Cloning And Expression Analysis Of CmGnT Gene In Cucumis Melo L.

Melon?Cucumis melo L.?belongs to the genus of Cucumis and the family of Cucurbitacea.It is one of the world's ten important fruits,which is also a kind of high efficient and economic fruit crops.In the melon cultivation,the adversity stress seriously affected the growth and development,yield and quality of melon,which hindered the sustainable development of melon industry.Study on N-acetylglucosaminyltransferase?CmGnT?in plants was less,and the regulation mechanism of this gene for melon under salt stress was still unclear.This study through the operation of cell engineering and genetic engineering technology of CmGnT gene from melon was cloned and analyzed.The expression vectors and RNAi interference vector were structured.This research willed lay a good technical foundation for future melon breeding work.Main results of this experiment were as follows:1.Primers were designed according to cDNA sequence of melon,and CmGnT was cloned by using RT-PCR technique.The full-length cDNA of CmGnT was 2193 bp,and the open reading frame?ORF?was 1179 bp,encoding 392 amino acids.The molecular weight of CmGnT protein was 46KD,and the theoretical isoelectric point?pI?was 7.93.The secondary structure comprised of 32.14%a-helix,26.53%extended strand,14.29%?-folding and 27.04%random coil.Protein structure analysis showed that CmGnT contained the beta-1,4-N-acety lglucosaminy transferase domain,which belonged to the glycosyltransferases 17 family.The protein had 1 strong transmembrane helices.Phylogenetic analysis showed that CmGnT and CsGnT?accession number:XP₀04135275.1?had the closest genetic relationship,and the homology was 99.74%.Homology with watermelon was 97.70%.2.The qRT-PCR analysis showed that the amount of gene expression in roots was higher than those in leaves and stems.And the expressions of CmGnT in leaf were significantly up-regulated under NaCl,PEG,H2O2 and ABA stresses,which indicated that CmGnT was induced by abiotic stresses.The results suggested that this gene in melon played an important role in response to abiotic stresses.3.The expression vector was built transformated into onion endepidermis cell by particle gun.The CmGnT protein was located in the cell nucleus.4.The CmGnT gene was subcloned into plasmid vector pBI121 by SmaI single enzyme digestion,and over expression vector pBII121-GnT was constructed.A 238 bp of CmGnT gene specific sequence was chosen to construct the RNAi vector pFGC1008-CmGnT.