Comparing the structural characteristics and expression of GA2ox gene in dwarf banana and its wild type

It is necessary to understand the molecular mechanism of banana dwarfing mutation in order to improve the high stem character of banana varieties and breed the new excellent dwarf lines. To elucidate the molecular-level regulation of banana dwarf mutations and identify the relevant genes, the complete cDNA sequence of the gibberellin 2-oxidase gene (GA2ox) in the dwarf banana and its wild type was cloned by RT-PCR and the encoded amino acid sequences bioinformatically was analyzed. Meanwhile, the expression levels of banana GA2ox gene in different tissues of dwarfed banana and its wild type were analyzed by qRT-PCR. Between the GA2ox gene sequences of the dwarf banana and its wildtype, there were 5 base pairs of variation, leading to the predicted GA2ox proteins having different molecular weights, isoelectric points, fat indices, total numbers of positive and negative charges, and hydrophilicity. In addition, the expression of GA2ox in the leaves, stalks, and fruits of dwarf banana plants was higher than that in the wild type in the early and middle stages of banana development. The results showed that the GA2ox gene may have important regulatory effects on banana stem dwarfing, the knowledge of which can help our understanding and manipulation of this important plant.


Introduction
Banana (Musa spp.) is one of the most important fruit trees in tropical and subtropical regions. Bananas are loved by consumers not only because of their pleasant taste, but plant height of dwarf banana mutations is generally 1.6-1.8 m high. In the seedling stage of dwarf banana mutants, the content of GA1 and GA3 in the pseudostem was significantly lower than that of wildtype parents, and the plant could be restored to wildtype height by applying exogenous GA3 and IAA (Chen et al. 2016). However, there has not been deep understanding or elucidation of the genes which play a major role in banana stem dwarfing, or their molecular mechanisms. This study aimed to clone and compare the full-length cDNA of the GA2ox of dwarf mutant bananas and the wild type, as well as analyze and compare their expression levels in different plant organs, to help reveal the molecular mechanism of banana dwarf mutation and lay the foundation for selecting superior dwarf banana strains.

Plant materials
Williams B6 dwarf mutants and their wildtype were used in the present study. From February 2020 to January 2021, the experiment was carried out in the Key Laboratory of Cultivation and Breeding of Agricultural and Forestry Crops at the Agricultural College of Guangxi University. In the early and mid-term growth stages of the Williams B6 dwarf mutant and its wild type, samples were taken from leaves and pseudostems at the 10th, 15th, 20th, and 25th leaf stages. Fruit finger samples from the 1st, 2nd, 3rd, 5th, 7th, and, 8th fruits were collected at the flowering stage, frozen in liquid nitrogen, and stored at − 80 °C.

Methods of gene cloning
The P1 5ʹ-ATGGTGGTCTTGACCAAACC-3ʹ and P2 5ʹ-CTACTTCTCGAACTGCC-3ʹ pair of primers were designed according to the open reading frame of the GA2ox gene (XM_009382221.2). Total RNA of banana leaves was extracted with RNAprep Pure Plant Plus Kit (Tian, Beijing, China). The cDNA was synthesized by M-MuLV First Strand cDNA Synthesis Kit (Sangon Biotech, Shanghai, China). The full-length cDNA sequences of the GA2ox gene in the banana dwarf mutant and its wild type were cloned using High-fidelity Taq Polymerase (Sangon Biotech, Shanghai, China) for RT-PCR amplification.

