ФИЗИОЛОГИЯ РАСТЕНИЙ, 2013, том 60, № 1, с. 113-119
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
y%K 581.1
Cloning and Expression Analysis of a CMS-Related Gene BcCoil
from Brassica campestris ssp. chinensis1 © 2013 T. Liu*, **, ***, 2, C. Zhang***, 2, L. Qi***, F. Sun***, X. Hou******
*State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University,
1# Weigang, Nanjing, Jiangsu, P.R. China **Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture, Nanjing Agricultural University, 1# Weigang, Nanjing, Jiangsu, P.R. China ***College of Horticulture, Nanjing Agricultural University, 1# Weigang, Nanjing, Jiangsu, P. R. China
Received March 11, 2012
BcCoil, a cytoplasmic male sterility related gene, which was isolated from flower buds of Brassica campestris ssp. chinensis Makino using the RACE technology, was characterized and submitted to the NCBI GenBank (accession no. GU263836). The gene encodes a 67.78-kD protein containing 16 leucine-rich repeats and an N-terminal F-box motif and is extremely similar to Arabidopsis thaliana Coil gene. The Southern blot showed that BcCoil belongs to a multigene family. In A. thaliana, the Coil gene is involved in jasmonate signaling, and Coil mutant displayed male sterility. In this study, qPCR results demonstrated that BcCoil was accumulated in stamens and was significantly higher expressed in flower organs of the maintainer line than in the CMS one. At microsporocyte development stage, the gene was expressed at a significantly lower extent in the CMS line than in the maintainer line. This expression profile presumes that BcCoil plays a role in early microspore development in non-heading Chinese cabbage.
Keywords: Brassica campestris ssp. chinensis — cytoplasmic male sterility — BcCoil — gene expression
DOI: 10.7868/S001533031206022X
INTRODUCTION
Cytoplasmic male sterility (CMS), a maternally inherited inability to produce functional pollen, is common in higher plants. The CMS phenotype is manifested by coordinated expression of certain mitochondrial and nuclear genes. The mitochondrial genes are thought to negatively regulate nuclear genes that restore a fertility phenotype. CMS is an important approach to utilize the heterosis of Brassica. In addition to its commercial use, CMS provides a convenient model to dissect genetic interactions between the mitochondria and the nucleus in plants. For instance, many researchers have concluded that an aberrant chimeric gene in mitochondria possibly induces CMS in various plant species [1]. The Ogura-type CMS, which is derived from wild radish (Raphanus sativus)
1 This text is published in original.
2 These authors contributed equally to the work.
Abbreviations'. CMS - cytoplasmic male sterility; cDNA-AFLP -cDNA-amplified fragment length polymorphism; JA - jasmonate; LRR - leucine-rich repeat; ORF - open reading frame; RACE - rapid amplification of cDNA ends; qPCR - quantitative real-time PCR.
Corresponding author. Xilin Hou. College of Horticulture, Nanjing Agricultural University, 1# Weigang, Nanjing, 210095 Jiangsu, P.R. China. Fax. +86-25-8439-5262; e-mail. hxl@njau.edu.cn
[2], is one of the most analyzed CMS cytoplasms. It was reported that the mitochondrial gene orfl38 was involved in CMS expression [3].
Cabbage is a widespread vegetable crop belonging to the Brassicaceae family. Non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) originated in China is one of the most widely cultivated vegetables in China, especially in the south of China. The male sterility is considered to be an ideal tool for hybrid seed production in non-heading Chinese cabbage, as manual cross-pollination for the production of large quantities of hybrid seeds is difficult and time-consuming. Because of the obvious heterosis, non-heading Chinese cabbage is the ideal material to produce hybrids by the male sterile line. A new OguCMS germplasm, obtained by asymmetric cell fusion technology [4], has a good prospect in the production and application.
Jasmonates (JAs) are cyclopentanone derivatives synthesized from linolenic acid via the octadecanoic pathway. They inhibit plant growth generally, but in addition they promote diverse processes, including fruit ripening, senescence, tuber formation, tendril coiling, pollen formation, and defense responses against insects and pathogens [5]. In Arabidopsis thaliana, JAs inhibit root elongation [6] and are re-
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quired for pollen development, anther dehiscence [7, 8], and defense against insects [9] and necrotrophic pathogens [10]. In Arabidopsis, a genetic screening has identified CORONATINEINSENSTIVE1 (COI1) as a key regulator in the jasmonate signaling pathway [7]. The COI1 gene encodes a 66-kD protein containing a leucine-rich repeat domain and an N-terminal F-box motif [11], which is characteristic of F-box proteins that form ubiquitin—ligase complexes for the ubiquit-ination of substrate proteins targeted for degradation. The coi1 mutant was first identified for its insensitivity to the bacterial toxin coronatine, but it also displays male sterility. The coi1 mutants are unresponsive to growth inhibition by MeJA, are male-sterile, fail to express JA-regulated genes, such as VSP [12], Thi2.1 [13], and plant defensin PDF1.2 [14], and are susceptible to insect herbivory and to pathogens [9, 10]. coi1-16 is a recently cloned allele that was isolated in a screen for failure to activate the VSPA promoter. coi1-16 exhibits fertility in a temperature-sensitive manner [15]. All JA mutants exhibit a similar characteristic pheno-type with reduced elongation of the filaments, delayed anther dehiscence, and reduced pollen viability resulting in male sterility. The male-sterile phenotype can be rendered fertile by application of JA or MeJA, except for the JA signal transduction mutant coi1 [11].
