Study On Cloning, Expression And Genetic Transformation Of Apomicxis-related SERK Genes In Malus

Pingyi Tiancha(Malus hupehensis Rehd. var. pingyiensis Jiang), a variety of crabapple (Malus hupehensis (Pamp.) Rehd.), which belongs to the Malus of Rosaceae, is a typical apomictic triploid plant. Pingyi Tiancha has high apomictic ability and is an important female parent in the apomictic dwarf rootstock breeding. Using the character of facultative apomixis in most apomictic plants of Malus, the tetraploid leaf-wrinkled and dwarf hybrids crossed by Pingyi Tiancha and Zha’ai Shandingzi (M. baccata) were obtained in the early study.The further research results showed that in most hybrids their apomictic ability declined significantly. Till now there are very little research and reports in molecular mechanism to reveal why apomixes rate declined significantly in the apomictic hybrids and in the reasons for this decline it is caused by some genes’controlling.In this research, using the triploid Pingyi Tiancha and tetraploid hybrid strain33#as paint materials, we investigated the mechanism of apomixes at the molecular level through cloning the apomixis-related genes, analysing the gene expression differences in the reproductive process, constructing vector and making genetic transformation.The main results are as follows:1.4homologous SERK genes’fragments were obtained by homologous clone from genome DNA of Pingyi Tiancha and hybrid strain33#, and the full-length of them were705bp,671bp,504bp,740bp and705bp,500bp,639bp,675bp, respectively.The homologous gene fragments had the similar structure of an intron, which had high homology with SERK homologous genes from other plants.2. Using the published genome sequence in Malus, cDNA full-length sequences of SERK1and SERK4were isolated from Pingyi Tiancha and the hybrid strain33#, named MhSERK1, MhdSERK, MhSERK4and MhdSERK4. The cDNA full-length sequences of MhSERK1and MhdSERK1were1899bp and1881bp, which encoded632and626amino acids, respectively. After comparing the cDNA sequences between MhSERKl and MhdSERK1it was found that there was18bp difference at696-714bp, which might be related with apomictic ability.The length of MhSERK4and MhdSERK4were both1836bp and had a99.4%homology, encoding611amino acids, respectively. The amino acids sequence has the highest homology of more than90%with coconut, and the kinase structure area was highly conserved which had the typical LRR-RLKs structural character of SERK gene family. 3. Based on the obtained cDNA sequence, the full-length sequences of MhSERKl and MhdSERK1in DNA coding region were obtained by PCR. The full-length sequences in DNA coding region of MhSERK1and MhdSERKl were6886bp and6719bp, both with11exons and10introns. The distribution of introns in MhSERKl and MhdSERKl were similar with that in Longan (accession number:HM773391) and Papaya (accession number: EF661025).4. SERKs expression was detected in different tissues, organs and flower’s different developmental stages of triploid Pingyi Tiancha (3n) and tetraploid hybrid strain33#(4n) through Real-time quantitative PCR methodThe expression of SERK1, SERK1, SERK1and SERK4were mainly occurred in the reproductive tissues, and expression was higher in Pingyi Tiancha than in hybrid strain, indicating that SERK gene was related to apomixes. Expression analysis on flower’s different developmental stages showed that SERK1expression was highest in the bud period of Pingyi Tiancha, then the full-blossom period, and it was lowest after flowering; while it was highest after flowering in hybrid strain which had lower expression in the bud period and the lowest expression in the full-blossom period. Expression of SERK1was the highest in the ovary of both Pingyi Tiancha and hybrid strain33#, in stamen was higher, In the petal, however, expression was the lowest in Pingyi Tiancha. Expression were occurred in the leaf, petal and pistil of hybrid strain33#, but it was nearly no expression. The highest expression of SERK2gene was occurred in the second day after flowering in Pingyi Tiancha, which was5times more than that in hybrids33#; and the highest expression was occurred in the third day after flowering in hybrid strain33#, which was2times bigger than that in Pingyi Tiancha. SERK3gene expression in Pingyi Tiancha reached the maximum in the second day after flowering and about4times larger than that in hybrid strain33#; the expression of SERK4gene was higher in1-5d in Pingyi Tiancha than in hybrid strain33#, respectively, which were inserted into agrobacterium strain EHA105successfully.5. Plant expression vectors of MhSERKl and MhdSERK1as well as MhSERK4and MhdSERK4were constructed, named pBI121-MhSERK1, pBI121-MhdSERKl, pBI121-MhSERK4and pBI121-MhdSERK4, and transformed the SERKl gene into tobacco successfully.6. Genetic transplant study was carried out based on Micro-Tom tomato and tobacco as materials by using agrobacterium infection method and constructed pBI121-MhSERKl and pBI121-MhdSERKl plant expression vectors,7and6tobacco resistant plants were obtained, and the PCR detection showed that transformation was successful.