Analysis of gene expression
The fluorescent quantitative RT-PCR (qRT-PCR) primers PGAq1 5ʹ-GTCGGTGATTCCTTACAG-3ʹ and PGAq2 5ʹ-CGAGACTTGAGACCATTG-3ʹ were designed for gene expression analysis according to the specific fragments with biosynthesis and signal transduction, via phytohormones such as gibberellins (GAs), brassinolide (BR), and indole-3-acetic acid ( IAA) Zhao et al. 2017;Li et al. 2018). In most dwarf phenotypes, plant height is closely related to the content and activity of endogenous gibberellin and the expression of related genes in the biosynthetic pathway . There are many key enzyme genes in the process of GA biosynthesis and metabolism, such as the Copay diophosphate synthase gene (CPS), inner root-kaurene synthase gene (KS), inner rootkaurene oxidase gene (KO), GA20 oxidation enzyme gene (GA20ox), GA3 oxidation enzyme gene (GA3ox), and GA2 oxidation enzyme gene (GA2ox) (Yamaguchi 2008). Among them, the geneencoded products CPS, KS, and KO, which are encoded by the single genes GA1, GA2, and GA3, respectively, play an important role in the early stage of GA biosynthesis. A severe dwarfing phenotype occurs when these gene sequences are mutated. GA2ox, GA3ox, and GA20ox are enzymes in the later stages of GA biosynthesis, which are encoded by multiple genes; when mutations occur in these gene sequences, that result in a semi-dwarf phenotype (Lo et al. 2008;Huang et al. 2010;Teplyakova et al. 2017;He et al. 2019). Studies have confirmed that a decrease in endogenous gibberellin content or activity in plants, and the mutation or expression of key enzyme genes in gibberellin biosynthesis and signal transduction, can lead to dwarfing phenotypes (Sasaki et al. 2002;Huang et al. 2010;Suo et al. 2012;Wei et al. 2012;Xu et al. 2016;Shen et al. 2019;Wang et al. 2019;Okada et al. 2020). The function of gibberellin 2-oxidase (GA2ox) is to convert the biologically active GA1 and GA4 into inactive GA8 and GA34, reducing the level of GA activity in plants and leading to the appearance of a dwarf phenotype (Sakamoto et al. 2001;Lo et al. 2008;Zhou and Underhill. 2016). For example, in barley, winter wheat, sesame, arabidopsis thaliana, and other plants, the elongation and growth of leaves and internodes is affected by decreased active gibberellin content or decreased expression levels of related genes in the gibberellin biosynthetic pathway, resulting in shorter plant heights (Barboza et al. 2013;Zhang et al. 2016;Teplyakova et al. 2017;Miao et al. 2020).
Studies have shown that GA2ox is encoded by multiple genes, and the expression patterns of different coding genes are different in different plants (Macmillan 1997;Sakamoto et al. 2001;Lo et al. 2008;Rieu et al. 2008;Wuddineh et al. 2015). The results of functional analysis also showed that overexpression of GA2ox in arabidopsis, rapeseed, tomato, potato, rice, and other crops could lead to the appearance of the dwarf phenotype (Sakamoto et al. 2001;Schomburg et al. 2003;Lee and Zeevaart 2005;Dijkstra et al. 2008;Lo et al. 2008;Huang et al. 2010;Zhao et al. 2010;Wuddineh et al. 2015;Hu et al. 2017Hu et al. , 2018Chen et al. 2019). The The length of both genes was 995 bp, and they were analysed and compared using DNAMAN software. It was found that they were 99.5% identical, with 5 base pairs that were different. The predicted amino acid sequence of the banana GA2ox gene is 270 aa. Through comparative analysis we found that there was 99.63% identity between the GA2ox-A and GA2ox-G sequences (Fig. 2).

