научная статья по теме CLONING AND EXPRESSION OF GHTM6, A GENE THAT ENCODES A B-CLASS MADS-BOX PROTEIN IN GOSSYPIUM HIRSUTUM Биология

Текст научной статьи на тему «CLONING AND EXPRESSION OF GHTM6, A GENE THAT ENCODES A B-CLASS MADS-BOX PROTEIN IN GOSSYPIUM HIRSUTUM»

ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ

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Cloning and Expression of GhTM6, a Gene That Encodes a B-Class MADS-Box Protein in Gossypium hirsutum

© 2011 M. Wu***, S. L. Fan**, M. Z. Song**, C. Y. Pang**, J. H. Wei***, J. Liu***, J. W. Yu**, J. F. Zhang***, S. X. Yu**

* College of Agronomy, Northwest Sci-Tech University of Agriculture and Forestry, Yangling, P. R. China ** Cotton Research Institute of CAAS, Key Laboratory of Cotton Genetic Improvement, Ministry of Agriculture, Anyang, Henan, P. R. China *** Department of Plant and Environmental Sciences, New Mexico State University, Las Cruces, USA

Received August 25, 2010

A full-length cDNA designated GhTM6, which encodes an organ differentiation-related B-class MADS-box protein, was isolated from Upland cotton (Gossipium hirsutum) by screening a normalized full-length cDNA library and using a RT-PCR strategy. The translated sequence analysis indicated that the polypeptide contained MADS-box and K domains and had a classic TM6 motif, i.e., the paleoAP3 in the C-terminal region. The phylogenetic analysis showed that GhTM6is closest to CeTM6, MaTM6, BuTM6, and PhTM6. Quantitative RT-PCR analysis showed that the GhTM6 gene was expressed at high levels in all tissues examined, such as those from squares, flowers, petals, stamens, and carpels under normal growth conditions. GhTM6 was expressed at high levels before floral initiation and declined thereafter. Furthermore, six stamens were seen in the transgenic tobacco flower as compared to five stamens in a wild-type flower. The results indicated that GhTM6 did not exhibit the full B-function spectrum, because it is only involved in the determination of stamen organ identity. However, its function in cotton will need to be examined in transgenic cotton plants.

Keywords: Gossipium hirsutum — B-class — MADS-box — GhTM6

INTRODUCTION

Flower development in higher plants requires the induction of a developmental shift in the shoot apex, which signals the termination of vegetative growth programs and the formation of a new reproductive organ, the floral buds [1]. The sequential differentiation of the floral organ due to the induction of apical cells has only recently become the focus of molecular genetic studies. In particular, homeotic genes in the final stages of differentiation of the floral organs were successfully determined [2]. In angiosperms, differential activities of homeotic genes in different regions of a developing flower are responsible for the specification of organ identities in the flower [3]. Most angiosperm flowers, including those of cotton (Gossypium), are made up of four types of organs that are arranged in concentric whorls [4]. The specification of floral organ identity is explained by the ABC model, which describes how floral organ identities are specified by the combined function of three classes of homeotic genes called A, B, and C [5]. That is, A — alone yields sepals; A in combination with B — yields petals; B with C — yields stamens; and C — alone yields carpels [6]. Thus,

Corresponding author. Shu-Xun Yu. Cotton Research Institute of CAAS, Key Laboratory of Cotton Genetic Improvement, Ministry of Agriculture, Anyang, Henan, 455000, P. R.China. Fax. +86-372-256-2256; e-mail. yu@cricaas.com.cn

the expression of B-class genes, such as the Arabidop-sis PISTILLATA (PI) and APETALA3 (AP3), as well as the Antirrhinum DEFICIENS (DEF) and GLOBOSA (GLO), is required for petal and stamen initiation and development [7].

The B-class genes AP3 and PI were derived from a duplication of an ancestral gene approximately 260 million years ago, shortly after the divergence of extant gymnosperms and angiosperms [8]. A second duplication event occurred in the AP3 lineage before the split of basal and core eudicots [9] and resulted in two paralogous lineages termed euAP3 and TM6, the latter named after the first-identified representative, Tomato MADS-Box Gene 6 [10]. In petunia and tomato, the functions of euAP3 and TM6 are diversified by sub-functionalization, an evolutionary process that partitions the original gene function into the two parts. The function of TM6 have been recently studied in tomato and petunia, and its function as a B-class gene is mainly in the determination of stamen identity [11]. The tomato TM6 gene is expressed in petals, stamens, and carpels. However, it is not clear whether the TM6 gene participates in petal development, since its ectopic expression was not observed at the petaloid sepals in tomato caused by its growth at low temperature [12]. In petunia, the function and mode of action of the paleoAP3-type PhTM6 differ significantly from those

of the euAP3-type PhDEF. PhTM6is expressed at high levels in stamens and carpels, but at very low levels in petals. Moreover, PhTM6 can rescue the phdef mutation in stamen development but not in petal development. These results indicated that PhTM6 is involved in stamen development, but not petal development [13].

In this study, we describe the isolation and characterization of a TM6 homologue in cotton with a paleoAP3 lineage, designated as GhTM6. We also evaluated the function of class-B genes in Upland cotton by the ectopic expression of TM6 homologues in tobacco.

