‘南通小方柿’赤霉素不敏感基因DkGAI2的克隆与功能分析

doi:10.3864/j.issn.0578-1752.2019.19.012

摘要/Abstract

摘要：

【目的】 对DkGAI2的亚细胞定位及表达特性进行分析,并将DkGAI2转化烟草,分析转基因烟草的生理和形态指标,为GAI的深入研究提供理论基础。【方法】 以‘南通小方柿’高通量转录组测序结果中标注的GAI2为原始序列（未发表）,利用RT-PCR,3′ RACE和5′ RACE技术从‘南通小方柿’中克隆得到DkGAI2的序列全长。利用生物信息学分析其序列特征,采用实时荧光定量PCR（qRT-PCR）分析DkGAI2在‘大方柿’及‘南通小方柿’5个不同物候期的表达特性以及DkGAI2在‘南通小方柿’不同组织中的表达特性,构建瞬时表达载体pCAMBIA-GFP-1302-DkGAI2,并瞬时侵染烟草分析DkGAI2的亚细胞定位,通过构建DkGAI2植物双元表达载体,利用农杆菌介导法转化烟草,经过GUS染色、RT-PCR对转基因烟草株系进行鉴定;并将野生型和转基因烟草移栽,于第一朵花开放时测定转基因植株的株高、节间长度、叶片长宽比以及GA₁和GA₄含量。【结果】 从‘南通小方柿’中克隆得到了1 827 bp的DkGAI2,DkGAI2核苷酸序列与猕猴桃（KF588651.1）、光皮烨（MF149049.1）、砂梨（KX078214.1）、苹果（FJ535245.1）、葡萄（MG738718.1）的相似度达72%-80%,DkGAI2具有DELLA蛋白家族特有的DELLA结构域,属于DELLA蛋白基因家族。DkGAI2编码608个氨基酸,相对分子质量为66.5 kD,理论等电点为5.54,不稳定系数为50.41,无明显疏水区,无跨膜区,无信号肽。序列系统进化分析结果显示,DkGAI2与葡萄亲缘关系最近。qRT-PCR结果显示,DkGAI2在‘南通小方柿’5个不同物候期的表达量都高于‘大方柿’;DkGAI2在‘南通小方柿’不同组织中表现出组织表达特异性,其中,在老叶中表达量最高,其次为茎尖和幼叶,而在幼果中几乎不表达。pCAMBIA-GFP-1302-DkGAI2融合蛋白的绿色荧光信号位于细胞核中,表明DkGAI2编码蛋白位于细胞核。经过GUS染色和RT-PCR检测,共获得5个转DkGAI2烟草株系,转基因烟草叶片GA₁含量增加,GA₄含量降低,GA₁和GA₄总含量降低,且转基因烟草植株出现植株矮化、节间缩短、叶片长宽比降低及花期延迟的表型。【结论】 DkGAI2具有组织表达特异性,定位于细胞核,DkGAI2在‘南通小方柿’5个不同物候期的表达量均高于‘大方柿’。推测DkGAI2可能通过降低GA₄的含量进而引起植株矮化。

关键词: 南通小方柿, DkGAI2, 基因克隆, 亚细胞定位, 组织特异性表达

Abstract:

【Objective】 In this study, the full-length sequence of gibberellin insensitive gene (GAI2) was obtained from the leaf of Nantongxiaofangshi (Diospyros kaki Linn. cv. nantongxiaofangshi), named as DkGAI2. The subcellular localization and expression characteristics of DkGAI2 gene were analyzed. Then DkGAI2 gene was transformed into plant tobacco, and the morphological and physiological indicators of the transgenic tobacco plants were determined. Our study would provide a theoretical basis for the future research of GAI gene.【Method】 The full-length sequence of DkGAI2 gene was cloned from Nantongxiaofangshi by RT-PCR, 3’ RACE and 5’ RACE using the labeled GAI2 gene as the original sequence of high-throughput transcriptome sequencing of Nantongxiaofangshi (Unpublished). Sequence characteristics were analyzed by bioinformatics. The expression characteristics of DkGAI2 gene in five different phenological stages of Dafangshi and Nantongxiaofangshi, and the expression level of DkGAI2 gene in different tissues of Nantongxiaofangshi was detected by real-time quantitative PCR (qRT-PCR). The transient expression vector pCAMBIA-GFP-1302-DkGAI2 was constructed and was transiently infected with tobacco to analyze the subcellular localization of DkGAI2 gene. DkGAI2 gene driven by the 35S promoter was constructed and delivered into plant tobacco by Agrobacterium-mediated transformation approach. Transgenic plants were identified by using GUS staining and RT-PCR. After transplanting, the plant height, internode length, leaf aspect ratio and the content of GA₁and GA₄ in transgenic tobacco plants were measured at the first flowering stage.【Result】 1 827 bp of DkGAI2 was cloned from Nantongxiaofangshi, the nucleotide sequence of DkGAI2 gene shared 72%-80% in homology compared with kiwifruit (KF588651.1), light skin (MF149049.1), pear (KX078214.1), apple (FJ535245.1) and grape (MG738718.1). DkGAI2 gene containing DELLA and GRAS conserved special domain, belonging to DELLA protein gene family. DkGAI2 gene encoding a putative protein about 608 amino acids, the relative molecular mass of DkGAI2 gene was 66.5 KD, the theoretical isoelectric point was 5.54, and the instability coefficient iwas 50.41, without obvious hydrophobic region, without transmembrane domain and signal peptide. Phylogenetic analysis showed that the DkGAI2 gene had close relationship with grape. qRT-PCR result showed that the expression level of DkGAI2 gene in five different phenological stages of Nantongxiaofangshi was higher than that of Dafangshi. DkGAI2 gene showed tissue expression specificity in different tissues of Nantongxiaofangshi, which had the highest expression in old leaves, followed by young leaves and shoot tips, while it had the lowest expression in fruitlet. The green fluorescent signal of the pCAMBIA-GFP- 1302-DkGAI2 fusion protein was located in the nucleus, indicating that DkGAI2 gene localized in the nucleus. After GUS staining and RT-PCR detection, 5 transgenic tobacco lines of DkGAI2 gene were obtained. The GA₁ content of transgenic tobacco leaves were increased, the GA₄ content were decreased, the total content of GA₁ and GA₄were decreased, and the transgenic tobacco plants showed plant dwarfing phenotypes with shortened internodes, reduced leaf aspect ratio, and delayed flowering.【Conclusion】 DkGAI2 gene had tissue expression specificity; DkGAI2 gene localized in the nucleus, the expression level of DkGAI2 gene in Nantongxiaofangshi was higher than that of Dafangshi at the five different phenological periods. It was speculated that DkGAI2 may cause plant dwarfing by reducing GA₄content.

