‘库尔勒香梨’自交不亲和S-RNase等位基因全长的克隆与分析

Cloning and Analysis of Self-incompatibility S-RNase allelic Genes in Korla Fragrant Pear

‘库尔勒香梨’是新疆重点发展的特色果树品种之一,具有自交不亲和性。本研究以新疆轮台国家果树资源圃栽培梨品种‘库尔勒香梨’为试材,利用梨S基因的保守序列设计合成的引物:‘FTQQYQ’/‘antiIIWPNV’初步确定‘库尔勒香梨’S-RNase等位基因型后,设计等位基因特异性引物,利用RT-PCR克隆两个花柱S-RNase等位基因cDNA片段,RACE(cDNA末端快速扩增)技术进行cDNA全长克隆,采用BLAST进行序列比对,Protparam等在线软件分析两个S-RNase等位基因的编码蛋白特性。根据NCBI上已登录的63条梨属S-RNase基因的全长序列,用DNAMAN软件对其氨基酸序列进行比对并用MEGA 4.0构建进化树。本试验从‘库尔勒香梨’中克隆得到了S_(22)-RNase(Gen Bank接受号:KX214125)/S_(28)-RNase(Gen Bank接受号:KX214124)等位基因cDNA的全长序列,为分子手段调控梨自交不亲和性状奠定了基础。S_(22)-RNase基因cDNA全长904 bp,ORF(开放阅读框)长684 bp,编码227个氨基酸;S_(28)-RNase基因cDNA全长921 bp,ORF长687 bp,编码228个氨基酸。BLASTP比对显示,这两个基因都具有保守的RNase-T2基因结构,属于RNase-T2家族。预测相对分子质量分别为25.6 k D和25.9 k D,等电点9.36和9.30,都属于亲水性蛋白。进化分析表明,‘库尔勒香梨’的S_(22)-RNase和S_(28)-RNase处在不同的两个分支上,表明两基因亲缘关系较远。

Korla fragrant pear, which exhibits S-RNase-based gametophytic self-incompatibility （GSI）, is a unique and famous cultivar in the Xinjiang Uyghur Autonomous Region. Pistils were collected from Korla fragrant pear growing at the Luntai National Fruit Germplasm Resource Garden in Xinjiang. Genomic DNA and RNA were extracted from leaves and pistils in Korla fragrant pear. The primers ＇FTQQYQ＇/＇anti-IIWPNV＇ were referenced to comfirm the S-RNase allelic genes of Korla fragrant pear, and gene-specific primers were designed. The S-RNase allelic genes cDNA fragments were obtained by RT-PCR technology and the full length sequences were obtained by RACE technology. The characteristics of putative proteins were analyzed using Protparam software. 63 complete sequences of S-RNase genes in all Rosaceae pyrus were selected from NCBI and a phylogenetic tree was constructed using MEGA 4.0 software. Two full length cDNA of S22-RNase gene （GenBank acceptance number： KX214125） and S28-RNase gene （GenBank acceptance number： KX214124） were cloned. This work laid a foundation for the improvement of self-incompatibility in Pyrus by molecular biology methods. The full length cDNA of S2cRNase was 904 bp which contained an ORF （open reading frame） of 684 bp and encoded a protein of 227 amino acids. The full length cDNA of S2s-RNase was 921 bp which contained an ORF of 687 bp and encoded a protein of 228 amino acids. It was predicted that both of the two genes had the RNase-T2 motif by BLASTP. The molecular mass of two S-RNase genes were 25.6 kD and 25.9 kD, and isoelectric point of two S-RNase genes were 9.36 and 9.30, respectively. They all were hydrophilic proteins. The phylogenetic analysis indicated that S22 and S28 from Korla fragrant pear laied different branch and possessed high polymorphism.