苹果赤霉素氧化酶基因MdGA2ox8的克隆及功能分析

doi:10.3864/j.issn.0578-1752.2018.22.012

摘要/Abstract

摘要：

【目的】从‘苏帅’苹果（Malus domestica）中分离克隆赤霉素氧化酶基因MdGA2ox8的开放阅读框（ORF）,分析其序列特征及在苹果中的组织表达特异性,通过在烟草中过表达MdGA2ox8研究其对烟草生长发育的影响,为该基因功能分析及应用提供理论参考。【方法】采用RT-PCR方法从苹果中克隆出MdGA2ox8的ORF区。利用NCBI、DNAMAN和Pfam在线软件进行氨基酸序列比对和保守结构域分析;利用Expasy在线软件对MdGA2ox8氨基酸组成、分子量、等电点进行分析;利用SignalP和TMHMM Server V.2.0分别在线分析蛋白质的信号肽和预测蛋白质的跨膜结构域;利用MEGA 7.0软件构建系统进化树。采用实时荧光定量PCR（RT-qPCR）方法检测MdGA2ox8在苹果不同组织中的表达量。构建MdGA2ox8过表达载体,采用农杆菌介导的遗传转化技术将MdGA2ox8导入烟草中,获得具有潮霉素抗性的转基因烟草植株,通过GUS染色、基因组PCR和RT-PCR鉴定转基因阳性植株;将野生型和转基因烟草移栽后,在第一朵花开放时测定转基因烟草的株高、节间长度和叶片长宽比以及烟草叶片叶绿素含量,激素GA₁和GA₄含量,并通过RT-qPCR方法分析烟草中相关基因的表达水平。【结果】成功克隆并获得了长为1 122 bp的MdGA2ox8 ORF区,编码373个氨基酸残基,蛋白相对分子质量为42.8 kD,理论等电点为5.44,不稳定系数为49.73,具有GA2ox的保守结构域DIOX_N和20G-Fell_Oxy,但无明显疏水区,无跨膜区,无信号肽,为非分泌蛋白。序列系统进化分析结果显示MdGA2ox8与白梨GA2ox8亲缘关系最近。RT-qPCR结果表明,MdGA2ox8在苹果各组织中差异表达,在花中的表达量最高,其次为老叶>幼叶>韧皮部,在木质部和幼果中几乎不表达。将MdGA2ox8转入模式植物烟草中,获得了5个转基因阳性株系。与野生型相比,过表达MdGA2ox8烟草植株GA₄含量降低,GA₁含量有所升高,其中野生型和转基因烟草GA₁平均含量分别为1.26和1.75 ng·g ^-1,GA₄平均含量分别为5.43和1.07 ng·g ^-1。与野生型相比,转基因烟草植株GA₁和GA₄总含量降低,使植株节间缩短、矮化以及花期推迟,其中野生型和转基因烟草植株平均高度分别为38.50和7.36 cm,节间长度分别为9.5和3.3 cm;转基因烟草叶片颜色深绿,叶片长宽比降低。烟草内源赤霉素合成途径相关基因NtGA3ox1和NtGA3ox2的表达水平受MdGA2ox8的正反馈调节。【结论】获得苹果赤霉素氧化酶基因MdGA2ox8的开放阅读框,MdGA2ox8具有组织表达差异性。MdGA2ox8过表达烟草植株中GA₁和GA₄总含量降低,导致植株节间长度缩短、植株矮化。

关键词: 苹果, 赤霉素氧化酶, 矮化, MdGA2ox8, 过表达, 烟草

Abstract:

【Objective】The objective of this study is to clone the open reading frame (ORF) of a gibberellin 2-oxidase gene (gibberellin2-oxidase 8, GA2ox8) from apple cultivar ‘Sushuai’, to analyze its sequence characteristics and tissue expression specificity. The effects of overexpression of MdGA2ox8 on tobacco growth and development were studied in order to provide a theoretical reference for the functional analysis and application of the gene.【Method】The ORF region of MdGA2ox8 was cloned from apple by RT-PCR method. The amino acid sequence alignment and conserved domain analysis were performed by NCBI, DNAMAN and Pfam online software. The composition, theoretical molecular weight and isoelectric point (pI) of the amino acid were deduced by Expasy software online. The protein signal peptide and transmembrane domain were analyzed by SignalP and TMHMM Server V.2.0. The phylogenetic tree was constructed by a neighbor-joining (NJ) method using MEGA 7.0 program. The expression level in different tissues of apple was detected by RT-qPCR. To characterize the function of MdGA2ox8, MdGA2ox8 ORF driven by the 35S promoter was delivered into tobacco by Agrobacterium-mediated transformation approach. Transgenic plants with hygromycin resistance were obtained and identified by using GUS staining, genomic PCR, and RT-PCR. After transplanting, the plant height, internode length, blade aspect ratio, content of chlorophyll, and content of GA₁, GA₄ in tobacco were measured at the first flowering stage. The expression level of related genes in tobacco was analyzed by RT-qPCR.【Result】The ORF sequence of MdGA2ox8 obtained from ‘Sushuai’ is 1 122 bp in length, encoding a putative protein about 373 amino acids. The predicted molecular weight of MdGA2ox8 is 42.8 kD, the theoretical pI is 5.44 and the instability coefficient is 49.73. MdGA2ox8 contains conserved domains DIOX_N and 20G-Fell_Oxy, without obvious hydrophobic region, transmembrane domain and signal peptide. The MdGA2ox8 protein is a non-secreted protein. Phylogenetic analysis showed that the MdGA2ox8 protein was closely related to Pyrus bretschneideri GA2ox8 protein. The RT-qPCR results showed that MdGA2ox8 was differentially expressed in apple tissues, with the highest expression in flowers, followed by old leaf>young leaf>phloem, but almost no expression in xylem and fruitlet. Five transgenic positive lines were obtained by transferring MdGA2ox8 into model plant tobacco. Compared with the wild-type, the GA₄content decreased and GA₁ content increased in overexpressed MdGA2ox8 tobacco plants. The average content of GA₁in wild-type and transgenic tobacco was 1.26 and 1.75 ng·g ^-1, respectively. The average content of GA₄ in wild-type and transgenic tobacco was 5.43 and 1.07 ng·g ^-1, respectively. Compared with the wild-type, the total content of GA₄ and GA₁in transgenic tobacco plants decreased, resulting in shorter internode length, dwarfing and delayed flowering. The average height of wild-type and transgenic tobacco plants was 38.50 and 7.36 cm, respectively. The average internode length of tobacco plants was 9.5 and 3.3 cm, respectively. The leaf of transgenic tobacco was dark green and the leaf aspect was reduced. Moreover, the expression level of endogenous gibberellin synthesis pathway-related genes NtGA3ox1 and NtGA3ox2 was positively regulated by MdGA2ox8 in transgenic tobacco.【Conclusion】The ORF of gibberellin 2-oxidase gene MdGA2ox8 was obtained. The difference of tissue expression in MdGA2ox8 was found. The total content of GA₁ and GA₄ in MdGA2ox8 overexpressed tobacco decreased, which resulted in the shortening of internode length and dwarfing of plants.

Key words: apple (Malus domestica), gibberellin oxidase, dwarf, MdGA2ox8, overexpression, tobacco

李飞鸿,侯应军,李雪涵,余心怡,渠慎春. 苹果赤霉素氧化酶基因MdGA2ox8的克隆及功能分析[J]. 中国农业科学, 2018, 51(22): 4339-4351.

LI FeiHong,HOU YingJun,LI XueHan,YU XinYi,QU ShenChun. Cloning and Function Analysis of Apple Gibberellin Oxidase Gene MdGA2ox8[J]. Scientia Agricultura Sinica, 2018, 51(22): 4339-4351.

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引物名 Primer name	引物序列Primer sequence (5′-3′)	产物Product
MdGA2ox8-F1	ATGTCAATGAAGATTCTA	开放阅读框 ORF
MdGA2ox8-R1	TTATACGACAAATCTAGG	开放阅读框 ORF
MdGA2ox8-F2	C/GAGCTC/ATGTCAATGAAGATTCTA	酶切位点SacⅠ/BamHⅠ Restriction site SacⅠ/BamHⅠ
MdGA2ox8-R2	CG/GGATCC/TTATACGACAAATCTAGG	酶切位点SacⅠ/BamHⅠ Restriction site SacⅠ/BamHⅠ
GA2ox8-RT-F	GAGGAGTGCATGACGGAGAT	特异扩增 Special amplification
GA2ox8-RT-R	ATGTGGCAGAAGGAGTTCCC	特异扩增 Special amplification
NtGA3ox1-RT-F	ATCGACCTCGACAACTACATCA	特异扩增 Special amplification
NtGA3ox1-RT-R	CAGGAGAACGAGCAGCCTTA	特异扩增 Special amplification
NtGA3ox2-RT-F	GTAGTGAACCGAACCCGACA	特异扩增 Special amplification
NtGA3ox2-RT-R	GCGCAAAGCTGAAAAGACGA	特异扩增 Special amplification
NtTubulin-F	AGATGTTCCGTCGTGTCAGTG	烟草内参基因 Tubulin in tobacco
NtTubulin-R	TGCTTCCTCTTCATCCTCATATCC	烟草内参基因 Tubulin in tobacco
MdTubulin-F	AGGATGCTACAGCCGATGAG	苹果内参基因 Tubulin in apple
MdTubulin-R	GCCGAAGAACTGACGAGAATC	苹果内参基因 Tubulin in apple