Bioinformatics analysis for GA2ox-A and GA2ox-G in banana
The physical and chemical properties of the predicted proteins GA2ox-G and GA2ox-A were analyzed using the ExPASY software. The results showed that the molecular formulas of GA2ox-G and GA2ox-A are C 1331 H 2089 N 365 O 400 S 15 and C 1332 H 2091 N 365 O 400 S 14 , respectively. Their molecular weights were 30085.33 Da and 30067.30 Da, the theoretical isoelectric points were both 5.82, and the lipid indices were 79.81 and 81.26, respectively. They were both unstable proteins, and the instability indices (II) were 48.27 and 49.27, respectively. The total number of negatively charged residues and positively charged residues in GA2ox-G and GA2ox-A were 34 and 30, respectively, and the mean hydrophilic values were -0.246 and -0.239, respectively. In brief, there are certain differences in the physical and chemical properties of the protein encoded by the dwarf banana and its wildtype GA2ox gene. a length of approximately 250 base pairs (bp) selected from the full-length cDNA sequence of the banana GA2ox gene. The RNA was extracted from banana leaves, stems, and fruits at different stages, and used as templates for qRT-PCR with the banana actin gene (ACT1) as the internal reference gene. The amplification primers of the ACT1 gene are PA1 5ʹ-GCCATACAGTGCCAATCTACGAGG-3ʹ and PA2 5ʹ-ATGTCACGAACAATTTCCCGCTCA-3ʹ. The expression levels of banana GA2ox gene in different tissues of dwarfed banana and its wild type were measured using qRT-PCR. The fluorescent dye SYBR Green was used for labeling in the qRT-PCR reaction. Each reaction was repeated three times, and the 2 −ΔΔCT method was used to calculate the relative gene expression level.

Structural analysis of the full-length cDNA sequence of the GA2ox gene in dwarf banana and its wild type
The cDNAs prepared from dwarf banana and wildtype young leaves were used as templates for PCR amplification and the amplified products were detected using 1% agarose gel electrophoresis, and two specific bands of approximately 1000 bp were obtained that corresponded to the expected length of the GA2ox gene (Fig. 1). The amplified products were collected and sequenced, and the full-length CDS sequences of the dwarf banana and wildtype GA2ox genes were obtained, named GA2ox-A and GA2ox-G, respectively.

Comparative analysis of GA2ox homology between banana and other species
The amino acid sequences of GA2ox-A were compared with those of other plants. The results showed that the amino acid sequences of GA2ox-A had high similarity with Cocos nucifera, Phoenix dactylifera, Ingiber officinale and Elaeis guineensis, with 77.04%, 75.93%, 75.75% and 75.19%,respectively (Fig. 6).
Based on the homologous evolution analysis of amino acid sequences, GA2ox-A and GA2ox-G were combined with GA2ox of arabidopsis, rice and other plants to construct a phylogenetic tree. The results showed that GA2ox-A and GA2ox-G clustered Zingiber officinale ZoGA2ox, Phoenix dactylifera PdGA2ox, Cocos nucifera CnGA2ox, Elaeis guineensis EgGA2ox, Ananas comosus AcGA2ox, Tetracentron sinense TsGA2ox, Juglans regia JrGA2ox, Carya illinoinensis CiGA2ox, Arabidopsis thaliana AtGA2ox1, The secondary structure of the banana GA2ox protein was analyzed and predicted to have α-helix, β-turn, extended strand, and random coil in both GA2ox-A and GA2ox-G, among which the proportions of α-helix were 24.62% and 33.13%, the extended strand was 15.50% and 17.93%, the β-turn was 5.47% and 5.17%, and the random coil was 54.41% and 43.77%, respectively (Fig. 3). The prediction results of protein phosphorylation sites for GA2ox-A and GA2ox-G showed that all proteins contained threonine, tyrosine, and serine phosphorylation sites, of which there were six threonine phosphate sites and two tyrosine phosphate sites, 17 serine phosphate sites in GA2ox-A, and 19 serine phosphate sites in GA2ox-G (Fig. 4). The GA2ox-G and GA2ox-A proteins were located in the cytoplasm according to the PSORT II Prediction software. The transmembrane region value was zero according to TMHMM Server v.2.0 analysis. Therefore, the proportion of secondary structure of GA2ox-A in the dwarf banana was different from that of GA2ox-G in the wild type, and GA2ox-A had two fewer serine phosphorylation sites than GA2ox-G. Domain analysis of GA2ox-A and GA2ox-G proteins revealed that both have 3 domains, including the 2OG-FeII-oxygenase superfamily, PLN02156 superfamily and secondary metabolic synthesis-related PcbC. This has a typical GA2ox domain. Therefore, it is indicated that GA2ox-A and GA2ox-G are members of the GA2ox gene family (Fig. 5 ).

