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Molecular cloning and expression analysis of a RanBP2 zinc finger protein gene in upland cotton (Gossypium hirsutum L.)
Colloids and Surfaces B: Biointerfaces 55 (2007) 153–158
Molecular cloning and expression analysis of a RanBP2 zinc finger protein gene in upland cotton (Gossypium hirsutum L.) Ping-An Chang ∗ , Biao Li ∗ , Xiao-Min Ni, Yong-Fang Xie, Ying-Fan Cai Key Laboratory of Molecular Biology, College of Bio-information, Chongqing University of Posts and Telecommunications, Chongqing 400065, PR China Received 13 September 2006; received in revised form 2 November 2006; accepted 29 November 2006 Available online 9 December 2006
Abstract Gossypol is an important resistant substance of Gossypium, and its storage organ is pigment gland. Although, the relationship between gossypol and pigment gland has been studied for a long time, the development mechanism of pigment gland has not been revealed up to now in molecular perspective. On the basis of differentially expressed cDNAs fragments at the stage of the cotton gland development using suppression subtractive hybridization (SSH), the complete cDNA sequence of a novel RanBP2 zinc finger protein (ZFP) gene was cloned by rapid amplification of cDNA ends (RACE) from upland cotton (Gossypium hirsutum L.), Xiangmian 18. The cotton RanBP2 ZFP cDNA (GenBank accession number: BQ173926) is 717 base pair (bp) long with an open reading frame encoding 139 amino acids, which encodes a 15.6 kDa protein. The cotton RanBP2 ZFP had three Ran-binding protein (RanBP) two zinc finger motifs and belonged to RanBP2 ZFP family. There is a 292-base non-coding sequence at 3 cDNA end, which includes polyA sequence. Sequence alignment analysis revealed that the cDNA nucleotide and its deduced amino acid sequence are moderately identical to the putative ZFP from other species. The mRNA expressing profiles of the novel ZFP gene was investigated by reverse transcription-polymerase chain reaction (RT-PCR). The result showed that it expressed at different development stages of gland, including the undeveloped stage, developing stage, developed stage and cotyledon stage. However, with the development of pigment gland, the mRNA levels in the gland-developed seed and cotyledon were increased to about 1.5 and 2 folds of that in gland-undeveloped seed, respectively, which suggested that the novel ZFP played a role in the development of the cotton gland. © 2006 Elsevier B.V. All rights reserved. Keywords: Upland cotton (Gossypium hirsutum L.); Zinc finger protein; Gene expression; Pigment gland
1. Introduction Gossypol, a polyphenolic binaphthyl dialdehyde stored in the pigment glands of cotton, is not only an important resistant substance for cotton but also an important phytochemical component of immense interest due to its several biological properties including anti-cancer, antimicrobial, anti-HIV, antioxidation and male contraceptive [1]. Gossypol content of cotton is mainly dependent on different genetic types of pigment glands [2]. The glanded cotton normally contains gossypol in both seeds and plants that is toxic to human and non-ruminant animals [3,4]. Ordinary glandless cotton contains no or low-gossypol in seeds, roots, stems as well as in leaves, but its resistance to diseases,
∗
Corresponding authors. Tel.: +86 23 62461884. E-mail addresses: [email protected] (P.-A. Chang), [email protected] (B. Li). 0927-7765/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2006.11.042
pests and even rats is reduced greatly [5,6]. Therefore, a cotton variety which is characterized by the presence of glanded roots, leaves and stems (to maintain the resistance trait) but glandless seeds (for safe utilization) is highly desired [7,8]. Biologists and agronomists have long been studying the glanded characters and the glandless characters of cotton as well as their gossypol content [9–13]. However, the relationship between gossypol and pigment gland was complicated and the molecular mechanism of pigment gland development has not been revealed up to now [7,8]. In order to screen the relative genes involving the development of cotton pigment gland, two differentially expressed cDNA libraries of forward and reverse subtraction were constructed using suppression subtractive hybridization (SSH) in the process of germinating of the seeds of Xiangmian 18, a cotton variety which is characterized by the presence of glanded roots, leaves and stems but glandless seeds [14]. The differentially expressed cDNA fragments between undeveloped gland
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and developing gland seeds were associated with regulation and control factors, apoptosis, protein synthesis, including a putative zinc finger protein (ZFP) cDNA fragment (GenBank accession number: AY162374) [14]. The ZFP cDNA fragment was not complete, but had the initial code, ATG, by alignment with ZFP genes from other species. For the reason that the zinc finger proteins belong to the largest family of regulatory transcription factors and play an important role in growth and development in animal and plant systems, the full length of a putative ZFP cDNA was first cloned using rapid amplification of cDNA ends (RACE), then the expressing profiles of the novel ZFP gene at the different gland development stage were investigated by reverse transcription-polymerase chain reaction (RT-PCR) to reveal the relation between development of cotton pigment gland and the expression of the ZFP gene in this paper.
