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1、Cloning, expression analysis, and sequence characters of a MADs-box gene associated with floral development in pineapple guava (Feijoa sellowiana Berg.) Chen Shenglin1, 2 1. College of Forestry, Central South University of Forestry and Technology, Changsha city, 410004, Peoples Republic of China (Em
2、ail: ) Zhang Riqing1, He Gongxiu1, Yuan Deyi1 2. China Forestry Industry Association, Beijing, 100714, Peoples Republic of China (Email: ) AbstractIn the ABC model, MADs-box proteins play an important role in floral development. As one of the MADs-box proteins, PI behaves as B function organ-identit
3、y genes acting in the second and third whorls of the flower to control petal and stamen identity. To find genes involved in the floral transition of pineapple guava, we cloned the full length cDNA encoding a MADs-box protein in Coolidge pineapple guava, using RT-PCR and RACE (Rapid Amplification of
4、cDNA Ends) strategy. The MADs-box gene was denominated FsMADs-box and belonged to a PI homolog. It was specifically expressed in flower buds, petal, and stamen, but not in leaves, sepal, carpel, and fruits in different developmental stages. The full length FsMADs-box cDNA contained a complete open r
5、eading frame (ORF) of 624 bp encoding 208 amino acids. The FsMADs-box gene showed a conserved MADS motif, a less conserved K-box motif, and MIKC structure that was typical for MADs-box proteins from high eudicots. The molecular weight (Mw) and isoelectric point (pI) of FsMADs-box were predicted to b
6、e 24.3 kDa and 8.83, respectively. In comparison with other PI homologs at the deduced amino acid level, the FsMADs-box showed the highest similarity (92%) with MqMADS-box of M. quinquenervia, and the lowest (65%) with HmPI of H. macrophylla. The present study provided the basis for further dissecti
7、on of the FsMADs-box gene function in governing the transition from the juvenile to the adult stage. Keywords-pineapple guava; floral development; MADs-box; PI homolog; RT-PCR; Rapid amplification of cDNA end I. INTRODUCTION Flowers of most flowering plants are made up of four types of organs that a
8、re arranged in concentric whorls1. To date, it has been known that a series of specific genes working together are responsible for the development of these whorls. Based on the widely accepted ABC model of floral organ development, expression of MADs-box genes may play a key role in specifying the i
9、dentities of floral organs. As one type of the MADs-box genes, the B-class gene, such as the Arabidopsis PISTILLATA (PI) and APETALA3 (AP3) and the Antirrhinum DEFICIENS (DEF) and GLOBOSA (GLO), is required for petal and stamen initiation and development2-4. The AP3 and PI proteins interact together
10、 to produce a heterodimer stabilizing both B-class proteins5, 6. The B-class heterodimer is necessary, not only for petal and stamen development, but may also play a role in establishing sex determination in both monoecious and dioecious flowers7. Pineapple guava (Feijoa sellowiana Berg.), a great c
11、ommercial plant for its fruit quality and unique flavor, has been introduced into China in recent years. Like many other woody plants, the pineapple guava has a long juvenile phase, which means that it will take a long time in breeding programs for favorable traits related to fruit production and fr
12、uit quality. It has been found that some genes govern the transition from the juvenile to the adult stage during which reproductive growth is initiated. Knowledge of these genes may facilitate selection of plants with shortened juvenile phases, and may enable interference in the processes governing
13、this transition. In the present study, we have cloned a full length cDNA encoding the MADs-box protein from pineapple guava, investigated its tissue expression specificity and performed a detailed characterization of sequence structural features. II. MATERIALS AND METHODS A. Plant materials Experime
14、nt materials consisted of leaves, floral organs including flower buds, sepal, petal, stamen and carpel, and fruits in different developmental stages of Coolidge pineapple guava. All of the plant materials were collected in test site of Central South University of Forestry and Technology, Changsha Ci
15、ty, Hunan Province. B. RT (Reverse transcription)-PCR Total RNA was isolated using Micro-to-Midi Total RNA Purification System (Invitrogen) according to the manufactures instructions. The quality of RNA was determined spectrophotometrically, and by agarose gel Author for correspondence. This work wa
16、s sponsored by “948 Project” of China State ForestryAdministration (2008-4-06). 978-1-4244-4713-8/10/$25.00 2010 Crownelectrophoresis. Purity was estimated from the absorbency ratios A260/A230 and A260/A280, a measure of contamination by the polyphenols/carbohydrates and proteins, respectively. To c
