Study On S-gene Of Pear Cultivated In The Protection District Of Dangshansu Pear Germplasm Resources

There were abundant pear germplasm resources in Dangshan County,Anhui province and the county was named the Pear City in China.The yield of Dangshansu pear held the great share in the world pear market.Many new biotypes of pear(Prus spp.)had been forming in the long history of pear evolution because of their complex genetic background and a lot variant from natural mutation and hybridization between species.Study of self-incompability gene(S-gene)in pears would be very significant to discuss the molecular mechanism of self-incompability and utilize the germplasm resources for accelerating the program of pear breeding.Specific primer PCR was a gene cloning technique with high efficiency,stability and accuracy.In the experiment the PCR specific primer(P1 and P2)were synthesized according to conserved region of S-gene two sides.The system suitable for pear S-gene was established according to contrasted experiment of the main factors involved in specific primer PCR technique system.The reaction system of specific primer PCR is in total volume of 50μl that including ExTaq DNA polymerase 1.0 U,Mg²⁺0.10μm,primer 0.02μm,template DNA 30 ng and dNTPs 0.02μm.The optimal reaction parameter of specific primer PCR were preliminary denature 3 min at 94℃,denature 15 sec at 94℃,anneal 30 sec at 52℃,extension 2 min at 72℃,30 cycles,extension 7 min at 72℃at last.The S-gene of 27 biotypes of pear cultivated in Dangshan Country were amplified and isolated by established specific primer PCR technique system.The results showed that there were 20 S-gene bands amplified in 17 pear cultivars That was Zisu,Niitaka,Kuerleixiangli, Xuehuali,Hosui,Cili,Nanguoli,jizhuahuang,Ehuang,P.betulaefolia Bge,Xiangmianli, Fusu,Red Bartlett,Xiusu,Matihuang,Xipisu and Lianglizais.Of which two S-gene bands had been amplified in Zisili,Laiyangcili and Kuerleixiangli.The longth of amplified fragment was 300～500 bp.No S-gene band had been amplified in the rest ten cultivars which was Hongxiangsu,Huangguan,Zaomeisu,Biansu,Baguaisu,Youpisu,Wangsu, Dangshansuli,Lensu and Yali.In the experiment S-gene production isolated primarily were amplified secondly then purified and cloned for sequence measuring.Sequence compared comparability using BLAST software and S-gene clustering analysis using ClustalW software.The result showed that there were 14 different S-gene types among 20 S-gene sequence and one S equipotential gene of 14 cultivars and S-gene types of three cultivars.In the experiment the methods including S-gene isolation purification and legation transform and PCR identification and so on were mended to provide new technique in order to improve studying on S-gene...