2.3.2. Synthesis of the first-strand cDNA A total 10 l reaction volume was generated containing 4 l (5 g) total RNA, 0.5 l 20 mol/l AP, 1 l 10 mmol/l dNTP mixture and 4.5 l DEPC-treated water, then incubated at 65 ◦ C for 5 min and placed on ice for at least 1 min to denature probable RNA secondary structure. The first-strand cDNA synthesis mixture was prepared by adding following components to the above 10 l reaction volume in the indicated order, 2 l 10× RT-PCR buffer, 4 l 25 mmol/l MgCl2 , 2 l 0.1 mol/l DTT, 1 l RNaseOUT (40 U) and 1 l (200 U) SuperScriptTM III reverse transcriptase, and mixed well. The first-strand cDNA synthesis mixture was incubated at 50 ◦ C for 50 min and terminated at 85 ◦ C for 5 min. Lastly, 1 l (2 U) RNase H was added to the first-strand cDNA synthesis mixture, which was incubated at 37 ◦ C for 20 min to destruct RNA template.
2. Experimental
2.3.3. Amplification of the full-length cDNA The first PCR reaction volume is 50 l, which combines 5 l 10× RT-PCR buffer, 3 l 25 mmol/l MgCl2 , 1 l 10 mmol/l dNTP mixture, 2 l above synthesized cDNA template, 1 l 120 mol/l GSP1 and AP, 1 l Ex TaqTM DNA polymerase, and 36 l sterilized H2 O. Then the cDNA was firstly amplified as following procedure: 94 ◦ C for 5 min to pre-denature cDNA template; 40 amplification cycles of 94 ◦ C for 1 min, 55 ◦ C for 1 min, 72 ◦ C for 1 min; 72 ◦ C for 7 min to extend. Then the cDNA was secondary amplified as the first amplification process, except that 2 l first amplification product was template, UAP substituted for AP and denaturing temperature was 58 ◦ C.
2.1. Materials Cotton seed of Xiangmian 18 was obtained from National Research Center for Cross-Cotton of China (Changsha, China). Total plant RNA extraction kit was from Watson Biotechnologies, Inc. (Shanghai, China). SuperScript III first-strand synthesis system for RT-PCR was purchased from Invitrogen Life Technologies (Groningen, The Netherlands). pMD18-T vector, T4 DNA ligase, DNA gel extraction kit, TaKaRa Ex TaqTM PCR kit (Hot start version) and TaKaRa RNA PCR kit (AMV) Version 3.0 was purchased from Takara (Dalian, China). 2.2. Total RNA extraction Cotton seeds were disinfected in 70% ethanol and 15% H2 O2 solution, then dipped in sterilized water, and began to bud in the plates containing sterilized filter paper and water. The pigment gland begins to develop after 36-h disinfection and several pigment glands developed after 52 h, which was verified by microscopy. So the stage within 36 h after disinfection was considered to be gland-undeveloped stage, from 36 to 52 h as developing gland stage, after 52 h until cotyledon developed as developed gland stage [14]. Total RNA was isolated with total plant RNA extraction kit from Watson Biotechnologies according to the manufacturer’s instructions and was quantified photometrically. In addition, total RNA was electrophoresed on a denatured agarose gel to investigate its integrality. 2.3. Molecular cloning of cotton zinc finger protein gene 2.3.1. Primer design According to the sequence of cotton putative ZFP cDNA fragments, a gene specific primer, 5 -ATGAGCAGGCCAGGAGATTGG-3 was designed, named as GSP1. At the same time, adapter primer (AP), 5 -GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3 and universal amplification primer (UAP), 5 -GGCCACGCGTCGACTAGTAC-3 was also synthesized.
2.3.4. Molecular cloning of the full-length cDNA and sequencing The secondary amplification product was electrophoresed on a 1% agarose gel and the expected length band was extracted with DNA gel extraction kit. The extracted full-length cDNA was cloned into pMD18-T vector and the recombinant plasmid was certified by PCR. The inserted cDNA was forwardly and reversely sequenced with ABI 377 DNA sequencing system. 2.4. Sequence analysis of cotton zinc finger protein gene Primers and open reading frames finding of the novel cotton ZFP cDNA sequence was assisted by DNAman software. Protein sequence is deduced and analyzed by the same program. Blastn and Blastp program were used to find out the homologous sequences of cotton ZFP cDNA sequence and protein sequence in GenBank online (http://www.ncbi.nlm.nih.gov/blast), respectively. Protein motif scanning was carried out with MotifScan program online (http://myhits.isb-sib.ch/cgi-bin/motif scan). Multi-sequences alignment was accomplished by Clustalx software. 2.5. Expressing analysis of cotton ZFP gene Total RNA of different stages was isolated as described above and mRNA levels of different stages were investigated
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by TaKaRa RNA PCR kit (AMV) according to the manufacturer’s instructions. The forward primer was GSP1 and the reverse primer was 5 -TGGAGCACTGCATCTGAAACATTC3 , named GSP2, and was synthesized according the cotton ZFP cDNA sequence. The reverse transcription system is 10 l, including 3.75 l RNase free dH2 O, 1 l 10× RT buffer, 2 l 25 mM MgCl2 , 0.5 l dNTPs (10mM each), 0.5 l GSP2 (10 M), 1 l total RNA, 0.25 l RNase inhibitor (40 U/l), 1 l AMV reverse transcriptase (5 U/l). The reaction procedure was 50 ◦ C for 30 min, 99 ◦ C for 5 min, 4 ◦ C for 5 min. The PCR amplification reaction system was generated by adding 10 l 5× PCR buffer, 0.5 l GSP1 (10 M), 0.5 l Ex Taq Hs (5 U/l), 29 l dH2 O to the above reverse transcription system. The target cDNA was amplified as following procedure: 94 ◦ C for 3 min to pre-denature cDNA template; 35 amplification cycles of 94 ◦ C for 45 s, 56 ◦ C for 45 s, 72 ◦ C for 45 s; 72 ◦ C for 10 min to extend. The amplification products and total RNA were electrophoresed on a 1% agarose gel. The density ratio of target cDNA band and corresponding 18S rRNA band was carried out by Quantity One program (Bio-Rad). The experiment was duplicated for three times and data are presented as a percentage of mRNA level at undeveloped gland stage. Results were generally expressed mean ± standard error (ER) from values three independent tests. Groups of data were compared by one-way ANOVA. A difference between means was considered significant at a value of P < 0.05. 