Comparative analysis of GA2ox gene expression in dwarf bananas and the wild type
The expression of the GA2ox gene in banana leaves, stems, and fruits was determined by qRT-PCR to compare the expression abundance of GA2ox in different tissues of AtGA2ox2 and AtGA2ox3, among which they clustered with ZoGA2ox, PdGA2ox, CnGA2ox and EgGA2ox are closely related (Fig. 7).

Fig. 8
Expression of GA2ox in dwarf banana and wild-type leaves. Uppercase letter represents a significant difference at the level of p<0.01 using least significant difference statistical analysis. Lower letter represents a significiant differnce at the level of p<0.05 using least significant differnce statistical analysis. Mean labeled by the same letter are not significantly different. The same below.  At the 15-and 20-leaf stages, expression levels of GA2ox-A were higher than in other periods in dwarf plants. The expression of GA2ox-A in the leaves of dwarf plants at the 15-leaf stage was 4.3 times higher than that of GA2ox-G in the wild type, and in the pseudostem of dwarf plants it was 11.6 times higher than in the wild type. At the 20-leaf stage, the expression of GA2ox-A in dwarf banana leaves was 4.7 times higher than that of GA2ox-G in wild type plants, and 28.9 times higher in stems (Figs. 8 and 9).
The pseudostem height and fruit finger length of dwarf banana are both shorter than those of the wild type, and the length of the fruit finger shortens with an increase in fruit dwarf banana and wild type. The results indicats that the expression level of GA2ox in the leaves and stems first increased and then decreased during the period from the 10th to the 25th leaf stage. GA2ox was expressed differently in the leaves and stems of dwarf plants and wild type plants at different leaf age stages. At the 10th leaf stage, the expression levels of GA2ox-A in the leaves of dwarf plants were lower than those of GA2ox-G in wild type plants, and the difference in expression abundance was not significant . However, the expression of GA2ox-A in dwarf plant leaves and stalks was significantly higher than that of GA2ox-G in wild type plants at the 15th, 20th, and 25th leaf ages.  (Helliwell et al. 1998), pear (Sasaki et al. 2002 and other plants that GA2 oxidase plays an important role in regulating plant stem dwarfing. The CDS length and expression pattern of GA2ox homologous genes in different plants were different, and the expression levels of GA2ox homologous genes were significantly different in dwarf mutants and wild types (Otani et al. 2013;Wuddineh et al. 2015;Hu et al. 2017;Hu et al. 2018;Chen et al. 2019;Hsieh et al. 2020). For example, in switchgrass, the expression patterns of GA2ox varied across different tissues; the dwarf and semi-dwarf phenotypes appeared in transgenic plants when GA2ox was overexpressed, and the tiller numbers and lignin content were reduced (Wuddineh et al. 2015). The expression of GA2ox in dwarf lychee was higher than in the normal-height plant (Hu et al. 2018). When the GA2ox gene of Jatropha curcas was transgenically transformed into Arabidopsis thaliana, the gene was overexpressed, leading to shorter plant height and smaller leaves, flowers, fruits, and seeds (Hu et al. 2017). When the AtGA2ox1 gene of Arabidopsis was transformed into maize, the GA1 content in the transgenic maize was reduced by 50-74%, and the leaf chlorophyll content and root crown were increased by about 2 times compared to the wild type . The present study showed that the expression pattern of GA2ox in dwarf banana and its wild type was similar to that of lychee, Arabidopsis thaliana and Jatropha, and that the expression level of GA2ox in the dwarf mutant was significantly higher than in the wild type. The difference in expression was significantly higher in the dwarf mutant than that in wild type. These differences could be explained in that there were multiple mutation sites in the GA2ox sequence of dwarf bananas, which led to the biochemical properties of the predicted protein it coded being altered. Therefore, we speculate that the phenotypes of dwarfed bananas might be at least partially caused by mutations in the sequence of GA2ox, which lead to changes in the function of its coding product GA2ox. Alternatively, these changes could be caused simply by the increase in its expression in the dwarf banana. The enzyme activity of GA2ox would be stimulated when the GA2ox expression is enhanced, and the content of endogenous GA1/GA4 in dwarf banana decreased, which might lead to the elongation of stem and other organs being slowed down during the development of dwarf bananas. The results of this study indicate that mutations and variations in expression of GA2ox in dwarf bananas may be a major factor that regulates the phenomenon of banana stem dwarfing. However, the function of GA2ox in banana should be confirmed by further research.

