Studies On Cloning, Expression And Regulation Of SOD Gene Family During Somatic Embryogenesis In Dimocarpus Longan Lour.

Longan(Dimocarpus longan Lour.) is one of the most important tropical and subtropical woody fruit trees in south China, the developmental degree of the embryo is closely related to the output and quality of fruit. Plant embryo development is a cell differentiation process, which undergoes the effects of oxidative stress. As the first line of defense against ROS, superoxide dismutase (SOD) plays an important role in the process of plant embryo development and cell differentiation. In this experiment, on the basis of the research on physiological and biochemical of SOD, the model system of somatic embryogenesis(SE) was used as materials to further study the cloning,expression and regulation mechanism of SOD gene family, which lay a foundation for better understanding the regulatory mechanism of SOD during longan somatic embryogenesis, and the results also provided better knowledge the roles of SOD for longan in vivo embryonic development. The main results were as follows:1. Analyses of activities and isozymatic patterns of SOD during longan early SEIn this study, SOD activities and patterns isozymes of longan late embryos in vivo and early embryos in vitro were firstly analyzed. The results indicated that SOD activity showed a gradually increase along with the development of longan embyro in vivo or in vitro, suggesting that SOD should promote the role of cell differentiation in early SE and the development in late embryo. It showed a much milder effect on the SOD activities of temperature treatments(20, 25 and 30℃),while it had a great impact on the SOD isozymes with the performance of the bands for the emergence and disappearance and the depth of band color, it might be concluded that the changes of SOD activities and isozymes in a law played a key role in cell differentiation of early SE, the removal of reactive oxygen species and facilitating development of SE in longan.2.Obtaining the full-length cDNA and DNA of SOD gene family from longan EC20 different mRNA transcripts of SOD gene family were cloned from longan embryogenic callus(EC)by RT-PCR combined with RACE, which encoded cytoplasmic DlCSD1a and DlCSD1b, chloroplast DlCSD2a, chloroplast DlFSD1a, DlFSD1b and mitochondrial DlMSD, Which provided a first large scale cloning of SOD gene family from plants (longan) SE. Every member of SOD gene family contained multiple polyadenylation sites, or poly(A) sites, which suggested that variations in the length of the 3' UTR sequence should significantly augment post-transcriptional control of gene expression during embryonic development, such as microRNA (miRNA) mediated regulation. In addition, seven splicing mRNA variants with different splicing mode, were named as DlCSD1a-4, DlCSD1a-5, DlCSD1b-variant1, DlFSD1b-variant1 ~ variant4 respectively, were cloned from longan EC, which suggested that such variation of transcripts be a consequence of alternative splicing and/or differences in intron splicing efficiency.DlCSD1a, DlCSD1b and DlCSD2a genes with the length genomic sequences of 2741 bp, 4592 bp, 3727 bp, were consisted of eight, six and seven introns, respectively; DlFSD1a and DlFSD1b genes with the length genomic sequences of 2171 bp and 2137 bp, included six and seven introns, respectively; DlMSD gene with the length genomic sequence of 1589 bp, contained five introns. All sequences at the exon-intron junctions were consistent with eukaryotic GT-AG rule. The length of introns and exons in each SOD family varied greatly. Among them, the length of the fifth intron in DlCSD1b gene and the sixth intron in DlCSD2a gene were 3539 bp and 2178 bp respectively, which suggested that large introns be a reservoir of genetic diversity, because they had a greater number of mutable sites than short introns.3. Analysis of bioinformatics of longan EC SOD gene familyAccording to bioinformatics analysis, proteins encoded by longan EC SOD gene family all belonged to superoxide dismutase large family and contained the typical conserved domain, they were highly conserved in evolution and in function, and they were all hydrophilic, acidic proteins; in addition to DlFSD1a and DlFSD1b, the rest were stable proteins. In addition, different types of SOD proteins also had their own characteristics.①Proteins encoded by DlCSD1a and DlCSD1b without the signal peptide and transmem- brane helices located in the cytoplasm, were mainly responsible for the removal of reactive oxygen species in cytoplasm, and their phosphorylation occurred maily on serine, followed by threonine residues.②Proteins encoded by DlCSD2a with the signal peptide and transmembrane helices located in the chloroplast, were mainly responsible for the removal of reactive oxygen species in chloroplast, and phosphorylation occurred maily on serine, followed by threonine residues.③Proteins encoded by DlFSD1a with the signal peptide located in the chloroplast, might be cooperate with DlCSD2a for the removal of reactive oxygen species in chloroplast, and phosphorylation occurred maily on serine, followed by threonine and thyosine residues.