3. Results and discussion 3.1. Cloning of the full-length ZFP cDNA Because the putative ZFP gene was differentially expressed between undeveloped gland stage and developed gland stage, total RNA of developed gland stage was isolated [14]. The A260 /A280 value of total RNA is between 2.0 and 2.1, which showed total RNA is very pure and free of protein and DNA contamination, and can be used in the following experiments. According to the sequence of the putative ZFP cDNA fragment (GenBank accession number: AY162374), 3 rapid amplification cDNA ends was used to clone the full-length cDNA with only one gene specific primer, but not two gene specific primers. After twice amplifications, an about 0.75 kb specific band was shown by 1% agarose gel electrophoresis (Fig. 1), which was near to the expected length on the basis of alignment between the putative ZFP cDNA fragment and ZFP gene from other species. 3.2. Sequence analysis of the full-length ZFP cDNA After molecular cloning, the amplified fragment was sequenced forwardly and reversely to guarantee sequence correctness. As shown in Fig. 2, the full-length ZFP gene was 717 base pair (bp) in length with a complete opening reading frame (ORF) of 420 bp containing initial code (ATG) and terminal code (TAA). There was a 292-base non-coding sequence at 3 cDNA end, including the polyA sequence. It has been deposited in GenBank with accession number: BQ173926.
Fig. 1. Amplification of the full-length cDNA of cotton putative ZFP by 3 RACE. 1: DNA marker; 2: the full-length cDNA of cotton putative ZFP.
The predicted protein of complete ORF comprised of 139 amino acids with a calculated molecular mass of 15.6 kDa and isoelectric point of 8.81. Protein motif scanning showed the novel protein had three Ran-binding protein (RanBP) zinc finger motifs and belonged to RanBP2 ZFP family (Fig. 3). The consensus pattern of zinc finger RanBP2-type motif is W-x-C-X (2, 4)-C-x (3)-N-X (6)-C-x (2)-C. In detail, there are three zinc finger RanBP2-type subtype 1 (ZF RANBP2 1) signature in the region of 7–26, 52–73, 106–127 and zinc finger RanBP2-type subtype 2 (ZF RANBP2 2) signature in the region of 3–32, 48–79, 102–133 (Fig. 4). Therefore, the newly found ZFP in upland cotton was named as GRanBP2 ZFP1 (Gossypium hirsutum L. Ran-binding protein 2-type zinc finger protein 1). As shown in Fig. 4, GRanBP2 ZFP1 had 55–66% of identities in amino acid with ZFPs from other plants, 66 and 55% identities with ZFP1 and ZFP2 from Arabidopsis (accession number: AAM66130.1, NP 17931.1), respectively; 60% with ZFP30 from Oryza sativa (accession number: XP 464544.1); and 56% with ZFP2 from Zea mays (accession number: AAT42128.1). However, the coding sequences of GRanBP2 ZFP1 gene was higher identity at nucleotide level than amino acid level, such as 76% identity in Arabidopsis thaliana and 68% identity in O. sativa at nucleotide level (data not shown). Because two ZFPs in Arabidopsis were moderate identity with GRanBP2 ZFP1, there may be at least two RanBP2 ZFPs in upland cotton.
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Fig. 2. Nucleotide and predicted amino acid sequences of cotton ZFP. Both nucleotide and predicted amino acid sequences are countered on the left side. The accession number is DQ173926.
Fig. 3. Protein motif scanning of cotton predicted ZFP. There are three zinc finger Ran-binding protein 2 type (ZF-RanBP2) motif in the GRanBP ZFP1. Red and yellow frame indicates the subtype 1 and subtype 2 of ZF-RanBP2, respectively, and their locations shows the corresponding regions were ZF-RanBP2 motifs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 4. The multiple sequence alignment of GRanBP ZFP1 with several plant ZFP proteins. Symbol ‘*’ shows the identity amino acid in all plants. GRanBP ZFP1 was shortened to Gh ZFP, At ZFP1 and At ZFP2 was on behalf of ZFP1 and ZFP2 from Arabidopsis (accession number: AAM66130.1, NP 17931.1), Os ZFP30 on behalf of ZFP30 from Oryza sativa (accession number: XP 464544.1), and Zm ZFP2 on behalf of ZFP2 from Zea mays (accession number: AAT42128.1).
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Fig. 6. Quantified analysis of the expression of GRanBP ZFP1 gene at different stages in cotton. Data are presented as a percentage of mRNA level at undeveloped gland stage. Results were generally expressed mean ± standard error (ER) from values in three independent tests. Groups of data were compared by one-way ANOVA, * P < 0.05, n = 3.
Fig. 5. Expression of GRanBP ZFP1 gene at different stages in cotton. 1: DNA marker; 2–5 indicates: undeveloped gland stage, developing gland stage, developed gland stage, and cotyledon, respectively.
