Cloning And Expression Analysis Of Apomixis-related SERK2 Genes In Malus

Pingyi Tiancha(Malus hupehensis Rehd. var. pingyiensis Jiang), which is a variaty of Malus hupehensis(Pamp.) Rchd. in Shandong province, is one of the important apple rootstocks in China, and it is a typical triploid apomixis type plant. SERK genes arc closely related with apomixis. Our research group found that the apomixis ability of Pingyi Tiancha is very high, but the apomixis ability of its hybrid offspring are significantly reduced. The reports of the study for the reasons at home and abroad are very few and the reasons need to be further studied.In order to validate the functions of SERK2 gene families in the process of apomixes and establish a good research foundation, this study takes triploid Pingyi Tiancha, diploid Zha’ai Shandingzi (M. baccatia Borkh.) which is a dwarfing and cold-resistant strain, and tetraploid hybrid offspring strain 28# which is the offspring from the cross of Pingyi Tiancha and Zha’ai Shandingzi as the research materials, isolating and cloning the SERK2 gene associated with apomixes, and also exploring the gene expression and analysis of different organs in different stages of flowers. The main research results are as follows:1. Using the partial SERK2 gene sequence fragment of the Malus plant formerly obtained by our group, degenerate primers were designed, then the sequences of 3’ ends and 5’ends were amplified out by RACE and sequenced. By splicing, cDNA sequence of SERK2 gene in Pingyi Tiancha was 1928bp, CDS area was 1881 bp. The SERK2 gene was high homology compared with other species in the nucleic acid sequences.2. Using the cDNA sequence of Pingyi Tiancha, the specific primers were designed to clone and amplify the full-length sequences of Zha’ai Shandingzi and 28# strain:The full-length sequence in SERK2 cDNA of Zha’ai Shandingzi was 2007bp, CDS was 1881bp, and compared with Pingyi Tiancha, the sequence homology was 99.15%; The full-length sequence in SERK2 cDNA of the hybrid strain 28# was 2000bp, CDS was 1881bp, and compared with Pingyi Tiancha, the homology was 99.42%;And compared with Zha’ai Shandingzi, the homology was 98.99%. Compared with other species, the nucleic acid sequences also had high homology.3. SERK2 gene amino acid sequences of Pingyi Tiancha, Zha’ai Shandingzi and offspring 28# strain were analyzed:SERK2 gene of Pingyi Tiancha, Zha’ai Shandingzi and offspring 28# strain were all composed by 626 amino acids, and the proteins were negatively charged, not stable and hydrophilic. By the comparison of amino acid sequences with other species, they all had high homology.4. Phylogenetic tree was constructed by the sequences of Pingyi Tiancha, Zha’ai Shandingzi and offspring 28# strain with others 10 plant species by MAGE version 6.0 and Clustal W2. The results showed:Pingyi Tiancha, Zha’ai Shandingzi, offspring strain 28# had the closest relationship with apple (Malus domestica) and pear (Pyrus bretschneideri), followed by cucumber, orange, soybean, etc. Results showed that the SERK2 gene may belong to the family of SERK2 gene in evolution.5. By fluorescence quantitative RT-PCR analysis of the expression of SERK2 gene in Pingyi Tiancha, Zha’ai Shandingzi and offspring 28# strain, we obtained that SERK2 gene was mainly expressed in the ovaries and sepals, and in the expression level in ovary triploid Pingyi Tiancha was obviously higher than tetraploid hybrids offspring 28#. Because the SERK2 gene expressed high in the reproductive organs and different in the different materials, it is speculated that the SERK2 gene is an important gene associated with the Malus apomixis.