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

摘要/Abstract

摘要： [目的]GA2-oxidase （GA2ox）是赤霉素合成过程中起负调控作用的一种关键酶，能够催化有活性的赤霉素为无活性的赤霉素，从而对植物生长起到一定的抑制作用。本研究主要对核桃中赤霉素氧化酶基因JrGA2ox进行克隆及功能验证，有利于进一步探究核桃JrGA2ox基因在植物生长和发育过程中，尤其是在植物株高调控中的作用，从而助力于挖掘与利用核桃中更多的优质基因，培育出更多优良的核桃品种。[方法]采用PCR扩增技术，克隆获得核桃JrGA2ox基因的全长编码序列，进一步通过In-Fusion克隆技术构建具有强启动子的35S：：JrGA2ox：：GFP过表达载体；利用BLAST网络在线工具得到其他植物中的JrGA2ox同源氨基酸序列，并对其进行氨基酸同源序列比对和系统进化分析；其后，通过亚细胞定位揭示其发挥功能的场所，进一步利用农杆菌介导法将构建好的过表达载体转化到核桃体细胞胚中，获得JrGA2ox超表达的阳性转化植株，深入分析JrGA2ox基因的生物学特性。[结果]通过基因克隆，得到1条JrGA2ox开放阅读框，其全长为1 056 bp，共编码351个氨基酸，分子量为39.25 kDa。通过比对发现，该基因编码的蛋白序列含有保守2OG-FeⅡ-Oxy蛋白结构域，具有GA2-氧化酶蛋白家族共同的结构特点，表明JrGA2ox属于GA2-氧化酶基因家族。氨基酸进化树比对分析结果显示，核桃JrGA2ox与毛白杨PtGA2ox聚为一个分支。且JrGA2ox蛋白与木本植物川桑MnGA2ox1、西洋梨PcGA2ox、桃PpGA2ox1及苹果MdGA2ox1蛋白序列同源性较高。烟草叶片表皮细胞亚细胞定位分析表明，JrGA2ox是定位于细胞核与细胞膜中的蛋白。对核桃体细胞胚进行基因遗传转化后经荧光检测及PCR验证表明，35S：：JrGA2ox：：GFP过表达载体被成功转入核桃体细胞胚中。阳性再生植株株高与对照苗相比具显著性差异，其平均株高为对照植株的1/2；且其株高与JrGA2ox基因表达量呈负相关。[结论]核桃JrGA2ox蛋白亚细胞定位于细胞核与细胞膜中。JrGA2ox基因调控核桃株高，主要起到负调节作用。阳性基因转化再生植株中JrGA2ox基因的表达量升高，并表现出明显的矮化特征。本研究结果可为进一步分析该基因在核桃生长发育过程中的作用提供技术参考，且为优良的矮化核桃品种选育奠定一定的基础。

关键词: 核桃, GA2ox基因, 过表达, 亚细胞定位, 同源转化

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

中图分类号:

S718.46

张佳琦, 胡恒康, 徐川梅, 胡渊渊, 黄有军, 夏国华, 黄坚钦, 常英英, 叶磊, 娄和强, 张启香. 核桃JrGA2ox基因的克隆、亚细胞定位及功能验证[J]. 林业科学, 2019, 55(2): 50-60.

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.

参考文献

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.

[1]	周乃富, 宋晓波, 张俊佩, 常英英, 裴东. 核桃芽接愈合的组织学机制[J]. 林业科学, 2019, 55(6): 37-43.
[2]	黄仁, 张韵, 张启香, 王正加, 夏国华, 黄坚钦, 胡渊渊. 异源授粉山核桃果皮光合能力差异的转录组分析[J]. 林业科学, 2019, 55(1): 128-137.
[3]	曾楚楚, 娄钧翼, 郭明, 王冰璇, 卢闻君. 防治核桃干腐病的新型纳米生物基农药缓释胶囊制备及缓释性能[J]. 林业科学, 2018, 54(5): 87-100.
[4]	包文泉, 乌云塔娜, 杜红岩, 李铁柱, 刘慧敏, 王淋, 白玉娥. 基于SSR标记的西藏光核桃群体遗传多样性和遗传结构分析[J]. 林业科学, 2018, 54(2): 30-41.
[5]	谷战英, 杨若楠, 陈昊. 油桐叶肉细胞原生质体分离及瞬时转化体系的建立[J]. 林业科学, 2018, 54(1): 46-53.
[6]	李世亮, 倪张林, 莫润宏, 屈明华, 汤富彬, 刘毅华. 云贵川主产区核桃中重金属污染水平及其风险评估[J]. 林业科学, 2017, 53(11): 52-59.
[7]	徐川梅, 黄坚钦, 王正加, 夏国华, 张启香, 黄有军, 张守攻. 山核桃花粉母细胞减数分裂及核型[J]. 林业科学, 2017, 53(11): 77-84.
[8]	陈丹丹, 李天凤, 王琦琦, 唐光辉. 核桃举肢蛾成虫触角的超微结构观察[J]. 林业科学, 2017, 53(10): 118-132.
[9]	徐沁怡, 王标, 赵建文, 吴建峰, 曹亦润, 杨先有, 夏国华, 王正加, 黄坚钦, 胡渊渊. 2种花粉授粉山核桃果皮光合特性的差异比较[J]. 林业科学, 2017, 53(1): 38-46.
[10]	沈一凡, 钱进芳, 郑小平, 袁紫倩, 黄坚钦, 温国胜, 吴家森. 山核桃中心产区林地土壤肥力的时空变化特征[J]. 林业科学, 2016, 52(7): 1-12.
[11]	王娇莉, 南小宁, 任争争, 明洁, 唐光辉. 核桃举肢蛾和桃蛀螟幼虫肠道细菌多样性的PCR-DGGE和T-RFLP分析[J]. 林业科学, 2016, 52(6): 76-85.
[12]	张俊佩, 王滋, 周贤武, 赵荣军, 徐慧鸽, 贾志明, 裴东. 不同品系美国黑核桃木材物理力学性质的差异[J]. 林业科学, 2016, 52(6): 108-114.
[13]	徐永杰, 韩华柏, 王滑, 陈凌娜, 马庆国, 裴东. 大巴山区核桃实生居群的坚果表型和遗传多样性[J]. 林业科学, 2016, 52(5): 111-119.
[14]	胡珍珠, 潘存德, 潘鑫, 朱白雪. 基于光谱水分指数的核桃叶片含水量估算模型[J]. 林业科学, 2016, 52(12): 39-49.
[15]	相昆, 徐颖, 李国田, 王晓芳, 张美勇. 外源NO对低温胁迫下核桃幼苗活性氧代谢的影响[J]. 林业科学, 2016, 52(1): 143-149.