Key words: Nantongxiaofangshi, DkGAI2, gene cloning, subcellular localization, tissue specific expression

蒋梦婷,朱宁,龚洪泳,侯应军,余心怡,渠慎春. ‘南通小方柿’赤霉素不敏感基因DkGAI2的克隆与功能分析[J]. 中国农业科学, 2019, 52(19): 3417-3429.

JIANG MengTing,ZHU Ning,GONG HongYong,HOU YingJun,YU XinYi,QU ShenChun. Cloning and Function Analysis of Gibberellin Insensitive DkGAI2 Gene in Nantongxiaofangshi (Diospyros kaki Linn. cv. nantongxiaofangshi)[J]. Scientia Agricultura Sinica, 2019, 52(19): 3417-3429.

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https://www.chinaagrisci.com/CN/Y2019/V52/I19/3417

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引物名 Primer name	引物序列 Primer sequence (5′-3′)	用途 Usage
DkGAI2-F	CACAGCTCGTCCAATCTCCA	中间片段扩增 Intermediate fragment amplification
DkGAI2-R	CTTCATCTCCTTCACCGCCA	中间片段扩增 Intermediate fragment amplification
DkGAI2 3′ GSP-1	CTTCTCCGACCTCCTCCAA	3′端扩增 3′-end amplification
DkGAI2 3′ GSP-2	CTGGATGCGTCAATGCTGG	3′端扩增 3′-end amplification
DkGAI2 5′ GSP-1	CTTTGGCACTGGAGGAGGTCATG	5′端扩增 5′-end amplification
DkGAI2 5′ GSP-2	GGTGCTGCGGAGGAAGTGGGTTG	5′端扩增 5′-end amplification
DkGAI2-ORF-F	ATGAAGCGCGACCGCCGCA	ORF扩增 ORF amplification
DkGAI2-ORF-R	TCACCTCTGGTGGGTCTGGA	ORF扩增 ORF amplification
DkGAI2-YG-F	CGTCAATGCTGGATCTTC	qRT-PCR Quantitative RT-PCR
DkGAI2-YG-R	CTTCACCGCCAATAACAC	qRT-PCR Quantitative RT-PCR
NtTubulin-F	AGATGTTCCGTCGTGTCAGTG	烟草内参基因 Reference gene in tobacco
NtTubulin-R	TGCTTCCTCTTCATCCTCATATCC	烟草内参基因 Reference gene in tobacco
Dkactin-F	GCCATCATTAATTGGAATGGAAGC	柿内参基因 Reference gene in persimmon
Dkactin-R	GTGCCACAACCTTGATCTTCA	柿内参基因 Reference gene in persimmon

测定指标 Determination index	野生型 WT	株系2 L-2	株系3 L-3	株系11 L-11
GA₁含量 GA₁ content (ng?g^-1)	1.22±0.05	1.75±0.05	1.5±0.05	1.65±0.05
GA₄含量 GA₄ content (ng?g^-1)	4.66±0.05	1.6±0.05	1.15±0.05	1.2±0.05
GA₁+GA₄含量 GA₁+GA₄ content (ng?g^-1)	5.88±0.05	3.35±0.05	2.65±0.05	2.85±0.05
叶绿素含量Chlorophyll content (mg?g^-1)	0.78±0.05	1.18±0.05	1.12±0.05	1.3±0.05
株高 Plant height (cm)	31.5±0.05	7.7±0.05	10.2±0.05	9.1±0.05
节间长度 Internode length (cm)	17.5±0.05	4.5±0.05	7.2±0.05	6.5±0.05
叶片长宽比 Length/width ratio	2.6±0.05	1.7±0.05	2±0.05	1.4±0.05