3.3. Expression analysis of GRanBP2 ZFP1gene Because GRanBP2 ZFP1 cDNA fragment was firstly found to be associated with the development of cotton pigment gland, the transcription level of GRanBP2 ZFP1gene was detected by semiquantitative RT-PCR assay at different stage of gland development. In addition to GSP1, another primer, GSP2, was synthesized to amplify the encoding region from 1 to 390 nucleotides. As shown in Fig. 5, the gene expressed at different development stages of gland, including the undeveloped stage, developing stage, developed stage and cotyledon stage. With the development of pigment gland, the mRNA level was increased significantly. The mRNA levels in the gland-developed seed and cotyledon were increased to about 1.5 and 2 folds of that in gland-undeveloped seed, respectively, which suggested that the novel ZFP played a role in the development of the cotton gland (Fig. 6). The genus Gossypium, as well as related genera, has small, though conspicuous, pigment-bearing glands in certain of the subepidermal tissues in many parts of the plant [2]. As the storage organ of gossypol, more researches were focused on the biosynthesis of gossypol for the importance role of gossypol in cotton growth [15–17], so that although these pigments glands have attracted the attention of research workers for a number of years, the molecular mechanism of its development has not been known yet. In this study, we cloned a GRanBP2 ZFP1 gene and found that its expression is relative to the pigments gland development. Until now, four kinds of ZFPs have been found in cotton, including SUPERMAN-like ZFP [18], LIM-
domain protein [19], ZFWD proteins containing a C3H zinc finger and seven WD40 repeats [20] and RanBP2 ZFP in this paper. Using 3 RACE with only one gene specific primer, the fulllength cDNA of GRanBP2 ZFP1 gene was cloned. GRanBP2 ZFP1 has three Ran-binding protein zinc finger motifs and belonged to RanBP2 ZFP family. The function of GRanBP2 has been studied in several animal and human cell lines and little is known in plant. Nucleocytoplasmic transport is mediated by large supramolecular structures that span the nuclear envelope called nuclear pore complexes (NPC). The export as well as the import pathway requires three major components, the substrate, an adaptor complex and a receptor. The adaptor complex and the receptor pass through the NPC with the substrate. The nuclear transport is also dependent on the GTPase cycle of the small GTPase protein Ran and a number of Ranbinding proteins [21,22]. RanBP2 is one of these proteins located in the NPC on the cytoplasmic side of the ring. The function of RanBP2 is not yet well understood. It could function in coupling RanGTP hydrolysis to NPC translocation through its Ran-binding domain (RanBD1) [23,24]. RanBP2 contains also a cluster of zinc fingers, the RanBP2 zinc fingers. It has been shown that these zinc fingers bind RanGDP (but not RanGTP) [25], the exportin-1 protein (a receptor of the export pathway) [26], two kinesin microtubule-based motor proteins, KIF5B and KIF5C [27]. The RanBP2 zinc finger is also found in different proteins that are mainly involved in nuclear transport or localized to the nuclear envelope. Therefore, GRanBP2 ZFP1 may play a role in nuclear transport or localized to the nuclear envelope. ZFPs belong to the largest family of regulatory transcription factors and play an important role in growth and development in animal and plant systems. The nucleoporin RanBP2 has SUMO1 E3 ligase activity and takes part in degradation of target proteins through ubiquitin proteasome pathway [28]. The mRNA level of GRanBP2 ZFP1 gene increased with the development of cotton pigment gland, which suggested that it is involved in cotton pigment gland development. However, its role and regulatory mechanism in pigment gland development need further be investigated.
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4. Conclusions On the basis the sequence of a putative ZFP cDNA fragment (GenBank accession number: AY162374), which was differentially expressed at the stage of the cotton gland development and found by suppression subtractive hybridization, the complete cDNA sequence of a novel RanBP2 zinc finger protein gene was cloned by rapid amplification of cDNA ends from upland cotton (G. hirsutum L.), Xiangmian 18, and was named GRanBP2 ZFP1 gene. GRanBP2 ZFP1 cDNA (GenBank accession number: BQ173926) is 717 bp in length with a complete reading frame encoding 139 amino acids. GRanBP2 ZFP1 is a 15.6 kDa protein and isoelectric point is 8.81. RanBP2 ZFP1 has three Ran-binding protein zinc finger motifs and belonged to RanBP2 ZFP family. GRanBP2 ZFP1 had moderate identity, 55–66%, with ZFPs from other plants in amino acid. GRanBP2 ZFP1 gene expressed at different development stages of pigment gland, including the undeveloped stage, developing stage, developed stage and cotyledon stage. With the development of pigment gland, the mRNA levels in the seed at the gland-developed stage and cotyledon were increased to 1.5 and 2 folds of that in glandundeveloped seed, respectively. These results suggested that the novel ZFP may play a role in the development of the cotton pigment gland. Acknowledgments This work was supported by grants from the National Nature Science Foundation of China (Nos. 30600329 and 30440032), Natural Sciences Foundation of Chongqing City (Nos. CSTC2005BB5072 and CSTC2005BB1094) and Initial Research Foundation for Ph.D. of Chongqing University of Posts and Telecommunications (A2005-13). References [1] Y. Cai, H. Zhang, Y. Zeng, J. Mo, J. Bao, C. Miao, J. Bai, F. Yan, F. Chen, J. Biosci. 29 (2004) 67–71.
