‘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

Abstract

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using the labeled GAI2 gene as the original sequence of high-throughput transcriptome sequencing of Nantongxiaofangshi (Unpublished).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

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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References 40

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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

Cloning and Function Analysis of Gibberellin Insensitive DkGAI2 Gene in Nantongxiaofangshi (Diospyros kaki Linn. cv. nantongxiaofangshi)

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