- Email: [email protected]
BcMF11, a putative pollen-specific non-coding RNA from Brassica campestris ssp. chinensis
ARTICLE IN PRESS Journal of Plant Physiology 164 (2007) 1097—1100
www.elsevier.de/jplph
SHORT COMMUNICATION
BcMF11, a putative pollen-specific non-coding RNA from Brassica campestris ssp. chinensis Jiang-Hua Song, Jia-Shu Cao, Xiao-Lin Yu, Xun Xiang Laboratory of Cell and Molecular Biology, Institute of Vegetable Science, Zhejiang University, Hangzhou 310029, PR China Received 7 September 2006; received in revised form 7 October 2006; accepted 9 October 2006
KEYWORDS Brassica campestris ssp. chinensis; Expression patterns; Molecular cloning; Non-coding RNA; Pollen development
Summary A full-length cDNA, BcMF11, has been cloned from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) using rapid amplification of the cDNA ends (RACE) based on a pollen-specific cDNA fragment (DN237921). The BcMF11 cDNA has a total length of 828 bp with poly (A) tail. Analysis of the sequence demonstrated that BcMF11 is a novel non-coding RNA which has no prominent open reading frame (ORF) or coding capacity. No significant similarities were observed between BcMF11 and previously published sequences in GenBank. Transcription analysis indicated that BcMF11 is a novel pollen-specific ncRNA and may be involved in the pollen development of Chinese cabbage. & 2006 Elsevier GmbH. All rights reserved.
Introduction Recently, a surprisingly large number of novel non-coding RNAs (ncRNAs) have been identified in eukaryotic cells by using experimental or computational approaches (Aspegren et al., 2004; Billoud et al., 2005; Zhang et al., 2005). These ncRNAs contain a high density of stop codons and lack any extensive ‘‘Open Reading Frame’’ (Costa, 2005). Abbreviations: ncRNAs, non-coding RNAs; ORF, open reading frame; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcriptase polymerase chain reaction Corresponding author. Tel./fax: +86 571 86971188. E-mail address: [email protected] (J.-S. Cao).
Unlike mRNAs, ncRNAs are not translated into proteins but function at an RNA level and are commonly found associated with specific proteins (Erdmann et al., 1999; Mattick and Makunin, 2006). Most of ncRNAs identified from plants play important roles in plant responses to environmental abiotic and biotic stresses as well as signal transduction (Burleigh and Harrison, 1998; Kouchi et al., 1999; Liu et al., 1997; Teramoto et al., 1996). However, very little work has been done on the isolation and characterization of a certain ncRNA involved in the plant development. Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) is one of the most important commercial vegetables in the world. We are interested in learning what molecular mechanisms
0176-1617/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2006.10.002
ARTICLE IN PRESS 1098 control the pollen development of Chinese cabbage, so as to make good use of its heterosis for the cross-breeding. There must be numerous genes involved in the pollen development process of Chinese cabbage (Park et al., 2002). We have previously isolated and identified some preferentially expressed cDNA sequences during pollen development (Cao et al., 2006; Wang et al., 2004, 2005; Ye et al., 2003; Yu et al., 2004). One of cDNA fragments with the length of 232 base pair, named BBS18, was proved to be pollen-specific. Here, we obtained a gene, BcMF11, based on the BBS18 cDNA fragment. In the present study, the isolation and characterization of a novel putative pollen-preferential ncRNA from Chinese cabbage is reported.
