Analysis Of Gene Expression And Isolation Genes Of Plantlet Formation In Kalanchoe Daigremontiana Leaves

Kalanchoe daigremontiana is commonly known as the "colorful butterfly", "mother of thousands" and is a member of the Crassulacae. Crassulacae species are named because of special photosynthetic carbon assimilation pathway to arid environments. The genus Kalanchoe is originated in Madagascar and became fashionable during the last century, with numerous horticultural forms, cultivars, and hybrids brought under cultivation. K.daigremontiana remains an important horticultural plant that produces plantlets at the edges of the thick and fleshy leaves. K.daigremontianan is perennial succulent herb with erect stem and woody base. The leaf is usually 15 to 20 cm long that is fleshy and irregular reddish brown markings on the blade back. The kalanchoe inflorescence is usually a terminal cyme with few or many flowers which are lavender and madding in the short day. In general, kalanchoe plants need very little attention and can be grown well in dry, warm and ventilated culture conditions. In china, K.daigremontianan are mainly distributed in Fujian, Taiwan, Guangdong, Guangxi and Yunnan province. Kalanchoe species are exceptionally easy to propagate which can reproduces asexually by forming plantlets on the leaves. The ability of K.daigremontianan species to form somatic embryos outside of a seed environment makes an ideal model system to study somatic embryogenesis, organogenesis and cell totipotency. In this study, The plantlet of K.daigremontianan was the plant material, and the first enrichment of differentially expressed genes of K. daigremontiana was performed. Our results provide a framework and unified platform on which future research on asexual reproduction in K. daigremontiana can be based.In order to understand the role of the plant hormone in plantlet formation, a thorough experiment was studied here. The plantlet is often formatted from the edge of the mother leaf about 2-3 mm cell layer and occurs symmetrically from leaf tip to base.2,4-D had limited effect on the asexual reproduction, and the key hormone is 6-BA that controlled the plantlet formation. Plantlet was completely inhibited by the PR treatment. At the mean time, Mean maximum leaf pair spam was bigger than the control and other treatments.The suppression subtractive hybridization technique was used to investigate gene expression during asexual reproduction. Leaves from plants subjected to drought stress provided the source of ’Tester’DNA, and leaves from plants grown under normal conditions provided the’Driver’DNA for subtractive hybridization. A total of 481 high quality ESTs were generated, which clustered into 390 unigenes. Of these unigenes,132 grouped into 12 functional categories, suggesting that asexual reproduction is a complicated process involving a large number of genes. The expression characteristics of selected genes from the SSH library were determined by real-time PCR and were classified into five groups, suggesting that gene expression patterns during asexual reproduction are complex. The large number of unigenes involved in asexual reproduction suggests that plantlet formation is a concerted process involving multiple cellular pathways controlled by a complex gene regulatory system. The initiation of plantlet formation and subsequent developmental programs is accompanied by dynamic changes in gene expression.Six genes, KdHDH, KdGST, KdF-box, KdSAHH, KdGAD and KdCDK were selected and cloned, by RT-PCR and RACE-PCR methods. All of the six genes had no signal peptide that these proteins were not secreted protein and worked inside cell. Among them, KdSAHH、KdF-box、KdCDK、KdGAD、 KdGST were hydrophilic proteins, while KdHDH was hydrophobic protein. KdSAHH, KdF-box, KdCDK, KdGAD, KdHDH were acidic proteins, and KdGST was alkaline protein. The open reading frame (ORF) of KdHDH was 1452 bp encoding a polypeptide of 483 amino acids, which had obvious hydrophobic regions and transmembrane structure. Use LOCtree software to analyze, and the result showed that KdHDH located in the chloroplast membrane. In addition, KdHDH had interaction with imidazole glycerol phosphate dehydratase. The open reading frame (ORF) of KdGST was 1272 bp encoding a polypeptide of 423 amino acids, which had obvious hydrophilic regions and located in endoplasmic reticulum membrane. KdGST had no signal peptide, thus it cannot be secreted protein. The open reading frame (ORF) of KdGAD was 1509 bp encoding a polypeptide of 502 amino acids, which had no obvious hydrophobic regions and transmembrane structure. Use LOCtree software to analyze, and the result showed that KdGAD located in the nucleus. The predicted molecular weight (MW) of KdGAD was about 56.57 kDa. The open reading frame (ORF) of KdCDK was 888 bp encoding a polypeptide of 295 amino acids, which is hydrophilic and had no obvious transmembrane structure. This protein is located in the cytoplasm and had no signal peptide. The open reading frame (ORF) of KdF-box was 987 bp encoding a polypeptide of 328 amino acids, which had no transmembrane structure and signal peptide. Use LOCtree software to analyze, and the result showed that KdHDH located in the cytoplasm. The open reading frame (ORF) of KdSAHH was 1458 bp encoding a polypeptide of 485 amino acids, which had no obvious hydrophobic regions, no transmembrane structure and located in endoplasmic reticulum membrane.KdSAHH protein had no signal peptide and transmembrane structure indicated that KdSAHH was a broad-spectum expression protein rather than a membrane localization protein. Moreover, the interaction protein of KdSAHH was associated with methyl transfer. And the expression of KdSAHH gene was 3 times higher than in the control. In conclusion, SAHH protein is an important factor in regulating the organism methylation level and is inhibitory to all SAM dependent methyltransferases. The overexpression and RNAi vectors of KdSAHH gene were constructed for transformation of tobacco and K. daigremontiana. The transgenic plants could be used for analysis of the role of KdSAHH gene in asexual reproduction.In this study, due to analyse the transcriptome of plantlet asexual reproduction, a large number of genes related to plantlet formation were identified.30 selected genes were performed real-time PCR in order to analyse expression patterns. In addition,6 genes were cloned and analyzed by RACE-PCR and bioinformatic software. KdSAHH gene, was chose for further research, the vectors of overexpression and RNAi were constructed and transgenic plants were obtained. Our results provide a framework and unified platform on which future research on asexual reproduction in K. daigremontiana can be based.