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Molecular cloning and expression analysis of a RanBP2 zinc finger protein gene in upland cotton (Gossypium hirsutum L.) Ping-An Chang ∗ , Biao Li ∗ , Xiao-Min Ni, Yong-Fang Xie, Ying-Fan Cai Key Laboratory of Molecular Biology, College of Bio-information, Chongqing University of Posts and Telecommunications, Chongqing 400065, PR China Received 13 September 2006; received in revised form 2 November 2006; accepted 29 November 2006 Available online 9 December 2006
Abstract Gossypol is an important resistant substance of Gossypium, and its storage organ is pigment gland. Although, the relationship between gossypol and pigment gland has been studied for a long time, the development mechanism of pigment gland has not been revealed up to now in molecular perspective. On the basis of differentially expressed cDNAs fragments at the stage of the cotton gland development using suppression subtractive hybridization (SSH), the complete cDNA sequence of a novel RanBP2 zinc finger protein (ZFP) gene was cloned by rapid amplification of cDNA ends (RACE) from upland cotton (Gossypium hirsutum L.), Xiangmian 18. The cotton RanBP2 ZFP cDNA (GenBank accession number: BQ173926) is 717 base pair (bp) long with an open reading frame encoding 139 amino acids, which encodes a 15.6 kDa protein. The cotton RanBP2 ZFP had three Ran-binding protein (RanBP) two zinc finger motifs and belonged to RanBP2 ZFP family. There is a 292-base non-coding sequence at 3 cDNA end, which includes polyA sequence. Sequence alignment analysis revealed that the cDNA nucleotide and its deduced amino acid sequence are moderately identical to the putative ZFP from other species. The mRNA expressing profiles of the novel ZFP gene was investigated by reverse transcription-polymerase chain reaction (RT-PCR). The result showed that it expressed at different development stages of gland, including the undeveloped stage, developing stage, developed stage and cotyledon stage. However, with the development of pigment gland, the mRNA levels in the gland-developed seed and cotyledon were increased to about 1.5 and 2 folds of that in gland-undeveloped seed, respectively, which suggested that the novel ZFP played a role in the development of the cotton gland. © 2006 Elsevier B.V. All rights reserved. Keywords: Upland cotton (Gossypium hirsutum L.); Zinc finger protein; Gene expression; Pigment gland
1. Introduction Gossypol, a polyphenolic binaphthyl dialdehyde stored in the pigment glands of cotton, is not only an important resistant substance for cotton but also an important phytochemical component of immense interest due to its several biological properties including anti-cancer, antimicrobial, anti-HIV, antioxidation and male contraceptive [1]. Gossypol content of cotton is mainly dependent on different genetic types of pigment glands [2]. The glanded cotton normally contains gossypol in both seeds and plants that is toxic to human and non-ruminant animals [3,4]. Ordinary glandless cotton contains no or low-gossypol in seeds, roots, stems as well as in leaves, but its resistance to diseases,
∗
Corresponding authors. Tel.: +86 23 62461884. E-mail addresses: [email protected] (P.-A. Chang), [email protected] (B. Li). 0927-7765/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2006.11.042
pests and even rats is reduced greatly [5,6]. Therefore, a cotton variety which is characterized by the presence of glanded roots, leaves and stems (to maintain the resistance trait) but glandless seeds (for safe utilization) is highly desired [7,8]. Biologists and agronomists have long been studying the glanded characters and the glandless characters of cotton as well as their gossypol content [9–13]. However, the relationship between gossypol and pigment gland was complicated and the molecular mechanism of pigment gland development has not been revealed up to now [7,8]. In order to screen the relative genes involving the development of cotton pigment gland, two differentially expressed cDNA libraries of forward and reverse subtraction were constructed using suppression subtractive hybridization (SSH) in the process of germinating of the seeds of Xiangmian 18, a cotton variety which is characterized by the presence of glanded roots, leaves and stems but glandless seeds [14]. The differentially expressed cDNA fragments between undeveloped gland
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P.-A. Chang et al. / Colloids and Surfaces B: Biointerfaces 55 (2007) 153–158
and developing gland seeds were associated with regulation and control factors, apoptosis, protein synthesis, including a putative zinc finger protein (ZFP) cDNA fragment (GenBank accession number: AY162374) [14]. The ZFP cDNA fragment was not complete, but had the initial code, ATG, by alignment with ZFP genes from other species. For the reason that the zinc finger proteins belong to the largest family of regulatory transcription factors and play an important role in growth and development in animal and plant systems, the full length of a putative ZFP cDNA was first cloned using rapid amplification of cDNA ends (RACE), then the expressing profiles of the novel ZFP gene at the different gland development stage were investigated by reverse transcription-polymerase chain reaction (RT-PCR) to reveal the relation between development of cotton pigment gland and the expression of the ZFP gene in this paper.