Materials and methods Total RNA was extracted from the different tissue samples of roots, stems, leaves, flower buds, flowers, anthers, pistils and siliques of Chinese cabbage using TRIzol reagents (Invitrogen, USA) according to the manufacturer’s instruction. Flower buds were stored arbitrarily into five size groups (1.0, 1.6, 2.2, 2.8 and 3.4 mm in length) corresponding to five stages of pollen development based on the method described previously (Park et al., 2002). The rapid amplification of cDNA ends (RACE) strategy was employed to isolate the 50 and 30 ends of BcMF11. The cDNA was synthesized using the SMARTTM PCR cDNA synthesis kit (Clontech, USA) according to the manufacturer’s instruction. Based on the sequence of cloned
J.-H. Song et al. cDNA fragment (DN237921), the gene specific primers 30 GSP (50 -AACCCGAAGCGAAAGAAGAGAGAC-30 ) and 30 N GSP (50 -CAGATGGGAGGAAACAAGACT-30 ), were designed and used for 30 RACE, and the specific primer 50 GSP (50 -GGTTTTCATCCCTTAAAGTTCC-30 ) for 50 RACE. PCRs were performed in accordance with the procedure of the SMARTTM RACE cDNA Amplification Kit (Clontech, USA). The full-length cDNA of BcMF11 was amplified with the forward and reverse primer (50 -GGCCATTACGGCCGG GGGTG-30 ) and (50 -CGGGTTTCTACATTCTCCCC-30 ). All of the PCR products were purified and cloned into pGEM-T Easy Vector (Promega, USA) followed by sequencing. The sequence was compared by the BLAST program and analyzed with DNAMAN (Ver. 5.2.2) and RNA structure software (Ver. 3.2). RT-PCR analyses were carried out to determine the expression pattern of BcMF11 gene with the specific forward and reverse primers of amplifying the full-length cDNA. PCR amplifications were performed at 94 1C for 3 min, followed by 25 cycles of amplification (94 1C for 30 s, 60 1C for 30 s and 72 1C for 1 min) and by 72 1C for 7 min. A actin-1 fragment was amplified for comparison under the same RT-PCR conditions. RNA gel blot was performed according to a standard method (Sambrook and Russell, 2001). Total RNAs extracted from tissues of Chinese cabbage were separated in 1.2% formaldehyde agarose gels, transferred to a Hybond-N+ nylon membrane and hybridized with the full length sequence probe of BcMF11.
Results and discussion Based on the cDNA fragment (DN237921), a 30 cDNA end and 50 cDNA end turned out to be 418 and
Figure 1. Nucleotide sequence of BcMF11 cDNA. The primers for RACE are underlined and the orientation of each primer is pointed out by bold arrowheads. The conserved cis-acting elements are shown in boxes. The start codon (ATG) and stop codon (TAA) in the longest potential ORF are indicated by bold letters.
ARTICLE IN PRESS Identification of a pollen-specific non-coding RNA from Brassica campestris 379 bp in length by RACE, respectively. The cloned full-length cDNA of BcMF11 gene was 828 bp (GenBank accession number DQ925484) (Fig. 1). No significant similarities were found between BcMF11 and previously published DNA and protein sequences. However, high similarity (94% identity over 186 bases) was found with a Brassica napus. EST cDNA clones (data not shown). Sequence analysis revealed that BcMF11 had a high A/T content of 58.7% and a considerably lower RNA secondary structure free energy value of 203.26 kcal/mol. Furthermore, the calculated Testcode value was under 0.5. The longest potential ORF was only 228 nucleotides long coding 75 amino acids. Multiple termination codons (TAA, TAG and TGA) appeared in the six possible reading frames in BcMF11. It revealed that BcMF11 has no capacity of coding mature protein like most of identified ncRNAs in plants. For instance, CR20, ENOD40 and GUT15 can form stable RNA secondary structure, but not contain a long ORF (Campalans et al., 2004; Crespi et al., 1994; Taylor and Green, 1995). Therefore, BcMF11 may be a member of a new category of ncRNA reported firstly in Chinese cabbage. Interestingly, some cis-acting elements containing the Quantitative element AAATGA and TGGTTA, and PB core motif TGTGGTT, noted to have the strongest gain-of-function effect on expression in pollen (Twell et al., 1991), were found in the upstream site of the sequence of BcMF11 (Fig. 1). This may be responsible for enhancing the pollenspecific expression of BcMF11. RNA gel blot analysis of various tissues of Chinese cabbage demonstrated that the BcMF11 was expressed in developing flower buds, flowers and anthers at a high level, but was not detected in roots, stems, leaves, pistils and developing siliques (Fig. 2A). Identical expression patterns were also showed in RT-PCR analysis (Fig. 2B). This indicated BcMF11 is a pollen-preferential expressed gene in Chinese cabbage. The similar expression character was found in the ncRNA ZM401 isolated from maize (Dai et al., 2004). RNA gel blot analysis of different stages of pollen development showed that BcMF11 transcript first appeared at the early stage of pollen development and kept slightly increase in steady state mRNA levels at the middle and late stage of pollen development (Fig. 3A). This result was consistent with RT-PCR analysis (Fig. 3B). It suggested that BcMF11 belongs to a constant expressed pollen gene category during anther development, which is similar to the expression patterns of the NelF4a8 mRNA (Ylstra and McCormick, 1999). Therefore, it is possible
A
R
S
B R BcMF11
S
1099 L
FB
F
P
A
SL
BcMF11 rRNA L
FB
F
P
A
SL
actin
Figure 2. RNA gel blot analysis (A) and RT-PCR expression analysis (B) of BcMF11 mRNA accumulation in various organs of Chinese cabbage. RNAs were isolated from the root (R), stem (S), leaf (L), flower bud (FB), flower (F), pistil (P), anther (A) and silique (S) of Chinese cabbage.
