2009. 核桃种质资源研究进展. 果树学报, 26 (4):539-545.
(Wu G L, Liu Q L, Zheng X B, et al. 2009. Advances in research on the worldwide walnut germplsam. Journal of Fruit Science, 26 (4):539-545.[in Chinese])
邢浩然,刘丽娟,刘国振. 2006. 植物蛋白质的亚细胞定位研究进展. 华北农学报,21 (s2):1-6.
(Xing H R, Liu L J, Liu G Z. 2006. Advancement of protein sub-cellular localization in plants. Acta Agriculturae Boreali-Sinica, 21 (s2):1-6.[in Chinese])
Achard P, Genschik P. 2009. Releasing the brakes of plant growth:how GAs shutdown DELLA proteins. Journal of Experimental Botany, 60(4):1085-1092.
Chen Q, Ya H M, Li S, et al. 2012. Isolation and analysis of homoeologous genes encoding gibberellin 2-oxidase 3 isozymes in common wheat. Journal of Genetics, 91(3):1-9.
Chiu W, Niwa Y, Zeng W, et al. 1996. Engineered GFP as a vital reporter in plants. Current Biology, 6(3):325-330.
Dong H L, Lee I C, Kim K J, et al. 2014. Expression of gibberellin 2-oxidase 4 from Arabidopsis under the control of a senescence-associated promoter results in a dominant semi-dwarf plant with normal flowering. Journal of Plant Biology, 57(2):106-116.
Gargul J M, Mibus H, Serek M. 2013. Constitutive overexpression of Nicotiana GA2ox leads to compact phenotypes and delayed flowering in Kalanchoe blossfeldiana and Petunia hybrida. Plant Cell, Tissue and Organ Culture, 115:407-418.
Guo Y, Li W, Sun H, et al. 2012. Detection and quantification of Rhizoctonia cerealis in soil using real-time PCR. Journal of General Plant Pathology, 78(4):247-254.
Han F, Zhu B. 2011. Evolutionary analysis of three gibberellin oxidase genes in rice, Arabidopsis, and soybean. Gene, 473(1):23-35.
Hedden P, Phillips A L. 2003. Gibberellin metabolism:new insights revealed by the genes. Trends in Plant Science, 5:523-530.
Hood E E, Gelvin S B, Melchers L S, et al. 1993. New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Research, 2(4):208-218.
Lee D H, Lee I C, Kim K J, et al. 2014. Expression of gibberellin 2-oxidase 4 from Arabidopsis under the control of a senescence-associated promoter results in a dominant semi-dwarf plant with normal flowering. Journal of Plant Biology, 57(2):106-116.
Leslie C A, Hackett W P, McGranahan G H, et al. 2010. Improved rooting methods for walnut (Juglans) microshoots. Acta Horticulturae, 861:365-372.
Martin D N, Proebsting W M, Hedden P. 1999. The SLENDER gene of pea encodes a gibberellin 2-oxidase. Plant Physiology, 121(3):775-781.
Nelson B K, Cai X, Nebenführ A. 2007. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. The Plant Journal, 51:1126-1136.
Pearce S, Huttly A K, Prosser I M, et al. 2015. Heterologous expression and transcript analysis of gibberellin biosynthetic genes of grasses reveals novel functionality in the GA3ox family. BMC Plant Biology, 15(1):1-30.
Phinney B O, West C A, Ritzel M, et al. 1957. Evidence for "gibberellin-like" substances from flowering plants. PNAS, 43:398-404.
Ross J J, Reid J B, Swain S M, et al. 1995. Genetic regulation of gibberellin deactivation in Pisum. The Plant Journal, 7:513-523.
Saito T, Bai S L, Ito A, et al. 2013. Expression and genomic structure of the dormancy-associated MADS box genes MADS13 in Japanese pears (Pyrus pyrifolia Nakai) that differ in their chilling requirement for endodormancy release. Tree Physiology, 33(6):654-667.
Sambrook J, Fritsch E F, Maniatis T. 1995. Molecular cloning:a laboratory manual. Beijing:Science Press, 96-99.
Schomburg F M, Bizzell C M, Lee D J, et al. 2003. Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants. The Plant Cell, 15:151-163.
Shan C, Mei Z, Duan J, et al. 2014. OsGA2ox5, a gibberellin metabolism enzyme, is involved in plant growth, the root gravity response and salt stress. PLoS One, 9(1):e87110.
Sharkawy I E, Kayal W E, Prasath D, et al. 2012. Identification and genetic characterization of a gibberellin 2-oxidase gene that controls tree stature and reproductive growth in plum. Journal of Experimental Botany, 63(3):1225-1239.
Tulecke W, Mcgranahan G. 1985. Somatic embryogenesis and plant regeneration from cotyledons of walnut, Juglans regia L. Plant Science, 40(1):57-63.
Vahdati K, Razaee R, Mirmasoomi M. 2009. Micropropagation of some dwarf and early mature walnut genotypes. Biotechnology, 8(1):171-175.
Xiao Z, Fu R, Li J, et al. 2016. Overexpression of the gibberellin 2-oxidase gene from Camellia lipoensis induces dwarfism and smaller flowers in Nicotiana tabacum. Plant Molecular Biology Reporter, 34(1):182-191.
Yamaguchi M, Sasaki T, Sivaguru M, et al. 2005. Evidence for the plasma membrane localization of Al-activated malate transporter (ALMT1). Plant and Cell Physiology, 46(5):812-816.
Yamaguchi S. 2008. Gibberelllin metabolism and its regulation. Annual Review of Plant Biology, 59(1):225-251.
Yamaji N, Huang C F, Nagao S, et al. 2009. A zinc finger transcription factor ART1 regulates multiple genes implicated in aluminum tolerance in rice. The Plant Cell, 21:3339-3349.
Zhang L, Li Y, Dai K, et al. 2015. Establishment of a successive markerless mutation system in Haemophilus parasuis through natural transformation. PLoS One, 10(1371):1-12.
Zhou B, Lin J, Peng W, et al. 2012. Dwarfism in Brassica napus L. induced by the over-expression of a gibberellin 2-oxidase gene from Arabidopsis thaliana. Molecular Breeding, 29(1):115-127.

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Cloning, Subcellular Localization and Function Verification of Gibberellin 2-Oxidase Gene in Walnut (Juglans regia)

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