17、lone partial MADs-box sequences, RT (reverse transcription)-PCR was carried out. First-strand cDNA was synthesized by RT from total RNA using the First Strand cDNA Synthesis Kit (Fermentas). The RT was performed in a total volume of 20 L containing 2 L 10RNA PCR Buffer, 4 L MgCl2 (25mM), 2 L dNTP Mi
18、xture (dATP, dCTP, dGTP and dTTP, each at 10 mM), 1L M-MuLV Reverse Transcriptase (5 U/L), 0.5 L RNase Inhibitor (40 U/L), 1 L Oligo(dT)18 primer (2.5 M), 2 L RNA (around 1 g), and 7.5 L RNase-free water. The analysis was programmed in a thermal cycler(PE-9600, USA) and conducted under condition of
19、10 min at 30 followed by 25 min at 50 and 5 min at 95 to stop the reaction. The second-step PCR amplification was performed using two newly designed primers FsMADSPF1 (Forward primer, 5- CGGAATTCATGGGNMGNGGNAARRT-3) and FsMADSPR1 (Reverse primer, 5- CGCTCGAGGATCCGGYTGNATNGGYTGNAC-3). The primers wer
20、e designed based on multiple amino acid sequences alignment of other MADs-box genes from Melaleuca quinquenervia (EU004206), Lotus corniculatus (AY770398), Betula pendula (AJ488589), Carica papaya (EF562500), Cucumis sativus (AF043255), Gossypium hirsutum (FJ409869), Eucalyptus grandis (AF029976), P
21、opulus deltoides (EU029172), Malus domestica (AJ291490), Vitis vinifera (DQ988043), Hydrangea macrophylla (AB454441). For expression examination of the putative MADs-box gene in different tissues of Coolidge pineapple guava, RT-PCR was conducted with specific primers FsMADSPF2 (5-CTCAAGTGTCTGTGATCAT
22、CTT-3)/FsMADSPR2 (5-GCTGGATTGGTTGGACACGGTA-3) that spanned the region of 484 bp in the MADs-box gene. C. RACE (Rapid Amplification of cDNA Ends) To clone the full length cDNA of the MADs-box gene, RACE strategy was adopted based on known partial sequences acquired by RT-PCR. The full length cDNA seq
23、uences were amplified with 3 RACE System for Rapid Amplification of cDNA Ends (Invitrogen, Version E). Total RNA was reverse transcribed using SuperScript II Reverse Transcriptase with Adapter Primer provided in the system under condition of 10 min at 70, followed by 50 min at 42, 15 min at 70 and 2
24、0 min 37 to stop the reaction. The second PCR amplification was conducted with primers FsMADS3F and AUAP. FsMADS3F (5- TTGAGAACGGCCTCGCATGT-3) was designed on the basis of the partial sequences for the putative FsMADs-box gene, and AUAP was provided in the3 RACE System. The amplification program con
25、sisted of 35 cycles of 30 s at 94, 30 s at 50, 3 min at 72, and a final extension of 7 min at 72. D. Bioinformatics analysis Target RT-PCR and RACE products were cloned and sequenced. Sequence data were analyzed with software Vector NTI. Proteotics analysis was conducted by using online ExPASy Prote
26、omics Server (http:/expasy.org/). Multiple amino acid sequence alignment was performed with the method of ClustalW by AlignX program (Vector NTI) and percentage differences were computed. III. RESULTS A. Amplification of partial sequence for F. sellowiana MADs-box Using Total RNA Purification System
27、 (Invitrogen), the total RNA was successfully isolated from flower buds of Coolidge pineapple guava. The RNA sample had a ratio OD 260/280 of 1.9037, suggesting that the RNA was not contaminated by DNA. The RNA quality was verified by visualization on the agarose gel, which showed no degradation and
28、 was of high quality, allowing us to use it in the next RT-PCR amplification. The two newly designed primers FsMADSPF1 and FsMADSPR1 were applied in RT-PCR, and were found to be successful. They efficiently amplified two different sized fragments that included one of around 630 bp and the other of a
29、round 450 bp. Taking into consideration the position of the primers FsMADSPF1 and FsMADSPR1 in the MADs-box genes, only the 630 bp-sized fragment were more likely to represent a putative MADs-box gene in Coolidge pineapple guava. After cloning and sequencing the two amplification fragments, two diff
30、erent nucleotide sequences were determined. When the 450 bp nucleotide sequences were used as the query by Blastn, no significant similar sequence was found in GenBank database. In contrast, several MADs-box genes were found in a Blast search of GenBank using the 630 bp sequence as the query. Refere
31、ncing the denomination of published MADs-box genes in other species, the 630 bp amplification fragment was designated FsMADs-box (F. sellowiana MADs-box). B. Full length cDNA cloning and sequence characters of FsMADs-box After 3 RACE-PCR, one fragment of approximately 550 bp was produced. As the FsM
32、ADSPF1 primer spanned the putative initiation codon ATG of MADs-box genes, the full length FsMADs-box cDNA sequence was determined by overlapping the RT-PCR and the 3 RACE-PCR sequences. The full length FsMADs-box cDNA was 891 bp long and contained a complete open reading frame (ORF) of 624 bp encod
33、ing 208 amino acids. The putative initiation codon ATG was at positions 7-9. The termination codon TGA was present at positions 631-633, followed by 3 untranslated region, which contained the consensus polyadenylation signal AATAAA at positions 741-746. The FsMADs-box gene showed the conserved MADS