A
1
2
3
4
5
BCMF11 rRNA
B
1
2
3
4
5
BCMF11 actin
Figure 3. RNA gel blot analysis (A) and RT-PCR expression analysis (B) of BcMF11 mRNA accumulation in different stages of pollen development in Chinese cabbage. RNAs were isolated from buds of five size groups (1.0, 1.6, 2.2, 2.8 and 3.4 mm in length), which correspond to five stages of pollen development in Chinese cabbage.
that BcMF11 play a regulatory role in the pollen development. In conclusion, we identified and characterized a novel putative ncRNA in Chinese cabbage. Our study indicated that BcMF11 expression was both pollen-specific and developmentally regulated. The cloned BcMF11 ncRNA will be a useful contribution to elucidating the gene regulation of sexual propagation in flowering plants.
Acknowledgements This work was supported by the National Natural Science Foundation of China (no. 30370975) and the Key Sci-technology Project of Zhejiang Province (no. 2005C12019-02).
ARTICLE IN PRESS 1100
References Aspegren A, Hinas A, Larsson P, Larsson A, Soderbom F. Nucleic Acids Res 2004;32:4646–56. Billoud B, De Paepe R, Baulcombe D, Boccara M. Biochimie 2005;87:905–10. Burleigh SM, Harrison MJ. Gene 1998;216:47–53. Campalans A, Kondorosi A, Crespi M. Plant Cell 2004;16: 1047–59. Cao JS, Yu XL, Ye WZ, Lu G, Xiang X. Plant Cell Rep 2006;24:715–23. Costa FF. Gene 2005;357:83–94. Crespi MD, Jurkevitch E, Poiret M, dAubenton-Carafa Y, Petrovics G, Kondorosi E, et al. EMBO J 1994;13: 5099–112. Dai XY, Yu JJ, Zhao Q, Zhu DY, AO GM. J Integr Plant Biol 2004;46:497–504. Erdmann VA, Szymanski M, Hochberg A, de Groot N, Barciszewski J. Nucleic Acids Res 1999;27:192–5. Kouchi H, Takane K, So RB, Ladha JK, Reddy PM. Plant J 1999;18:121–9. Liu C, Muchhal US, Raghothama KG. Plant Mol Biol 1997;33:867–74.
J.-H. Song et al. Mattick JS, Makunin LV. Hum Mol Genet 2006;15: 17–29. Park BS, Park YD, Kim HU, Jin YM, Kim HI. Mol Cells 2002;14:150–7. Sambrook J, Russell DW. Molecular cloning: a laboratory manual, 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2001. Taylor CB, Green PJ. Plant Mol Biol 1995;28:27–38. Teramoto H, Toyama T, Takeba G, Tsuji H. Plant Mol Biol 1996;32:797–808. Twell D, Yamaguchi J, Wing RA, Ushiba J, McCormick S. Genes Dev 1991;5:496–507. Wang YQ, Ye WZ, Cao JS, Yu XL, Xiang X, Lu G. J Integr Plant Biol 2005;47:863–72. Wang YQ, Yu XL, Cao JS. Acta Genet Sin 2004;31: 1302–8. Ye WZ, Cao JS, Xiang X, Zeng GW. J Hortic Sci Biotech 2003;78:319–23. Ylstra B, McCormick S. Plant J 1999;20:101–8. Yu XL, Cao JS, Ye WZ, Wang YQ. J Hortic Sci Biotech 2004;79:833–9. Zhang BH, Pan XP, Wang QL, Cobb GP, Anderson TA. Cell Res 2005;15:336–60.