MATERIALS AND METHODS

Plant materials and growth conditions. Cotton (Gossypium hirsutum) plants CCRI36 (a short-season cotton cultivar with a whole growth period of 107 days) and TM-1 (the genetic standard in Upland cotton, which is a late-maturing genotype with a whole growth period of 132 days), and tobacco (Nicotiana tabacum cv. NC89) plants were grown under standard field conditions in Anyang, Henan province, China, during the summer of 2009. For the quantitative RT-PCR (qRT-PCR) analysis, shoot apices of CCRI36 were collected from the field-grown cotton plants, exposed to the natural day-length conditions for 10, 20, and 25 days from planting, immediately frozen in liquid nitrogen after tissue harvesting, and stored at —70°C until analysis. The same method was used to collect other tissue samples from CCRI36 during the flowering stage.

PCR cloning and cDNA library screening of MADS-box genes. We used the same cDNA library from Upland cotton that was described by Wu et al. [14]. Briefly, the cDNA libraries were constructed from the mRNA of mature flowers and floral meristems and used for screening for TM6 cDNA clones in cotton. GhTM6 cDNA clones were identified using PCR with primers 5' - GAGTTCATCAGCCCT-AATATC-3' and 5'- GGCGAAGAGCATGTAAGT-TA-3'. To amplify the full-length of GhTM6, PCR was further carried out using primers designed as: GhTM6-full-3L (5'-TTCAAGGGAAAAGAAAAT-GGGTCGT-3') and GhTM6-full-3R (5'-AGAAAA-GAAAGAGTCGGTAGCAAGA- 3'). PCR products were first checked using electrophoresis on 1.2% agarose gels in 1X TAE buffer, and then cloned into the pGEM-T Easy Vector ("Promega", United States). The determination of DNA sequences in the clones was performed on an ABI Prism 3700 Sequencer (Beijng Genomics Institute, China), and sequence analyses were carried out using LASERGENE sequence analysis software ("DNASTAR", United States).

Sequence comparative and phylogenetic analysis.

The full-length of amino acid alignment of 33 published MADS-box homologue genes and GhTM6 was performed using ClustalW. A phylogenetic tree was

obtained by the neighbor-joining method of the PHYLOGENY package and visualized using MEGA3.1. The protein sequences used in this study were retrieved from GenBank, and their accession numbers are listed below: GhTM6 (HM006911); ScTM6 (ABG20633.1); IaTM6 (ABF56132.1); CeTM6 (ABG20634.1); GdEF (CAA08802.1); VaTM6 (ACA47117.1); CpTM6 (ABQ51321.1); PtAP3 (AAO49713.1); TaAP3 (ABE11601.1); VvAP3 (ABN71371.1); LjAP3 (AAX13301.1); SmDEF (ABG20626.1); AtAP3 (AAT46098.1); PhTM6 (AAF73933.1); MaTM6 (ABG20631.1); SmTM6 (ABG20635.1); SpTM6 (ABG20636.1); BuTM6 (ABG20632.1); JaTM6 (ABG20630.1); HmAP3 (BAG68950.1); SvDEF (AAS45979.1); RcDEF (XP_002533305.1); AvAP3 (ABP01804.1); AnDEF (P23706.1); ZmPI (CAC33848.1); NtPI (CAA48142.1); PhPI (CAA49568.1); VvAP1 (AAT07447.1); AtAP1 (AAM28457.1); AmAP1 (CAA45228.1); AmAG (CAB42988.1); PhAG (BAB79434.1); and LeAG (AAM33099.1). For a bootstrap analysis, 1000 replications were conducted for each branch. CpTM6, IaTM6, GhTM6, PhTM6, VaTM6, MaTM6, ScTM6, SmTM6, SpTM6, BuTM6, CeTM6, and JaTM6 were used to align conserved regions.

Gene expression analysis. The total mRNA was isolated from mature floral buds (bracts, sepals, petals, stamens, and carpels), roots, stems, fibers, ovules, apices, squares, flowers, and leaves of the CCRI36 cultivar based on CTAB method [15]. Each organ type was sampled in duplicate and combined in RNA extraction. RNA samples were quality-checked by measuring A260/A280 ratios and confirmed using agarose gel electrophoresis. An oligo(dT) primer was used to synthesize the first-strand cDNA from 5 ^g of total RNA using the SuperScript III reverse transcriptase enzyme ("Invitrogen", United States) according to the manufacturer's instructions. A single reverse transcription (RT) reaction was performed for each RNA sample.

qRT-PCR assays were performed on a LightCy-cler®480 Real-time Cycler ("Roche Applied Science", Germany) using PCR Master Mix ("Applied Biosystems", United States) with SYBR® Green. Cycling conditions were as follows: one cycle at 95°C for 10 min, followed by 42 cycles of 95°C for 15 s, and 60°C for 1 min. A melting curve analysis was conducted for each reaction to confirm the specificity of each PCR in addition to RT-PCR products resolved on an agarose gel. Three replicate reactions for each cDNA-primer combination were performed for each sample in the same run. For each cDNA sample, 18S levels were also quantified in the same run in three replicate reactions and used as an internal control. All quantifications were performed based on five-point calibration curves. The transcription level of each gene was normalized according to the transcription level of the 18S gene as a co-variabl

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