④Proteins encoded by DlFSD1b and DlFSD1b(JF316733)without the signal peptide may located in peroxisome or cytoplasm, containing two transmembrane helices and phosphorylation occurred maily on thyosine, followed by serine and threonine residues.⑤Proteins encoded by DlMSD without the signal peptide located in mitochondria, containing two transmembrane helices, were mainly responsible for the removal of reactive oxygen species in mitochondria, and phosphorylation occurred maily on serine, followed by threonine and thyosine residues.4. Cloning and identification of the promoter of longan EC SOD gene familyTo better understand how the SOD gene family are controlled at the gene transcription level, the 5′-flanking regions of DlCSD1a, DlCSD2a, DlFSD1a and DlMSD were isolated by TAIL-PCR and IPCR from longan EC DNA, and the length of them were 2252 bp, 164 bp, 1080 bp and 843 bp, respectively. In addition, the transcription start site(TSS) of longan SOD gene family were identified by RLM-RACE. The results showed that SOD gene family were transcribed from multiple start sites, the number of their TSS ranged from 3 to 10, 5'UTR length varied from 11 to 193 bp and the base composition of the first base of TSS was mostly composed by A, followed by G and C. It maybe implied that the interplay between polymerases and regulators over broad promoter region controlled the efficiency of transcription for the SOD gene family.Bioinformatic analysis showed that the consensus sequences of TATA box and CAAT box were found in DlCSD1a, DlCSD2a, and DlFSD1a promoters, but not in DlMSD promoter; in addition, there were a lot of putative cis-elements that respond to light, GA, auxin, JA, MeJA, as well as elements that responded to different environmental stresses, such as dehydration, coldness, wounding, etc. Also, there were some specific cis-elements for different members of SOD gene with the respective functions. Further more, DlCSD1a and DlFSD1a genes maybe regulated by the bZIP transcription factor, and DlMSD might be subject to the regulation of WRKY transcription factors. On this basis, The 35S promoter in the plant expression vector-pCAMBIA1301 containing GUS gene was replaced forward by the deletion fragments of regulatory sequences in the 3'/5'terminal of DlSOD genes with different lengths, respectively. The mentioned above results were expected to be the basis for further studies on the regulation and expression of longan SOD gene family.5. Cloning and identification of miRNAs regulating DlSODs from longan SEIn this study, to investigate the number of miRNAs related to SOD gene family, Solexa sequecing technology was employed to identify miRNAs from a short RNA library generated from synchronized longan embryogenic cultures at different developmental stages. Solexa sequencing showed that longan had a complex small RNA population and the length of small RNAs varied, 24 nt being the predominant. 12, 554, 858 reads produced 13,151 unique miRNAs representing 367 conserved miRNAs as well as 23 novel poential miRNAs were obtained. This research provided a first large scale cloning and characterization of plants(longan) SE related miRNAs. The newly identified miRNAs greatly enriched the repertoire of somatic embryo miRNAs and provided insights into miRNA biogenesis and expression in plant somatic embryo development. Based on the above results, 11 dlo-miRNAs were evaluated by regulating DlSODs expression at the posttranscriptional level during longan SE by RLM-RACE, including dlo- miR156 (target: DlCSD1a); dlo-miR159, 863-3p, 1023b-3p, 1223 and 2643 (target: DlCSD1b); dlo-miR159, 398, 1171a, and 1171b (target: DlCSD2a) and dlo-miR808 and 2089 (target: Dl FSD1a). According to the reads of Solexa, Their expression level in longan SE were varied greatly. It suggested that differentially expressed dlo-miRNAs lead to the different expression patterns for different types of SOD during longan SE. In addition, The miR398a/b precursor were isolated by RT-PCR from longan EC and were highly conserved among different species.6. Analysis of SOD gene family and miRNA expression profiles during longan SE①Analysis of SOD gene family expression profiles during longan SEThe analysis of expression patterns of different types of DlSODs was based on reference gene selection for qPCR analysis during longan SE, and the main results were as follows: firstly, different types of DlSODs showed a similar expression change tendency during longan SE, and played an important role in the development of SE at middle and later developmental stages. From the friable-embryogenic callus to mature embryo, the expression variation tendency of DlSODs was mainly displayed as"increase (embryogenic callus II)-decrease (incomplete compact pro- embryogenic cultures)-decrease (compact pro-embryogenic cultures)- decrease (globular embryos) -increase (heart-shaped embryos)-decrease(torpedo-shaped embryos)-decrease (coty- ledonary embryos) - increase (mature embryo)"mode. Secondly, the role of different types of DlSODs at different developmental stages of longan SE was different. All types of DlSODs mediated the development of longan SE at