Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (22): 4339-4351.doi: 10.3864/j.issn.0578-1752.2018.22.012

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Function Analysis of Apple Gibberellin Oxidase Gene MdGA2ox8

LI FeiHong(),HOU YingJun,LI XueHan,YU XinYi,QU ShenChun()   

  1. College of Horticulture, Nanjing Agricultural University, Nanjing 210095
  • Received:2018-06-04 Accepted:2018-07-16 Online:2018-11-16 Published:2018-11-16

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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 GA4 content decreased and GA1 content increased in overexpressed MdGA2ox8 tobacco plants. The average content of GA1 in 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 GA1 in 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

Table 1

Primers used for cloning, expression and functional analysis of MdGA2ox8"

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
MdGA2ox8-F2 C/GAGCTC/ATGTCAATGAAGATTCTA 酶切位点SacⅠ/BamHⅠ
Restriction site SacⅠ/BamHⅠ
MdGA2ox8-R2 CG/GGATCC/TTATACGACAAATCTAGG
GA2ox8-RT-F GAGGAGTGCATGACGGAGAT 特异扩增
Special amplification
GA2ox8-RT-R ATGTGGCAGAAGGAGTTCCC
NtGA3ox1-RT-F ATCGACCTCGACAACTACATCA 特异扩增
Special amplification
NtGA3ox1-RT-R CAGGAGAACGAGCAGCCTTA
NtGA3ox2-RT-F GTAGTGAACCGAACCCGACA 特异扩增
Special amplification
NtGA3ox2-RT-R GCGCAAAGCTGAAAAGACGA
NtTubulin-F AGATGTTCCGTCGTGTCAGTG 烟草内参基因
Tubulin in tobacco
NtTubulin-R TGCTTCCTCTTCATCCTCATATCC
MdTubulin-F AGGATGCTACAGCCGATGAG 苹果内参基因
Tubulin in apple
MdTubulin-R GCCGAAGAACTGACGAGAATC

Fig. 1

Vector map of pCAMBIA1301-35SN-MdGA2ox8 (A) and agarose gel electrophoresis of MdGA2ox8 (B)"

Fig. 2

Alignment of amino acid sequences of MdGA2ox8 Red pane indicates the DIOX_N domain; black pane indicates the 2OG-FeII_Oxy domain"

Fig. 3

Phylogenetic tree of GA2ox8 from different plant species"

Fig. 4

Expression of MdGA2ox8 in different tissues of apple The different lowercases indicate significant difference (P<0.05). The same as below"

Fig. 5

GUS staining (A), PCR (B) and RT-PCR (C) amplification of transformation tobacco with MdGA2ox8"

Fig. 6

Determination of phenotype in transgenic tobacco plants"

Fig. 7

Effect of MdGA2ox8 overexpression on chlorophyll content in tobacco plants"

Fig. 8

Effect of MdGA2ox8 overexpression on gibberellin content in transgenic tobacco leaves"

Fig. 9

Detection of MdGA2ox8-overexpressed lines using RT-qPCR"

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