34、motif from amino 1 to 60, a less conserved K-box motif from amino acid 76 to 164. It also displayed the MIKC structure that was typical for MADS-box proteins from high eudicots. In the K motif, moreover, the sequence (KHENL) of the FsMADS-box matched well with the highly conserved sequence KHExL in
35、PI homologs. In the C terminal region of the FsMADS-box, the sequence AYRVQPIQPNLQQ shared higher identity to PI motif MPFxFRVQPxQPNLQE conserved in the PI family, than to PI-derived motif FxFRLQPSQPNLH (Fig. 1). Fig. 1 Alignment of the deduced amino acid sequence of FsMADs-box with those of other r
36、elated MADs-box genes of plants. The MADs-box and K-box domains are underlined. The solid lined box indicates conserved motif of PI homologs and conserved motif of AP3 homologs. The molecular weight (Mw) and isoelectric point (pI) of FsMADs-box were predicted to be 24.3 kDa and 8.83, respectively. I
37、n comparison with other PI homologs at the deduced amino acid level, the FsMADs-box shared the highest similarity (92%) with MqMADS-box of M. quinquenervia, the lowest (65%) with HmPI of H. macrophylla. Fig. 2 Phylogenetic tree of plant MADs-box sequences using NJ method based on the multiple alignm
38、ent of the MADs-box amino acid sequences. The newly cloned FsMADs-box gene is boxed. A total of 23 amino acid sequences for MADs-box proteins were aligned by clustalx. Similarity analysis indicated that FsMADs-box exhibited the highest similarity (92%) with MqMADS-box gene, and the lowest (25%) with
39、 PatSEP1 gene. Based on the multiple amino acid sequence alignment, a neighbour-joining phylogenetic tree was generated. In accordance with similarity scores, FsMADs-box displayed the closest relatedness with MqMADS-box of M. quinquenervia on the phylogenetic tree (Fig. 2). The phylogenetic tree als
40、o showed that FsMADs-box belonged to the PI clade of B class. C. Tissue expression specificity of FsMADs-box To examine tissue expression specificity of FsMADs-box, total RNA was isolated from Coolidge pineapple guava leaves, flower buds, sepal, petal, stamen, carpel, fruits of 20 FS (fruit setting)
41、, 30 FS and 50 FS. RT-PCR revealed that a 500 bp fragment with expected size was generated only from the total RNA of flower buds, petal, and stamen, but not from leaves, sepal, carpel, and fruits in different developmental stages. The 500 bp-sized fragment was cloned and sequenced. The nucleotide s
42、equences of the amplification fragment well matched FsMADs-box gene, suggesting that the FsMADs-box gene was specifically expressed in specific floral organs. The expression pattern of FsMADs-box gene is consistent with that of peach PpMADs10 and Arabidopsis PI8, 9. IV. DISCUSSION It was reported th
43、at B-class genes are derived from a duplication of the ancestor of these genes approximately 260 million years ago, shortly after the divergence of extant gymnosperms and angiosperms10. A second duplication event occurred in the AP3 lineage before the split of basal eudicots and core eudicots11 and
44、produced two paralogous lineages named euAP3 and TM6, the latter named after the first identified representative, TM612. The PI lineage does not have major duplications, whereas several recent duplications were detected in genus or species level. The two AP3 sublineages differ by their typical motif
45、s in the C-terminal region caused by ancestral frameshift mutation13. The C-terminal motifs of the core eudicot TM6 sublineage are similar to those of the paleoAP3 lineage which are present in basal angiosperm, monocots, magnoliids, and basal eudicots. However, the C-terminal motifs of euAP3 subline
46、age differ from those of the paleoAP3 lineage and are found exclusively in core eudicots. Here, we successfully cloned a FsMADs-box gene associated with floral development by degenerate primers design and RACE-PCR from flower buds of Coolidge pineapple guava. It has exhibited the characteristic stru
47、ctural features, i.e. a conserved MADS motif, K-box motif, and MIKC structure. Blastp and phylogenetic analysis indicated that the FsMADs-box gene belonged to PI type, B-class of the MADs-box gene. RT-PCR analysis revealed that FsMADs-box gene was expressed in both the second and the third whorl in
48、specific floral organs such as flower buds, petal, and stamen, not in leaves, sepal, carpel, and fruits, which confirmed that this gene was a PI homolog. The FsMADs-box sequences cloned herein provide a molecular basis for governing the transition from the juvenile to the adult stage by genetic engi
49、neering-based method in woody plants. This transition may shorten juvenile phases, which is useful in breeding programs for favorable traits related to fruit production and fruit quality. And further study needs to be done on the function of the FsMADs-box gene. ACKNOWLEDGMENT The authors thank Prof
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