Cloning And Expression Analysis Of CpNOD From Chimonanthus Praecox(L.)

The interaction of legume plants and nodule bacteria (azotobacter) leads to form a speacial organ where the nitrogen fixation process takes place---odule in roots, the bacteria absorb the nutrition from the host plants for thennself and fixed N2in amsphere into ammonia to supply to plants, so it formatted a legume-rhizobium symbiosis. Many specific plant genes play an important role by nodule development and nitrogen fixation. During rhizobium infection several plant protein involved in the nodules, these proteins are collectively termed nodulins,The nodulins have been classified as early nodulins anad late nodulins according to the timing of their expression during root nodule development. Early nodulins are involved in initial signaling events, infection development and nodule organogenesis. Such ENOD93is a early nodulin. ENOD93family protein have been discovered in different plants but the function is still unclear.The purpose of the present study was to clone and express the NOD gene from Chimonanthus praecox(L.), the main works are as follows:1. Cloning of CpNOD gene from Chimonanthus praecox(L.)By randomly selection sequencing of the cDNA library of Chimonanthus praecox(L.), an EST of300bp was obtained. Then by using hiTAIL PCR and RACE methods, the full length sequence of the cDNA was cloned, The full length of CpNOD cDNA sequence was611bp, with an open reading frame (ORF) of333bp. This encoded protein was consisted of111amino acids, among them17basic amino acids,9acid amino acids,49hydrophobic amino acids,26polar amino acids, and the content of C+G is47.45, the isoelectric point is9.87, so the protenin belongs to basis protein. The secondary structure is composed of69.09alpha-helix,6.36%beta-sheet, and37.3%coil. Sequence alignment and phylogenetic analysis showed this gene was high identical to early nodulin ENOD, belonging to ENOD93superfamily early nodulin protein gene, named as CpNOD(Gen Bank accession No.JQ622290).2. Relative expression analysis of a quantitative real-time PCRThe expression studies by real-time quantitative PCR showed that CpNOD was constitutively expressed in roots, stems and flowers under normal conditions, and it expressed higher in flowers than in other organs, whereas it was expressed the highest in middle flaps; during blooming stage the expression is correlation with the mature of the flower and it expressed the highest in wither period. All these transcriptional expression analysis results suggested that CpNOD may play a positive role in blooming physiology of Chimonanthus praecox(L.) and N responses.3. Construction of expression vectorLink the ORF fragment of CpNOD into vector pMD-19T, then using restriction Proteinase BamHI and Sacl clone and construct expression vector PCAMBIA2301, PCR and digestase revealed that the target gene has been transformed successfully into expression vector.4. Obtaining the positive transgenic tobacco plantUsing Agrobacterian tumefacieus the expression vector was transformed into wild tobacco plants, it showed positive by GUS staining and PCR detection.