Cloning Of The Suspensor-specific Genes

In Angiosperm, zygote's first division is asymmetric, forming the apical cell with dense cytoplasm and the large, highly vacuolate basal cell, which form the suspensor after transverse division. The apical cell differeninates into the embyro proper ,which will eventually differeninates into the apical meristem, cotyledon, hypocotyl, radicle and part of the radicle tip meristem. Differentiation of embryonic development and pattern formation in different regions are complex, many genes are involved in this process.What causes the apical and basal cells to become specified and follow different developmental pathways is not known. By mutants, it has identified and cloned a number of genes that play key roles in embryo development in plants .It revealed that the embryo development in plant are regulated by multiple genes.Known to all, regulation of the gene expression mainly occurs at the transcriptional level . Transcription factors become the determinants of transcription efficiency through binding and interacting with promoter of the target genes or the cis-acting element in enhancer region. At present, it has identified several suspensor-specific gene like WOX8, WOX9, PtNIP1, G564, etc.Some studies showed that a Divergent 10-bp Motif Sequences (GAAAAGCGAA short sequences) upstream the G564 gene can program transcription in the Suspensor.The purpose of this study is to identify the regulatory factors combine with the A section (GAAAAGCGAA short motif) of the G564 suspensor-specific gene promoter by yeast one-hybrid technology.we construct the bait vector pHIS2.1-G564-5A (known as P5A)of the suspensor-specific cis-acting elements (5,-GAAAAGCGAA-3,) and test the bait vector in different types of SD media; we also construct the cDNA library of Arabidopsis thaliana immature seed. Double-stranded cDNA with SMARTTMIII and CDSIII anchored in the end ,linear pGADT7-Rec2and pHIS2.1-G564-5A were transformed into the Y187 yeast competent cells.Positive clones were analyzed. The major findings are as follows:1.synthesize two antiparallel oligo-5A nucleotides(GAAAAGCGAA-GAAAAGCGAA-GAAAAGCGAA-GAAAAGCGAA-GAAAAGCGAA) and connected the DNA sequence to the pHIS2.1 vector, named P5A bait vector. Reverse sequenceanalysis on the P5A , DNA sequencing results show that 5 repeat of the core sequence (GAAAAGCGAA) section have been constructed onto pHIS2.1 vector.Plate diluted transformed cellson SD/-Trp;SD/-His/-Trp;SD/-His/-Trp /50mM 3-AT agar medium. The results show that the bait vector P5A can induce the expression of His marker gene, but the 3-AT can inhibit the expression of His. P5A can be the bait vector for the yeast one-hybrid next step.2. RNA extraction from Arabidopsis thaliana and construct cDNA library(1)Total RNA was extracted from immature seed of Arabidopsis thaliana and Nicotiana tobaccum using four different methods . Trizol reagent resulted in low quantity and poor quality of RNA. The other three methods could isolate RNA from immature seed with quality that match the downstream experiments. Among these methods, CTAB-Isopropyl alcohol method could get the highest yield of total RNA, with the advantages of time saving and low cost. Bioteke kit is also a convenient method to isolate immature seed RNA under room temperature. CTAB-LiCl method could get higher quality total RNA without genome DNA contamination. However, LiCl couldn't precipitate small molecule RNA well. The quality of total RNA isolated using the above three methods was analyzed with UV spectrophotometer and agarose gel electrophoresis. The OD260/OD280 ratio was 1.80-1.97 and OD260/OD230 ratio was 2.03-2.37. 28S rRNA and 18S rRNA were clearly detected in the RNA electrophoresis.The results showed that , CTAB-Isopropyl alcohol method could get the highest yield and quality of total RNA. Using this method to extract total RNA, and then prepared with SMARTTMIII and CDSIII anchored at the end of double-stranded cDNA.ds cDNA clearly distributed in the 100 bp ~ 5000 bp.thecDNA library construction can be the next step.(2)Double-stranded cDNA with SMARTTMIII and CDSIII anchored at the end 20μl (2-5μg),6μl linear pGADT7-Rec2(3μg)and pHIS2.1-G564-5A (5μg) were transformed into the Y187 yeast competent cells.Diluted the transformation in different auxotrophic medium to verify the results show that the bait vector P5A, both protein expression vector pGADT7 vector was transformed into the yeast Y187 cells.3.Pick the single colone on the third time restreaked SD /-Trp /-His /-Leu +100 mM3-AT agar plate,isolate plasmid DNA and for PCR verification. Results showed that the size of all fragments between 500bp and 2000bp.Recyle target segment and diggest the target segment onto the pMD-18 vector and transformed into E. coli DH5α. Test the positive clones and analyze.