Cloning And Expression Analysis Of Pyrus Spp Genes Related To The Aromatic Constituents Biosynthesis

Pyrus spp,is one of fruits loved by comsumer in the world,in China,it has a long history of cultivation,it has the very high nutritional value and health care effect. Volatile compounds is an important index of fruit quality,it is also a main factor of attract consumers and strengthen market competitiveness.By means of analyzing the volatile compounds of the7Pyrus ussuriensis Maxim and6Pyrus serotina germplasm resources, we excavated some excellent pear germplasms which had high content of main monoterpenes,and on this basis, we cloned two genes PuFS and PpAAT which related to the monoterpene biosynthetic pathway by using ’Nan guo’ and ’Jin er shi shi ji’ which had high content of main monoterpenes for test material, then expression patterns of the two genes in different developmental stages and different organs of Pyrus spp were researched, and the relationship of the Pyrus spp volatile substances accumulation and the gene expression were analyzed too. The main results are as follows:1^The floral headspace compounds of the commercial stage fruit of7Pyrus ussuriensis Maxim、6Pyrus serotina germplasms were isolated by an automated headspace sampler with built-in trap, and followed by gas chromatography-mass spectrometry for identification and quantification. The results show that,(1)10、22、25、19、26and25volatile compounds were identified from the fruit of7Pyrus ussuriensis Maxim germplasms.The main floral components identified were aldehydes、esters、alcohol and alkenes.Aldehydes were the main floral components of’Rong xiang’、’Long xiang、’Xiang shui’ and ’Wan xiang’, esters were the main floral components of ’Nan guo’、’Da nan guo’ and ’Han xiang’.The fruit of ’Nan guo’ had more α-farnesene,but the others were without or had a little.(2)21、15、17、23、19and18volatile compounds were identified from the6Pyrus serotina germplasms commercial stage fruit, ethyl caproate is the main floral component in ’Jin er shi shi ji’, on the contrary, hexanal were the main floral components in other5pears.(3) The different mature period(green stage、commercial stage、ripe stage) of ’Er shi shi ji’ and ’Jin er shi shi ji’ fruit were used to do more research for the different of volatile compounds.Analysis by synthesis,hexanal was the main compounds in the3different mature period of ’Er shi shi ji’ and the green stage of ’Jin er shi shi ji’, however,ethyl caproate becomed the most compounds in the commercial stage of ’Jin er shi shi ji’, and the full ripe stage had more,this may was the reason of ’Jin er shi shi ji’ was the excellent pear germplasms which had high content of main monoterpenes. 2、Total RNA of ’Nan guo’ and "Jin er shi shi ji’were extracted using the CTAB method, then the full sequence of PpAAT and PuFS gene which related to the mnoterpene biosynthetic pathway were cloned by RACE method. The full length of PpAAT was1648bp, with the start codon, the complete open reading frame (1316bp), termination codon, the5’non-coding region (79bp), the3’non-coding region (242bp) and poly (A) tail (11bp), which encodes473amino acids, and the Genbank No. is KF887152; the full length of PuFS was1505bp, with the start codon, the complete open reading frame (1240bp), termination codon, the5’non-coding region (145bp), the3’non-coding region (104bp) and poly (A) tail (11bp), which encodes460amino acids, and the Genbank No. is KF887153.3、’Nan guo’ and ’Jin er shi shi ji’ were used as plant material to study the expression patterns of PuFS and PpAAT in different developmental stages and different organs of Pyrus spp by RT-PCR.(1)We found that the expression level of PpAAT in ’Jin er shi shi ji’ was,gradual decline along with fruit ripening in pericarp,the green stage was the highest,the commercial was begin to drop,and dropped rapidly to ripe stage, the expression level was a little rise from green stage to commercial stage,and then dropped rapidly to ripe stage in sarcocarp.As a whole, the expression level of PpAAT in commercial stage was higher than the other two stages,and the pericarp was higher than the sarcocarp.(2)The expression level of PuFS in ’Nan guo’ was the highest in green stage and the lowest in ripe stage among the3stage in pericarp, from green stage to commercial stage, the expression level was a little drop,and to ripe stage dropped rapidly; The expression level in sarcocarp was no change in the3satge,and all of them was very low. As a whole,the expression level in green stage was higher than the other two stages,and the pericarp was obviously higher than the sarcocarp.