In our previous work, we exploited the cDNA-AFLP technique to determine differential gene expression in the buds of newly bred Ogura CMS and its maintainer line [16] and found a fragment significantly discerning these two plant lines. The putative cDNA fragment was isolated and found homologous of Arabidopsis Coi1. In order to understand the role of Coi1 gene in CMS of non-heading Chinese cabbage, the full length cDNA of BcCoi1 gene was isolated from flower buds using 3'- and 5'-RACE according to the reported sequences ofArabidopsis coi1 gene. The presence of BcCoi1 gene in non-heading Chinese cabbage genome was examined by the Southern blot. Expression of BcCoi1 gene was followed using real-time quantitative PCR (qPCR) to reveal the temporal and spatial expression patterns.
MATERIALS AND METHODS
Plant materials. The male-sterile plants (Brassica campestris ssp. chinensis Makino) were created by electrofusion of protoplasts from the hypocotyl of Ogura CMS line (91HQiu-100) and the cotyledon of its maintainer line (91HQiu-21). Briefly, the hypocotyl and cotyledon protoplasts were treated with X-ray and R-6G to induce nuclear inactivation and mitochondrial bluntness, respectively. Then two treated protoplasts were fused in a Cell electrofusion device ("BTX", United States). After regeneration, the F1 generation was obtained after pollinating with main-tainer pollen [4, 17].
New CMS line (98HQiu-45) and its maintainer (91HQiu-21) of non-heading Chinese cabbage were
grown on the Jiangpu Farm of the Nanjing Agricultural University. Flower buds from the maintainer line were obtained for BcCoi1 gene cloning and Southern blot analysis. Different tissues (pistils, stamens, petals, calyx, flower stalks, bolting stems, leaf, siliques, and roots) from the new CMS line and its maintainer line at the full flowering stage were collected for qPCR. Flower buds were isolated from male sterile CMS and fertile maintainer lines and were grouped according to four developmental stages: microsporocyte development stage (<0.5 mm in diameter), dyad stage (0.5-1.0 mm), tetrad stage (1.0-1.5 mm), and mature microspore stage (1.5-2.0 mm) according to [18]. At least two biological replicates were collected from each line.
Total RNA extraction. Total RNA was extracted using a Qiagen RNeasy Plant Mini Kit ("Qiagen", China) according to the manufacturer's instructions.
cDNA cloning and sequence analysis of BcCoil gene. RNA extracted from the flower buds was used for RT-PCR using the TaKaRa RNA PCR Kit (AMV) v. 3.0 ("TaKaRa", Japan). Conserved domains were identified by alignment of the amino acid sequences of several Coi1 genes searched from the NCBI GenBank. According to the conserved domains, two pairs of PCR primers for the fragments, two nested PCR primers for 3'-RACE and two nested PCR primers for 5'-RACE (table 1) were designed using the Primer Premier 5 program to obtain the full-length BcCoi1. The 3'-Full RACE Core Set v. 2.0 and 5'-Full RACE kits ("TaKaRa") were applied according to the manufacturer's instructions. The PCR product was puried, cloned into the pGEM-T vector ("Tiangen", China), and sequenced.
Homology analysis and database search were performed using the BLAST network service (http:// www.ncbi.nlm.nih.gov/). ORF finding was carried out using online server program ORF finder. Multiple sequence alignment and splicing site recognition were performed with the DNAman software package (v. 5.2.2). The molecular weight of deduced proteins of BcCoi1 was calculated using the Bioedit software package. The motif analysis was performed using the online server program MotifScan (http://myhits.isb-sib.ch/cgi-bin/motif_scan).
DNA gel blot analysis. Genomic DNA was isolated from non-heading Chinese cabbage flower buds by the CTAB method [19]. Two primers for BcCoi1 gel blot were designed (table 2), and PCR amplified DNA fragments of each gene with DIG-label were used as probes according to the DIG-High Prime DNA Labeling kit ("Roche", Germany). The genomic DNA (15 ^g) was digested with four restriction enzymes (EcoRI, EcoRV, Hind
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