| DELLA proteins located in cell uncleus in botany, fuction be a repressor in GA cell signaling process. More than that, DELLA proteins widely participate in physiology processes such as bonany photomorphogenesis, plant environment cell singnaling. and some other phytohormones responing to plant growth regulation. Relatedgenes about DELLA proteins research relatively late in China, but it is played close attention to by the researchist. For the past few years,a batch of DELLA genes were cloned, GA receptor gene in Maize, DELLA downstream gene GASA gene family in Arabidopsis. SLR1 gene in rice, SmGAI gene in Eggplant, GhRGL gene (RGL-LIKE) in Gossypium barbadense, and so on. Furthermore, correlational research about the construction of Vector, gene expression and functional analysis were carrying out. At present, there have not been relative research reports about DELLA gene. We cloned DELLA genes in loquat of different varieties or different-ploidy in the same variety by the method of Homologous sequence cloning. Then we expanded the overall length by RACE, and analysed the sequences by alignment. And then we did the preliminarily quantitative analysis about DELLA genes express in loquat by RT-PCR amplification.The results of the study will establish the theoretical foundation for further study of conducting functional identification, space-time gene expression and some other relative researches about DELLA proteins in loquat. The results showed as following.1. Improved the extracting method of DNA and RNA in LoquatExtracting the DNA by the method of CTAB improved method I and CTAB improved methodⅡ, and extracting the RNA by the method of improved CTAB-LiCl method of precipitation, we obtained DNA and RNA with higher quality, basically overcoming negative influences of epidermis with more fuzz and secondary metabolites, such as polysaccharide. polyphenol. protein, and so on, totally meeting the demand of basical molecular biological experiments, and guaranteeing the experiments follow-up to go to well.2. Homologous sequence cloning of DELLA genes in loquat (1) Obtained the optimum system PrimeSTARTM HS DNA Polymerase specific PCR amplification by orthogonal test screening (Total 20μL):5×PrimeSTARTM Buffer(Mg2+ plus) 3μL, dNTP Mixture (each 2.5μL) 1.6μL, Forward primer 0.4μL, Reverse primer 0.4μL, templat DNA /cDNA 0.4μL, PrimeSTARTM HS DNA Polymerase (2.5 U/μL) 0.2μL. ddH2O up to 20μL.(2) Devising a pair of degenerate primers according to the DELLA gene sequences in Malus logined in NCBI, we gained there fragments (temporarily named Sequence Al-A3)in "dawuxing"(A), and other there fragments(temporarily named Sequence B1-B3) in "longquanNO.1"(B),almost with 700bp.by DNA PCR amplification and clone. The results of blast showed there had been more than 90% identities between these fragments and DELLA genes sequences in Malus,and they were exon sequences, not containing introns.(3) Devising a pair of peculiar primer according to Sequence B3 in "longquanNO.1", we gained two specific fragments, with 731 bp. by PCR amplification of gDNA and cDNA in diploid and triploid of "longquanNO.1". The results of blast showed they both were DELLA genes fragments with mononucleotide variation, one of them was accordance with the SequenceB3. and another had the 94% identities with it. temporarily named SequenceB3a.(4) Devising a primer according to Sequence B3a for RACE amplification, we gained 3' fragments with 646bp. The results of blast showed that there were 97% identities between MdRGL3a (GenBank accession:DQ007887) with it, and 92% identities between MdRGL3b (GenBank accession:DQ007888) with it.3. Sequences analysis about DELLA genes in loquat(1) By alignment of DELLA fragments in loquat from different varieties, the results showed that there were more than 60% identities among the 6 fragments in "dawuxing" and "longquan No.1" There were multiple mutants nucleotide among the 3 fragments between the two varieties, and also been among the 3 fragments in the same variety.(2) By alignment of DELLA fragments in loquat from different-ploidy in the same variety, the results showed that both SequenceB3 and SequenceB3a were cloned from gDNA in "longquanNo.1" diploid. while only SequenceB3a from cDNA from it, and both of them were cloned from both gDNA and cDNA in "longquanNo.1" triploid. On these grounds, we inferred that SequenceB3 should occur gene silence in diploid, and should express in triploid.(3) Structuring molecular evolutionary tree of together these 7 fragments in loquat with DELLA genes sequences in Malus in Rosaceae login in Genbank, the results showed that the tree separated into three evolution branches, these 7 fragments in loquat nested into them.(4) We gained SequenceB3a 3' fragments in "longquan No.1" by RACE amplification, with 1095 bp. containing terminator codon TAA. and a no translation region. The effective translation region translated into 304 amino acids in C terminal, with 915bp nucleic acids. There were highly identities between other varieties reported and this C terminal.4. Preliminary expressing analysis of DELLA genes in loquatSemi-quantitative RT-PCR was used to validate the actual expression of DELLA homologous genes of diploidy and triploidy "Longquan No.1" in leaves and flowers at different times during development. The results indicated that the DELLA homologous genes have expression in all tissues. The expression in triploidy was higher than in diploidy and increases in flowers during the development. The DELLA homologous genes were over expressed in the ending blooming compared with diploidy.
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