www.elsevier.de/jplph
SHORT COMMUNICATION
BcMF11, a putative pollen-specific non-coding RNA from Brassica campestris ssp. chinensis Jiang-Hua Song, Jia-Shu Cao, Xiao-Lin Yu, Xun Xiang Laboratory of Cell and Molecular Biology, Institute of Vegetable Science, Zhejiang University, Hangzhou 310029, PR China Received 7 September 2006; received in revised form 7 October 2006; accepted 9 October 2006
KEYWORDS Brassica campestris ssp. chinensis; Expression patterns; Molecular cloning; Non-coding RNA; Pollen development
Summary A full-length cDNA, BcMF11, has been cloned from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) using rapid amplification of the cDNA ends (RACE) based on a pollen-specific cDNA fragment (DN237921). The BcMF11 cDNA has a total length of 828 bp with poly (A) tail. Analysis of the sequence demonstrated that BcMF11 is a novel non-coding RNA which has no prominent open reading frame (ORF) or coding capacity. No significant similarities were observed between BcMF11 and previously published sequences in GenBank. Transcription analysis indicated that BcMF11 is a novel pollen-specific ncRNA and may be involved in the pollen development of Chinese cabbage. & 2006 Elsevier GmbH. All rights reserved.
Introduction Recently, a surprisingly large number of novel non-coding RNAs (ncRNAs) have been identified in eukaryotic cells by using experimental or computational approaches (Aspegren et al., 2004; Billoud et al., 2005; Zhang et al., 2005). These ncRNAs contain a high density of stop codons and lack any extensive ‘‘Open Reading Frame’’ (Costa, 2005). Abbreviations: ncRNAs, non-coding RNAs; ORF, open reading frame; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcriptase polymerase chain reaction Corresponding author. Tel./fax: +86 571 86971188. E-mail address: [email protected] (J.-S. Cao).
Unlike mRNAs, ncRNAs are not translated into proteins but function at an RNA level and are commonly found associated with specific proteins (Erdmann et al., 1999; Mattick and Makunin, 2006). Most of ncRNAs identified from plants play important roles in plant responses to environmental abiotic and biotic stresses as well as signal transduction (Burleigh and Harrison, 1998; Kouchi et al., 1999; Liu et al., 1997; Teramoto et al., 1996). However, very little work has been done on the isolation and characterization of a certain ncRNA involved in the plant development. Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) is one of the most important commercial vegetables in the world. We are interested in learning what molecular mechanisms
0176-1617/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2006.10.002
ARTICLE IN PRESS 1098 control the pollen development of Chinese cabbage, so as to make good use of its heterosis for the cross-breeding. There must be numerous genes involved in the pollen development process of Chinese cabbage (Park et al., 2002). We have previously isolated and identified some preferentially expressed cDNA sequences during pollen development (Cao et al., 2006; Wang et al., 2004, 2005; Ye et al., 2003; Yu et al., 2004). One of cDNA fragments with the length of 232 base pair, named BBS18, was proved to be pollen-specific. Here, we obtained a gene, BcMF11, based on the BBS18 cDNA fragment. In the present study, the isolation and characterization of a novel putative pollen-preferential ncRNA from Chinese cabbage is reported.
