Cloning And Expression Of Genes Related To Polyamine Metabolism In Exocarp Of Russet Skine Mutant Pear (Pyrus Bretschneideri Rheid.) And Construction Of The Transgenic Vector PBll21-SPMS

Pear is a nutrient-rich,very popular fruit.It's appearance quality including the shape,the skin color and the smooth surface.However,the color of the shin is a important factor in the fruit qualty.A good skin color could have a stronger market competition.The polyamine is an important substances in the plant growth.It is involved in all aspects about the growth and development in plant.What's more,it also has an important role in the resistant of the bad environment.To investigate the formation mechanism of the russet mutant of Dangshansuli.The peels of‘Dangshansuli' pear and its russet mutant ‘Xiusu' in different days after full bloom(DAFB)were used to determine the contents of polyamine by High Performance Liquid Chromatography(HPLC)in present study.The key genes in polyamine biosynthesis were cloned by rapid amplification of cDNA ends(RACE),and their bioinformatic analysis were conducted by the protocol of Protparam website(http://web.expasy.org/protparam/)and the phynogenic tree of putative proteins was constructed by MEGA5.0 software.The relative expressions of ADC,SPDS,SPMS and ACL5 genes at different stages were analyzed by using real-time quantitative reverse transcription-PCR(qRT-PCR).The expression vector of SPMS gene was also constructed.The main results had been achieved were as follows.1.The putrescine contents in the exocarp of ‘Dangshansuli' were significantly higher than in ‘Xiusu' except for 50 DAFB and 150 DAFB.The results showed that the trendline of Spd and Spm in exocarps of ‘Dangshansuli' and the russet mutant‘Xiusu' at different days after full bloom increased at first,then decreased after.At the 75 d,the contents of Spd and Spm in the exocarp of ‘Xiusu' were higher than‘Dangshansuli'.The contents of Put were high in the early days after the full bloom,and then decreased after.Later,the Put increased.At the 75 d,the contents of Put in in the exocarp of ‘Xiusu' were higher than that in ‘Dangshansuli'.2.The exocarp of ‘Xiusu' cDNA as template,RACE was used to get the sequence of ADC?ODC?SPDS SPMS?SAMS.The full length of ADC is 2,193 bp.527 amino acids were coded.The Gen Bank accession of ADC is KM923903.The full length of ODC is 1,278 bp and 527 amino acids were coded.The GenBank accession of ODC is KP144199.The full length of SPMS is 1,110 bp and 242 amino acids were coded.The GenBank accession of SPMS is KM923906.The full length of SPDS is 909 bp and 221 amino acids were coded.The Gen Bank accession of SPDS is KM923905.The full length of SAMS is 1,182 bp and 521 amino acids were coded.The GenBank accession of ADC is KM923904.The structure of putative proteins of ADC,ODC,SPDS,SPMS and SAMS gene in pear was closer to that in apple with the homonogy of91.9%,96.6%,97.3%,90.8% and 94.6%,respectively.ADC?SPMS?SAMS were all water-soluble by the prediction of their physicochemical properties.3.The results of real-time PCR showed that the expression of synthesis genes of polyamine in the exocarp of ‘Xiusu' were higher than that in ‘Dangshansuli' at 50 d.It had significant difference in the polyamine contents between in the exocarp of‘Dangshansuli' and its mutant ‘Xiusu'.ADC,SPDS and SPMS increased at first,then decreased later.This trend was in agreement with the contents of polyamin.The expression of ACL5 was different from the other genes,which was higher in the exocarp ‘Xiusu' than that in ‘Dangshansuli' at 75 d.4.Based on the full lenth of SPMS,the expression vector of SPMS was constructed.We inserted two restriction enzyme cutting sites on both sides of SPMS and the seeds were collected.