2.3.2. Synthesis of the first-strand cDNA A total 10 l reaction volume was generated containing 4 l (5 g) total RNA, 0.5 l 20 mol/l AP, 1 l 10 mmol/l dNTP mixture and 4.5 l DEPC-treated water, then incubated at 65 ◦ C for 5 min and placed on ice for at least 1 min to denature probable RNA secondary structure. The first-strand cDNA synthesis mixture was prepared by adding following components to the above 10 l reaction volume in the indicated order, 2 l 10× RT-PCR buffer, 4 l 25 mmol/l MgCl2 , 2 l 0.1 mol/l DTT, 1 l RNaseOUT (40 U) and 1 l (200 U) SuperScriptTM III reverse transcriptase, and mixed well. The first-strand cDNA synthesis mixture was incubated at 50 ◦ C for 50 min and terminated at 85 ◦ C for 5 min. Lastly, 1 l (2 U) RNase H was added to the first-strand cDNA synthesis mixture, which was incubated at 37 ◦ C for 20 min to destruct RNA template.
2. Experimental
2.3.3. Amplification of the full-length cDNA The first PCR reaction volume is 50 l, which combines 5 l 10× RT-PCR buffer, 3 l 25 mmol/l MgCl2 , 1 l 10 mmol/l dNTP mixture, 2 l above synthesized cDNA template, 1 l 120 mol/l GSP1 and AP, 1 l Ex TaqTM DNA polymerase, and 36 l sterilized H2 O. Then the cDNA was firstly amplified as following procedure: 94 ◦ C for 5 min to pre-denature cDNA template; 40 amplification cycles of 94 ◦ C for 1 min, 55 ◦ C for 1 min, 72 ◦ C for 1 min; 72 ◦ C for 7 min to extend. Then the cDNA was secondary amplified as the first amplification process, except that 2 l first amplification product was template, UAP substituted for AP and denaturing temperature was 58 ◦ C.
2.1. Materials Cotton seed of Xiangmian 18 was obtained from National Research Center for Cross-Cotton of China (Changsha, China). Total plant RNA extraction kit was from Watson Biotechnologies, Inc. (Shanghai, China). SuperScript III first-strand synthesis system for RT-PCR was purchased from Invitrogen Life Technologies (Groningen, The Netherlands). pMD18-T vector, T4 DNA ligase, DNA gel extraction kit, TaKaRa Ex TaqTM PCR kit (Hot start version) and TaKaRa RNA PCR kit (AMV) Version 3.0 was purchased from Takara (Dalian, China). 2.2. Total RNA extraction Cotton seeds were disinfected in 70% ethanol and 15% H2 O2 solution, then dipped in sterilized water, and began to bud in the plates containing sterilized filter paper and water. The pigment gland begins to develop after 36-h disinfection and several pigment glands developed after 52 h, which was verified by microscopy. So the stage within 36 h after disinfection was considered to be gland-undeveloped stage, from 36 to 52 h as developing gland stage, after 52 h until cotyledon developed as developed gland stage [14]. Total RNA was isolated with total plant RNA extraction kit from Watson Biotechnologies according to the manufacturer’s instructions and was quantified photometrically. In addition, total RNA was electrophoresed on a denatured agarose gel to investigate its integrality. 2.3. Molecular cloning of cotton zinc finger protein gene 2.3.1. Primer design According to the sequence of cotton putative ZFP cDNA fragments, a gene specific primer, 5 -ATGAGCAGGCCAGGAGATTGG-3 was designed, named as GSP1. At the same time, adapter primer (AP), 5 -GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3 and universal amplification primer (UAP), 5 -GGCCACGCGTCGACTAGTAC-3 was also synthesized.
2.3.4. Molecular cloning of the full-length cDNA and sequencing The secondary amplification product was electrophoresed on a 1% agarose gel and the expected length band was extracted with DNA gel extraction kit. The extracted full-length cDNA was cloned into pMD18-T vector and the recombinant plasmid was certified by PCR. The inserted cDNA was forwardly and reversely sequenced with ABI 377 DNA sequencing system. 2.4. Sequence analysis of cotton zinc finger protein gene Primers and open reading frames finding of the novel cotton ZFP cDNA sequence was assisted by DNAman software. Protein sequence is deduced and analyzed by the same program. Blastn and Blastp program were used to find out the homologous sequences of cotton ZFP cDNA sequence and protein sequence in GenBank online (http://www.ncbi.nlm.nih.gov/blast), respectively. Protein motif scanning was carried out with MotifScan program online (http://myhits.isb-sib.ch/cgi-bin/motif scan). Multi-sequences alignment was accomplished by Clustalx software. 2.5. Expressing analysis of cotton ZFP gene Total RNA of different stages was isolated as described above and mRNA levels of different stages were investigated
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by TaKaRa RNA PCR kit (AMV) according to the manufacturer’s instructions. The forward primer was GSP1 and the reverse primer was 5 -TGGAGCACTGCATCTGAAACATTC3 , named GSP2, and was synthesized according the cotton ZFP cDNA sequence. The reverse transcription system is 10 l, including 3.75 l RNase free dH2 O, 1 l 10× RT buffer, 2 l 25 mM MgCl2 , 0.5 l dNTPs (10mM each), 0.5 l GSP2 (10 M), 1 l total RNA, 0.25 l RNase inhibitor (40 U/l), 1 l AMV reverse transcriptase (5 U/l). The reaction procedure was 50 ◦ C for 30 min, 99 ◦ C for 5 min, 4 ◦ C for 5 min. The PCR amplification reaction system was generated by adding 10 l 5× PCR buffer, 0.5 l GSP1 (10 M), 0.5 l Ex Taq Hs (5 U/l), 29 l dH2 O to the above reverse transcription system. The target cDNA was amplified as following procedure: 94 ◦ C for 3 min to pre-denature cDNA template; 35 amplification cycles of 94 ◦ C for 45 s, 56 ◦ C for 45 s, 72 ◦ C for 45 s; 72 ◦ C for 10 min to extend. The amplification products and total RNA were electrophoresed on a 1% agarose gel. The density ratio of target cDNA band and corresponding 18S rRNA band was carried out by Quantity One program (Bio-Rad). The experiment was duplicated for three times and data are presented as a percentage of mRNA level at undeveloped gland stage. Results were generally expressed mean ± standard error (ER) from values three independent tests. Groups of data were compared by one-way ANOVA. A difference between means was considered significant at a value of P < 0.05. 