Materials and methods Total RNA was extracted from the different tissue samples of roots, stems, leaves, flower buds, flowers, anthers, pistils and siliques of Chinese cabbage using TRIzol reagents (Invitrogen, USA) according to the manufacturer’s instruction. Flower buds were stored arbitrarily into five size groups (1.0, 1.6, 2.2, 2.8 and 3.4 mm in length) corresponding to five stages of pollen development based on the method described previously (Park et al., 2002). The rapid amplification of cDNA ends (RACE) strategy was employed to isolate the 50 and 30 ends of BcMF11. The cDNA was synthesized using the SMARTTM PCR cDNA synthesis kit (Clontech, USA) according to the manufacturer’s instruction. Based on the sequence of cloned
J.-H. Song et al. cDNA fragment (DN237921), the gene specific primers 30 GSP (50 -AACCCGAAGCGAAAGAAGAGAGAC-30 ) and 30 N GSP (50 -CAGATGGGAGGAAACAAGACT-30 ), were designed and used for 30 RACE, and the specific primer 50 GSP (50 -GGTTTTCATCCCTTAAAGTTCC-30 ) for 50 RACE. PCRs were performed in accordance with the procedure of the SMARTTM RACE cDNA Amplification Kit (Clontech, USA). The full-length cDNA of BcMF11 was amplified with the forward and reverse primer (50 -GGCCATTACGGCCGG GGGTG-30 ) and (50 -CGGGTTTCTACATTCTCCCC-30 ). All of the PCR products were purified and cloned into pGEM-T Easy Vector (Promega, USA) followed by sequencing. The sequence was compared by the BLAST program and analyzed with DNAMAN (Ver. 5.2.2) and RNA structure software (Ver. 3.2). RT-PCR analyses were carried out to determine the expression pattern of BcMF11 gene with the specific forward and reverse primers of amplifying the full-length cDNA. PCR amplifications were performed at 94 1C for 3 min, followed by 25 cycles of amplification (94 1C for 30 s, 60 1C for 30 s and 72 1C for 1 min) and by 72 1C for 7 min. A actin-1 fragment was amplified for comparison under the same RT-PCR conditions. RNA gel blot was performed according to a standard method (Sambrook and Russell, 2001). Total RNAs extracted from tissues of Chinese cabbage were separated in 1.2% formaldehyde agarose gels, transferred to a Hybond-N+ nylon membrane and hybridized with the full length sequence probe of BcMF11.
Results and discussion Based on the cDNA fragment (DN237921), a 30 cDNA end and 50 cDNA end turned out to be 418 and
Figure 1. Nucleotide sequence of BcMF11 cDNA. The primers for RACE are underlined and the orientation of each primer is pointed out by bold arrowheads. The conserved cis-acting elements are shown in boxes. The start codon (ATG) and stop codon (TAA) in the longest potential ORF are indicated by bold letters.
ARTICLE IN PRESS Identification of a pollen-specific non-coding RNA from Brassica campestris 379 bp in length by RACE, respectively. The cloned full-length cDNA of BcMF11 gene was 828 bp (GenBank accession number DQ925484) (Fig. 1). No significant similarities were found between BcMF11 and previously published DNA and protein sequences. However, high similarity (94% identity over 186 bases) was found with a Brassica napus. EST cDNA clones (data not shown). Sequence analysis revealed that BcMF11 had a high A/T content of 58.7% and a considerably lower RNA secondary structure free energy value of 203.26 kcal/mol. Furthermore, the calculated Testcode value was under 0.5. The longest potential ORF was only 228 nucleotides long coding 75 amino acids. Multiple termination codons (TAA, TAG and TGA) appeared in the six possible reading frames in BcMF11. It revealed that BcMF11 has no capacity of coding mature protein like most of identified ncRNAs in plants. For instance, CR20, ENOD40 and GUT15 can form stable RNA secondary structure, but not contain a long ORF (Campalans et al., 2004; Crespi et al., 1994; Taylor and Green, 1995). Therefore, BcMF11 may be a member of a new category of ncRNA reported firstly in Chinese cabbage. Interestingly, some cis-acting elements containing the Quantitative element AAATGA and TGGTTA, and PB core motif TGTGGTT, noted to have the strongest gain-of-function effect on expression in pollen (Twell et al., 1991), were found in the upstream site of the sequence of BcMF11 (Fig. 1). This may be responsible for enhancing the pollenspecific expression of BcMF11. RNA gel blot analysis of various tissues of Chinese cabbage demonstrated that the BcMF11 was expressed in developing flower buds, flowers and anthers at a high level, but was not detected in roots, stems, leaves, pistils and developing siliques (Fig. 2A). Identical expression patterns were also showed in RT-PCR analysis (Fig. 2B). This indicated BcMF11 is a pollen-preferential expressed gene in Chinese cabbage. The similar expression character was found in the ncRNA ZM401 isolated from maize (Dai et al., 2004). RNA gel blot analysis of different stages of pollen development showed that BcMF11 transcript first appeared at the early stage of pollen development and kept slightly increase in steady state mRNA levels at the middle and late stage of pollen development (Fig. 3A). This result was consistent with RT-PCR analysis (Fig. 3B). It suggested that BcMF11 belongs to a constant expressed pollen gene category during anther development, which is similar to the expression patterns of the NelF4a8 mRNA (Ylstra and McCormick, 1999). Therefore, it is possible
A
R
S
B R BcMF11
S
1099 L
FB
F
P
A
SL
BcMF11 rRNA L
FB
F
P
A
SL
actin
Figure 2. RNA gel blot analysis (A) and RT-PCR expression analysis (B) of BcMF11 mRNA accumulation in various organs of Chinese cabbage. RNAs were isolated from the root (R), stem (S), leaf (L), flower bud (FB), flower (F), pistil (P), anther (A) and silique (S) of Chinese cabbage.
