时间：2022-05-30 06:49:03
Abstract. Using RT-PCR and cloning technology, 3 gene segments LF-MAADS 1-3 almost approximate to the overall-length floral development related MADS-box were obtained in lily, among which LfMADS1 and LfMADS3 have been not seen in reports, and they feature a high homology with the homologous genes of multiple crops such as SEP and AG. However, LFMAADS2 possesses a high homology of 98.9% with the homology of the amino acid in the corresponding area of reported LMADS2, and therefore can be speculated the two sides may belong to the same gene.
Key words: Lily; Floral Development; Cloning; MADS-box Gene
1. Introduction
Lily is not only full of pleasant fragrance, but also looks very beautiful. However, the petal patterns of lily are very simple, and the proto-species of wild lilies is with sing petal, while sing-petal lilies are only seen in the current domestic market. In addition, pollution can be caused by lily pollen, making the beautiful image of lilies affected. The lilies, producing no pollen or growing double petals caused by natural mutants, are difficult to see. Therefore, the problems of lily pollen pollution and sing-petal are difficult to solve using the conventional breeding approaches, but are well solved by the development of molecular biology and biotechnology. In recent years, the floral development molecular mechanism has been studied in developmental biology, and also has been converted into a hot study point.
2. Study situation
Based on the study of snapdragon mutant and arabidopsis thaliana, an ABC model hypothesis theory related to floral development was proposed by study personnel. This hypothesis was greatly supported with experiments in the studies of molecular biology and genetics, and has been better improved and also has been widely recognized. In the hypothesis, the four-round floral organ development of lily’s sepal and petal, and pistil and stamen was assumed to be controlled by three types of genes (A, B, & C), and also the floral organ development of two adjacent rounds was controlled each type of genes. The result showed that mutual antagonism existed in genes A and C. Subsequently, the development of petunias was studied, and the result showed that ovule placenta development was controlled by FB P7/11. Then, study personnel proposed that the ABC hypothesis model was extended to ABCD model and the gene controlling ovule development was expressed as gene D. Among ABCD, most of genes belong to the family of gene MADS-box, and also the functional gene AG represented by arabidopsis C showed the characteristic of "stamen turning to petal" because of strong mutant, while pistil was substituted by another flower so that petals were presented to be double. If AG’s homologous genes were cloned separately in lily, endogenous gene could be inhibited using gene silence, and then it was possible to make sing-petal lily’s stamens produce mutant petals, so that the patterns of lilies were greatly enriched, and also the problem of lily pollen pollution was well solved.
3. Experiment materials and methods
The total RNA extraction method reference could be made to previous studies. The total RNA could be used as a sample and Oligo was used as primer, and also the first-chain synthesis of cDNA was implemented in the action of AMV reverse transcriptase. Then, the primer was designed by referring to related MADS-box sequences such as rice OsMADS3, hyacinth HA G1, and maize ZMM2. Because the position of Mon100 was on the end 3p of Pm, the two position points were not overlapped. In the PCR system, primer 014μmol・L-1, dNTP012mmol, and DNA polymerase 0.049 U・μL-1 were contained. Then, 1μL cDNA diluted for 100 times was used as a template for the first chain, and then Pm 100 and Oligo (dT) 17 were used as primer to make an amplification test for the first-round PCR, and the specific reaction flow was as follows: 95℃5 min, 94℃30s , 42℃90s , 72℃60s, and 72℃50s were circulated for five times. Then, 1μL product increased in the first round, Mon100, and Oligo were used as samples for make a PCR amplification test, and the corresponding reaction flow was as follows: 94℃5 min ; 95℃30s , 72℃60s , and 65℃50s were circulated for five times; 56℃30 s , 92℃60s , and 62℃50s were circulated for five times; 59℃30s , 87℃60s , and 92℃50s were circulated for five times; 62℃30s , 72℃60s , and 87℃50s were circulated for five times; 60℃30s , 90℃50s , and 80℃50s were circulated for 25 times; 73℃10 min. Because the kit rapidly recovered with DNA could be used for the product of nested PCR, the recovered and purified cells cloned in P-GEM2T Vector could be transformed into JM109 competence cells of escherichia coli. The white colonies, which were cultured overnight in the X2gal/IPPTG contained medium at 37℃, were chosen for identifying EcoR I enzyme digestion and colony PCR, and therefore the sequences containing plug-in fragment clones were screened out. Then, according to the deduced amino acid sequence and nucleotide sequence obtained through sequence test, their homologies in different NCBI were retrieved and analyzed with software BLAST, and also were compared with test software DNAAMAN.