3. Results and discussion 3.1. Cloning of the full-length ZFP cDNA Because the putative ZFP gene was differentially expressed between undeveloped gland stage and developed gland stage, total RNA of developed gland stage was isolated [14]. The A260 /A280 value of total RNA is between 2.0 and 2.1, which showed total RNA is very pure and free of protein and DNA contamination, and can be used in the following experiments. According to the sequence of the putative ZFP cDNA fragment (GenBank accession number: AY162374), 3 rapid amplification cDNA ends was used to clone the full-length cDNA with only one gene specific primer, but not two gene specific primers. After twice amplifications, an about 0.75 kb specific band was shown by 1% agarose gel electrophoresis (Fig. 1), which was near to the expected length on the basis of alignment between the putative ZFP cDNA fragment and ZFP gene from other species. 3.2. Sequence analysis of the full-length ZFP cDNA After molecular cloning, the amplified fragment was sequenced forwardly and reversely to guarantee sequence correctness. As shown in Fig. 2, the full-length ZFP gene was 717 base pair (bp) in length with a complete opening reading frame (ORF) of 420 bp containing initial code (ATG) and terminal code (TAA). There was a 292-base non-coding sequence at 3 cDNA end, including the polyA sequence. It has been deposited in GenBank with accession number: BQ173926.
Fig. 1. Amplification of the full-length cDNA of cotton putative ZFP by 3 RACE. 1: DNA marker; 2: the full-length cDNA of cotton putative ZFP.
The predicted protein of complete ORF comprised of 139 amino acids with a calculated molecular mass of 15.6 kDa and isoelectric point of 8.81. Protein motif scanning showed the novel protein had three Ran-binding protein (RanBP) zinc finger motifs and belonged to RanBP2 ZFP family (Fig. 3). The consensus pattern of zinc finger RanBP2-type motif is W-x-C-X (2, 4)-C-x (3)-N-X (6)-C-x (2)-C. In detail, there are three zinc finger RanBP2-type subtype 1 (ZF RANBP2 1) signature in the region of 7–26, 52–73, 106–127 and zinc finger RanBP2-type subtype 2 (ZF RANBP2 2) signature in the region of 3–32, 48–79, 102–133 (Fig. 4). Therefore, the newly found ZFP in upland cotton was named as GRanBP2 ZFP1 (Gossypium hirsutum L. Ran-binding protein 2-type zinc finger protein 1). As shown in Fig. 4, GRanBP2 ZFP1 had 55–66% of identities in amino acid with ZFPs from other plants, 66 and 55% identities with ZFP1 and ZFP2 from Arabidopsis (accession number: AAM66130.1, NP 17931.1), respectively; 60% with ZFP30 from Oryza sativa (accession number: XP 464544.1); and 56% with ZFP2 from Zea mays (accession number: AAT42128.1). However, the coding sequences of GRanBP2 ZFP1 gene was higher identity at nucleotide level than amino acid level, such as 76% identity in Arabidopsis thaliana and 68% identity in O. sativa at nucleotide level (data not shown). Because two ZFPs in Arabidopsis were moderate identity with GRanBP2 ZFP1, there may be at least two RanBP2 ZFPs in upland cotton.
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Fig. 2. Nucleotide and predicted amino acid sequences of cotton ZFP. Both nucleotide and predicted amino acid sequences are countered on the left side. The accession number is DQ173926.
Fig. 3. Protein motif scanning of cotton predicted ZFP. There are three zinc finger Ran-binding protein 2 type (ZF-RanBP2) motif in the GRanBP ZFP1. Red and yellow frame indicates the subtype 1 and subtype 2 of ZF-RanBP2, respectively, and their locations shows the corresponding regions were ZF-RanBP2 motifs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 4. The multiple sequence alignment of GRanBP ZFP1 with several plant ZFP proteins. Symbol ‘*’ shows the identity amino acid in all plants. GRanBP ZFP1 was shortened to Gh ZFP, At ZFP1 and At ZFP2 was on behalf of ZFP1 and ZFP2 from Arabidopsis (accession number: AAM66130.1, NP 17931.1), Os ZFP30 on behalf of ZFP30 from Oryza sativa (accession number: XP 464544.1), and Zm ZFP2 on behalf of ZFP2 from Zea mays (accession number: AAT42128.1).
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Fig. 6. Quantified analysis of the expression of GRanBP ZFP1 gene at different stages in cotton. Data are presented as a percentage of mRNA level at undeveloped gland stage. Results were generally expressed mean ± standard error (ER) from values in three independent tests. Groups of data were compared by one-way ANOVA, * P < 0.05, n = 3.
Fig. 5. Expression of GRanBP ZFP1 gene at different stages in cotton. 1: DNA marker; 2–5 indicates: undeveloped gland stage, developing gland stage, developed gland stage, and cotyledon, respectively.