Declarations
Conflict of interest All authors declare that they have no conflict of interest. pointing position. The expression abundance of GA2ox was determined by qRT-PCR in the first, second, third, fifth, seventh, and eighth row fruit fingers in both dwarf and wild type plants at the fruit flowering stage, to elucidate the expression pattern of the GA2ox gene. Results revealed that the expression level of GA2ox in corresponding fruit fingers differed between dwarf and wild-type plants, but there was a common expression trend: the expression level of GA2ox in the seventh row of fruit fingers was the highest, followed by the fifth row fruit fingers, then the third row, then the eighth, and the lowest was in the first row of fruit fingers. Except in the third row, the relative expression levels of GA2ox-A in different fruit fingers of dwarf plants were all significantly higher than those of GA2ox-G in the wild type (Fig. 10). This indicates that the GA2ox gene may play an important regulatory role in stem dwarfing variation in banana, as the expression levels of GA2ox in the stem, leaf, and fruit of dwarf banana were significantly higher than in the wild type.

Discussion
Gibberellin 2-oxidase is the encoded product of the GA2ox gene in plants. It is a dioxygenase that relies on 2-ketoglutarate and oxygen molecules as cosubstrates and Fe 2+ and ascorbic acid as cofactors. The GA2ox gene belongs to the 20G-Fe (II) oxygenase gene superfamily (Helliwell et al.1998). In this paper, the protein domains of GA2ox-A and GA2ox-G were analyzed and found to include the conserved 20G-FeII-Oxy protein domain, 2-ketoglutarate binding domain and Fe 2+ binding site. This indicates that GA2ox-A and GA2ox-G have the common structural characteristics of GA2ox protein family and belong to GA2ox gene family.
The GA2-oxidase gene family is divided into three categories. The first and second categories use C19-GAs as catalytic substrates, such as arabidopsis AtGA2ox1-4, AtGA2ox6, rice OsGA2ox1-4, and OsGA2ox7-8 belong to the first and second categeries. The third category use C20-GAs as catalytic substrates, such as arabidopsis AtGA2ox7, AtGA2ox8, rice OsGA2ox5, OsGA2ox9, OsGA2ox6, OsGA2ox11 belong to the third class of enzymes. The optimal substrates for GA2-oxidase are mainly C19-GAs, which convert active GA1 and GA4 into inactive GA8 and GA34 (Lee and Zeevaart, 2005). Amino acid homology evolution analysis found that GA2ox-G and GA2ox-A cluster with arabidopsis AtGA2ox1, AtGA2ox2 and AtGA2ox3, and it is speculated that GA2ox-A uses C19-GAs as the reaction substrate, and directly catalytically active GAs as inactive substances, thus reducing the content of active GAs, inhibiting plant stalk elongation growth, and leading to plant dwarfing. At present, it has been identified in arabidopsis semi-dwarf phenotype in rice (Oryza sativa L. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/.