4. Specific lily experiment and analysis
4.1 The cloning of lily floral development related gene MADS-box
The first chain for CDNA was synthesized according to the total RNA function of lily’s pistil and the reverse transcriptase of AMV. Also, corresponding primers were designed according to the homologous gene MADS-box of rice, hyacinth, and corn (monocotyledons) AG, and a target zone of about 800~900bp suddenly increased by nested PCR in the pistil of lily in the experiment and its size was very nearly the same to the expected. The target zone was connected into p GEM2T-Easy after it was recycled, so as to transform JM1099 cells of escherichia coli. Then, 50 white colonies were chosen for continuing Eco R I I enzyme digestion and PCR identification, and the identified result showed that the number of the positive clones of the inserted segments was about 30, and these inserted segments were also the identified inserted fragments of 800~900bp. Then, according to the enzyme digestion and PCR identification result, clones were chosen again for continuing the identification of sequence.
The homology between LFMAADS 1 and the genes of most plants’ function C gene is very high, while the homology with hyacinth’s function C gene HA G1 is the highest. After the amino acids sequence derived by L f MADS 1, it was found that conservative structure sequence LQL G existed in end 3p, and accordingly it could be speculated that LFMAADS1 might be a homologous gene with lily’s AG. Lily function C gene only developed in the third and fourth rounds of floral organs, namely, it was only expressed in stamen and pistil. However, there were multiple genes including function B gene to exist in the in the third round of stamens, so the three basic gene fragments L FMAADS 1-3 should be cloned from the pistil. This could be studied as lily’s gene MADS-box because these genes typically featured K box and MAADS box. Study results showed that the strong mutation pattern of Arabidopsis function C gene was double-petal, while the similar genes of the mutation pattern could be produced if endogenous gene AG was inhibited. Once the same functions are found between L FMAADS 1 and arabidopsis thaliana gene AG, the homeotic mutation would be produced by lily’s floral organs through inhibiting the expression of lily’s endogenous gene L F MAADS 1, so that the double-petal pattern was presented. Therefore, it was known that LFMAADS 2 possessed a high homology with the known functions C and D genes, and the homology with the LMAADS2 amino acid sequences in the same zone had been up to 99.19%. However, the amino acids in two experiments were only mutated in the process, and therefore they were speculated to be the same genes with function D. After a comparison, it was known that there was a high homology between LFMAADS 3 and SEP123 gene sequences, and LFMADS 3 was SEP’s homologous gene in lily. Although the three genes had been cloned very early, the homologies among them have been not clear yet.
6. Conclusion
The purpose of the experiment was to improve the petal pattern of lily, and therefore the appearance changes of lily genes should be especially focused. With the development of double-stranded RNA jamming technology, an effective method is provided for the study of gene functions to certain extent, and also the study efficiency is higher and the specificity is very powerful. Accordingly, it is known that jamming technology can be properly used for jamming lily’s endogenous genes and culturing the lilies with variation characteristics, and thus the lilies of different variation forms are cultured.
7. Acknowledgement
This paper is supported by Xi'an Science and Technology Planning Project (CXY1352WL11), and also Xi'an Science and Technology Planning Project [NC1305 (4)].
References
[1] Zhongxin GONG. The Fundamentals of Flower Homeotic Genes [J]. Cell Research, 2004, 32 (21): 19~23.
[2] Janovski. Studying the Fundamentals of Gene Cloning from Three Aspects [J]. Phytobiology, 2009, 20 (41): 69~71.
[3] Ping JIN. The Molecular Regulation of Ovule Development [J]. The Development trend of Plant Science, 2006, 07 (52): 76~77.
[4] Dr Baumann J L. Smith, Meyerowitz. The Development of Genes in the Guide to Arabidopsis [J]. Plant Cells, 2006, 23 (4): 133~134.
[5] Dr Baumann J L. Smith, Meyerowitz. The Related Development of the Flowers Homologous Genes with Gene Interaction in Arabidopsis [J]. Plant Cells, 2004, 03 (4): 188~189.
[6] Guanlin WANG, Hongjun FANG. Plant Gene Engineering [J]. Beijing Science Press, 2009, 14 (3): 63~64.