The Cloning,Expression And Function Researches Of SERK Genes In Malus

Malus hupehensis, common names Chinese crab apple, Hupeh crab or tea crabapple, is a species of flowering plant in the apple genus Malus of the family Rosaceae. It is native to China. Malus hupehensis (Pamp.) Rehd. is naturally distributed in different provinces in China and cultivated as a rootstock and an ornamental plant. Mainly we had used the apomictic plant of 'Pingyi Tiancha' (Malus hupehensis (Pamp.) Rehd. var. pingyiensis Jiang),which originates from Pingyi County in the Shandong province of China, and the dwarf and leaf-wrinkled tetraploid hybrids, which were obtained in early study from the cross of 'Pingyi Tiancha' × 'Zha'ai Shandingzi' (Malus baccata Borkh.), were cultivated in the experimental farm at Shenyang Agricultural University, Shenyang, China. Former studies had found that apomixes rate in the offspring hybrids declined significantly, but the molecular mechanisms of this decline are rarely reported. In this study, the mechanism of apomixes through gene cloning, different expression in different organs during floral development stages, vector construction and genetic transformation were demonstrated. The main findings were as follows:1. The cDNA full length sequences of SERK2, SERK3 from 'Pingyi Tiancha' and the hybrid strain '33#' were obtained. These genes have been named MhdSERK2, MhSERK3 and MhdSERK3, respectively. The cDNA full length sequences of MhdSERK2, MhSERK3 and MhdSERK3 were 2,058 bp, 2,386 bp and 1,979 bp which encoded proteins with 626, 616 and 616 amino acids, respectively. The cDNA sequence similarity between MhSERK2 and MhdSERK2 in triploid 'Pingyi Tiancha' and tetraploid hybrid strain '33#' was 99.51%, with only 8 bp differences, whereas MhSERK3 and MhdSERK3 were 98.06% with only 36 bp differences, which might be related to apomictic ability. The cDNA sequences in SERK2 and SERK3 had an integral ORF with the initial codon ATG at the 5'-end and the terminal codon TGA at the 3'-end. The MhSERK2, MhdSERK2, MhSERK3 and MhdSERK3 were shared all the characteristic domains of SERK gene plant family, including a signal peptide, leucine zipper domain, five LRRs, SPP motif, TM and kinase domains.2. The expression patterns of MhdSERK2, MhSERK3 and MhdSERK3 genes were investigated by qRT-PCR in different floral organs and developmental stages, including ovary,stamen, sepal, petal, and stigma. The results showed that, the highest expression level was observed in the ovary while the lowest level in the stamen. Moreover, the expression levels of MhSERK2 and MhSERK3 of 'Pingyi Tiancha' in the ovary, sepal, petal, stamen and stigma were higher than those of MhdSERK2 and MhdSERK3 genes of the hybrid strain '33#'. Our results provided some indications that MhSERK2 and MhSERK3 might be related to apomixis in 'Pingyi Tiancha'.3. Vector construction of SERK2 and SERK3 in triploid 'Pingyi Tiancha' was successful which were inserted into the binary vector pRI 101-AN DNA. Then recombinant plasmids SERK2-pRI 101-AN and SERK3-pRI 101-AN were introduced into Agrobacterium tumefaciens strain EHA105.4. Genetic transformation mainly was conducted in MhSERK2 to Micro-Tom tomato(Solanum esculentum) and tobacco (Nicotiana benthamiana) using EHA105 (Agrobacterium tumefaciens),which harbor the binary vector pRI101. Finally,23 plants of tomato and 7 plants of tobacco were obtained; and the PCR analysis proved that the 18 and 5 plants from tomato and tobacco respectively were positively and successfully transformed.