A
1
2
3
4
5
BCMF11 rRNA
B
1
2
3
4
5
BCMF11 actin
Figure 3. RNA gel blot analysis (A) and RT-PCR expression analysis (B) of BcMF11 mRNA accumulation in different stages of pollen development in Chinese cabbage. RNAs were isolated from buds of five size groups (1.0, 1.6, 2.2, 2.8 and 3.4 mm in length), which correspond to five stages of pollen development in Chinese cabbage.
that BcMF11 play a regulatory role in the pollen development. In conclusion, we identified and characterized a novel putative ncRNA in Chinese cabbage. Our study indicated that BcMF11 expression was both pollen-specific and developmentally regulated. The cloned BcMF11 ncRNA will be a useful contribution to elucidating the gene regulation of sexual propagation in flowering plants.
Acknowledgements This work was supported by the National Natural Science Foundation of China (no. 30370975) and the Key Sci-technology Project of Zhejiang Province (no. 2005C12019-02).
ARTICLE IN PRESS 1100
References Aspegren A, Hinas A, Larsson P, Larsson A, Soderbom F. Nucleic Acids Res 2004;32:4646–56. Billoud B, De Paepe R, Baulcombe D, Boccara M. Biochimie 2005;87:905–10. Burleigh SM, Harrison MJ. Gene 1998;216:47–53. Campalans A, Kondorosi A, Crespi M. Plant Cell 2004;16: 1047–59. Cao JS, Yu XL, Ye WZ, Lu G, Xiang X. Plant Cell Rep 2006;24:715–23. Costa FF. Gene 2005;357:83–94. Crespi MD, Jurkevitch E, Poiret M, dAubenton-Carafa Y, Petrovics G, Kondorosi E, et al. EMBO J 1994;13: 5099–112. Dai XY, Yu JJ, Zhao Q, Zhu DY, AO GM. J Integr Plant Biol 2004;46:497–504. Erdmann VA, Szymanski M, Hochberg A, de Groot N, Barciszewski J. Nucleic Acids Res 1999;27:192–5. Kouchi H, Takane K, So RB, Ladha JK, Reddy PM. Plant J 1999;18:121–9. Liu C, Muchhal US, Raghothama KG. Plant Mol Biol 1997;33:867–74.
J.-H. Song et al. Mattick JS, Makunin LV. Hum Mol Genet 2006;15: 17–29. Park BS, Park YD, Kim HU, Jin YM, Kim HI. Mol Cells 2002;14:150–7. Sambrook J, Russell DW. Molecular cloning: a laboratory manual, 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2001. Taylor CB, Green PJ. Plant Mol Biol 1995;28:27–38. Teramoto H, Toyama T, Takeba G, Tsuji H. Plant Mol Biol 1996;32:797–808. Twell D, Yamaguchi J, Wing RA, Ushiba J, McCormick S. Genes Dev 1991;5:496–507. Wang YQ, Ye WZ, Cao JS, Yu XL, Xiang X, Lu G. J Integr Plant Biol 2005;47:863–72. Wang YQ, Yu XL, Cao JS. Acta Genet Sin 2004;31: 1302–8. Ye WZ, Cao JS, Xiang X, Zeng GW. J Hortic Sci Biotech 2003;78:319–23. Ylstra B, McCormick S. Plant J 1999;20:101–8. Yu XL, Cao JS, Ye WZ, Wang YQ. J Hortic Sci Biotech 2004;79:833–9. Zhang BH, Pan XP, Wang QL, Cobb GP, Anderson TA. Cell Res 2005;15:336–60.