embryogenic callus II and heart-shaped embryos; DlCSD1a-5 and DlCSD1b-2 were related to globular-shaped embryos formation; DlFSD1a, DlFSD1b and DlMSD were required for cotyledon-shaped embryos morphogenesis; however, the expression levels of DlSODs were very low at cotyledon embryos stage; DlCSD1a and DlMSD were mainly responsible for the development of mature embryo. In addition, the relationship of the expression patterns among DlSODs gene family, other genes related to antioxidant system(CAT and POD) and DlCCS (molecular chaperone) were also studied in this experiment. The results showed that the mutual coordination in gene expression of CAT, POD and other genes related to antioxidant system and DlSODs were responsible for the normal operation of the ROS network; moreover, some developmental stages of longan SE rely on DlCCS for activating DlCSDs, but some developmental stages i.e. embryogenic callus II could activate DlCSDs independently of DlCCS.②Analysis of miRNAs expression profiles during longan SETo better understand the role of dlo-miRNAs during longan SE, the analysis of expression patterns of twenty dlo-miRNAs were carried out based on reference gene selection for qPCR analysis during longan SE, and the main results were as follows: stage and tissue-specific expressions of twenty dlo-miRNAs suggested their possible modulation on longan SE. dlo-miR3, 5, 10, 12, 13, 156a, 156c, 397a, 398b.1, 398b.2, 398b.3, 808 and 2089*, which were expressed at high levels at early developmental stages (before globular embryos) and were expressed at low levels at later developmental stages of SE, might play coordination of regulatory role in morphogenesis of Early SE in longan; dlo-miR6, 160a and 167a may play a major role in the development of SE at middle and later developmental stages; dlo-miR6 and 160a accumulation might be required for heart and torpedo-shaped embryos formation and morphogenesis; dlo-miR167a maybe played a major role in the development of SE at cotyledon and mature embryo stages; and the expression levels of dlo-miR159a.1, 159a.2, 159 c and 159f during longan SE were stable.③Comparative analysis of expression patterns of miRNA and DlSODs during longan SEdlo-miRNAs and its targets(DlSODs) were involved in the regulation of development longan SE by coordinating expression. Firstly, the expression profiles were compared between DlSODs and dlo-miRNAs, the analysis found that the transcription level of dlo-miR156 family, 159 family, 398b family, 808 and 2089* were in reverse correlation with their targets level; However, there were non-specific products of dlo-miR863-3p, 1023b-23p, 1171a/b, 1223 and 2643 after amplifi- cation using SYBR qPCR, which suggestted that there be other potential miRNAs or endogenous siRNA direct DlSODs cleavage in the same binding sites during longan SE.In addition, different miRNA members played a diffent role in different developmental stages during longan SE, and each member might control the expression of the corresponding target mRNA transcript. Such as dlo-miR159 family, four members with a clear division of labor at different developmental stages regulated the expression of DlCSD1b and DlCSD2a. During Early SE in longan (before globular embryos), the expression of DlCSD1b and DlCSD2a were mainly controled by dlo-miR159a.1; from globular to heart-shaped embryos, their expression were regulated by dlo-miR159c and 159f; from heart-shaped embryos to mature embryos, the expression were mainly controled by dlo-miR159a.2 and 159c, whcih was the same as dlo- miR156 family and 398b family. This complementation of gene expression appeared to be due to each member inducing functions not induced by the other, so that the combined miRNA family regulated target gene expression at post-transcriptional level. dlo-miR159 regulate DlCSD1b and DlCSD2a simultaneously, and might be responsible for coordinating the expression profiles between its targets; In addition, the expression pattern of dlo-miR398b and pre-miR398b was similar, exhibiting a reverse correlation with the expression of target DlCSD2a, indicating that the expression level of miRNA precursors might be representive of mature miRNA level in a certain extent.In summary, the expression and regulation mechanism of SOD gene family during longan SE might be as follows: When environmental factors changed (the levels of endogenous hormones or environmental stress), the level of intracellular ROS was affected, which led to changes of the relevant regulatory factors levels [(transcription factor bZIP, WRKY, miRNAs (dlo-miR156, 159, 398, 808, and 2089*), phosphorylation)] , and further led to the regulation of SOD gene familiy. In addition, the coordination in gene expression of CAT, POD and other genes related to antioxidant system and DlSODs played important roles in various aspects of biological and metabolic processes during longan SE, including cell differentiation, cell apoptosis, hormone signal trans- duction, and their reponse to enviromental stress. In short, the regulation mechanism of SOD gene family expression was accomplished by complex regulatory network involving a variety of regulatory factors.