Cloning And Expression Analysis Of Lmp5CS Gene From Lycium Chinense Miller

Lycium chinense Miller in northwest China is the characteristic ofthe salt-tolerant plants, has a strong resistance. Cloning the key enzyme gene ofcoding proline, has important significance to Lyceum chinense Miller resistancemechanism, improvement and evolution of P5CS gene. And this provides material forDNA shuffling, using molecular techniques to complete gene evolution of thousandsof years, to lay the foundation of obtaining powerful resistance gene. The results wereas follows.1. After salt stress, extracted total RNA，full length cDNA sequence of a putativeΔ′-pyrroline-5-carboxylate synthase gene（P5CS）was cloned from Lyceum chinenseMiller leaves using RT-PCR and3′rapid amplification of cDNA ends（RACE）,namedLmP5CS,then connected to clone vector pMD-18T and transferred to E.coli.Sequenceanalysis showed that full-length cDNA of LmP5CS is2245bp,the complete openreading frame（ORF）of this gene is2154bp, encoding for a protein of717aminoacids with an isoelectric point of6.07and a molecular weight of77.5kD. LmP5CScontain6main functional domains of higher plant P5CS potein:ATP binding site,conserved Leu zipper, conserved Glu-kinase domain, NADPH binding domain,putative Leu domain and conserved GSA-PH domain.Potein module analysis showedthat P5CS conserved domains had been predicted, and significance module is alsoconserved Glu-kinase domain and conserved GSA-PH domain. This shows thatLmP5CS has Glu-kinase and GSA-PH domains.2. Constructed plant expression vector pH7m24GW,3rc-LmP5CS, by bacterialcolony PCR, extracting plasmid and enzyme digestion, proved purpose gene hadconnected to expression vector correctly. Obtained Agrobacterium tumefaciens C58that cotains plant expression vector pH7m24GW,3rc-LmP5CS and prepared forsubsequent transformation.3. By leaf discs transform plant expression vector pH7m24GW,3rc-LmP5CS totobacco, obtained PCR and RT-PCR positive transgenetic lines. Through the prolinecontent drawing standard curve for linear regression equation, usingspectrophotometry to measure free proline in transgenic positive plants underdifferent conditions,the result showed that proline rate of transformation plants werehigher than that of wild-type plants under NaCl stress, indicating that the transgenicplants have salt resistance.