3.3. Expression analysis of GRanBP2 ZFP1gene Because GRanBP2 ZFP1 cDNA fragment was firstly found to be associated with the development of cotton pigment gland, the transcription level of GRanBP2 ZFP1gene was detected by semiquantitative RT-PCR assay at different stage of gland development. In addition to GSP1, another primer, GSP2, was synthesized to amplify the encoding region from 1 to 390 nucleotides. As shown in Fig. 5, the gene expressed at different development stages of gland, including the undeveloped stage, developing stage, developed stage and cotyledon stage. With the development of pigment gland, the mRNA level was increased significantly. The mRNA levels in the gland-developed seed and cotyledon were increased to about 1.5 and 2 folds of that in gland-undeveloped seed, respectively, which suggested that the novel ZFP played a role in the development of the cotton gland (Fig. 6). The genus Gossypium, as well as related genera, has small, though conspicuous, pigment-bearing glands in certain of the subepidermal tissues in many parts of the plant [2]. As the storage organ of gossypol, more researches were focused on the biosynthesis of gossypol for the importance role of gossypol in cotton growth [15–17], so that although these pigments glands have attracted the attention of research workers for a number of years, the molecular mechanism of its development has not been known yet. In this study, we cloned a GRanBP2 ZFP1 gene and found that its expression is relative to the pigments gland development. Until now, four kinds of ZFPs have been found in cotton, including SUPERMAN-like ZFP [18], LIM-
domain protein [19], ZFWD proteins containing a C3H zinc finger and seven WD40 repeats [20] and RanBP2 ZFP in this paper. Using 3 RACE with only one gene specific primer, the fulllength cDNA of GRanBP2 ZFP1 gene was cloned. GRanBP2 ZFP1 has three Ran-binding protein zinc finger motifs and belonged to RanBP2 ZFP family. The function of GRanBP2 has been studied in several animal and human cell lines and little is known in plant. Nucleocytoplasmic transport is mediated by large supramolecular structures that span the nuclear envelope called nuclear pore complexes (NPC). The export as well as the import pathway requires three major components, the substrate, an adaptor complex and a receptor. The adaptor complex and the receptor pass through the NPC with the substrate. The nuclear transport is also dependent on the GTPase cycle of the small GTPase protein Ran and a number of Ranbinding proteins [21,22]. RanBP2 is one of these proteins located in the NPC on the cytoplasmic side of the ring. The function of RanBP2 is not yet well understood. It could function in coupling RanGTP hydrolysis to NPC translocation through its Ran-binding domain (RanBD1) [23,24]. RanBP2 contains also a cluster of zinc fingers, the RanBP2 zinc fingers. It has been shown that these zinc fingers bind RanGDP (but not RanGTP) [25], the exportin-1 protein (a receptor of the export pathway) [26], two kinesin microtubule-based motor proteins, KIF5B and KIF5C [27]. The RanBP2 zinc finger is also found in different proteins that are mainly involved in nuclear transport or localized to the nuclear envelope. Therefore, GRanBP2 ZFP1 may play a role in nuclear transport or localized to the nuclear envelope. ZFPs belong to the largest family of regulatory transcription factors and play an important role in growth and development in animal and plant systems. The nucleoporin RanBP2 has SUMO1 E3 ligase activity and takes part in degradation of target proteins through ubiquitin proteasome pathway [28]. The mRNA level of GRanBP2 ZFP1 gene increased with the development of cotton pigment gland, which suggested that it is involved in cotton pigment gland development. However, its role and regulatory mechanism in pigment gland development need further be investigated.
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4. Conclusions On the basis the sequence of a putative ZFP cDNA fragment (GenBank accession number: AY162374), which was differentially expressed at the stage of the cotton gland development and found by suppression subtractive hybridization, the complete cDNA sequence of a novel RanBP2 zinc finger protein gene was cloned by rapid amplification of cDNA ends from upland cotton (G. hirsutum L.), Xiangmian 18, and was named GRanBP2 ZFP1 gene. GRanBP2 ZFP1 cDNA (GenBank accession number: BQ173926) is 717 bp in length with a complete reading frame encoding 139 amino acids. GRanBP2 ZFP1 is a 15.6 kDa protein and isoelectric point is 8.81. RanBP2 ZFP1 has three Ran-binding protein zinc finger motifs and belonged to RanBP2 ZFP family. GRanBP2 ZFP1 had moderate identity, 55–66%, with ZFPs from other plants in amino acid. GRanBP2 ZFP1 gene expressed at different development stages of pigment gland, including the undeveloped stage, developing stage, developed stage and cotyledon stage. With the development of pigment gland, the mRNA levels in the seed at the gland-developed stage and cotyledon were increased to 1.5 and 2 folds of that in glandundeveloped seed, respectively. These results suggested that the novel ZFP may play a role in the development of the cotton pigment gland. Acknowledgments This work was supported by grants from the National Nature Science Foundation of China (Nos. 30600329 and 30440032), Natural Sciences Foundation of Chongqing City (Nos. CSTC2005BB5072 and CSTC2005BB1094) and Initial Research Foundation for Ph.D. of Chongqing University of Posts and Telecommunications (A2005-13). References [1] Y. Cai, H. Zhang, Y. Zeng, J. Mo, J. Bao, C. Miao, J. Bai, F. Yan, F. Chen, J. Biosci. 29 (2004) 67–71.
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