Proteome And Transcriptome Analysis Of Cotton Somatic Embryo And Study Of JA And ABA Mediated Cotton Somatic Embryogenesis

Cotton somatic embryo development (SED), an important part of cotton somatic embryogenesis, is a limiting step for increasing the number of regenerated plantlets. Gossypium hirsutum cv. CCRI24 was used as the experimental material. Somatic embryos were collected and then extracted protein and RNA was used to perform the proteome and transcriptome analysis to obtain the dynamic protein and gene profiles. Some specially expressed proteins and genes in different development stages were deeply analyzed to reveal the molecular mechanism underlying cotton SED. The preliminary research on the molecular mechanisms of JA and ABA regulating cotton somatic embryogenesis were performed. The main results are described as follows:1. Identification of the key pathways regulating cotton somatic embryo development and effects of hormones on SEDA global analysis of proteome dynamics between globular and cotyledonary embryos was performed using isobaric tags for relative and absolute quantitation (iTRAQ). Of 207 differentially expressed proteins identified by a mass spectrometric analysis,102 proteins were up-regulated and 107 were down-regulated in cotyledonary embryos compared with globular embryos. Gene ontology annotation classified the differentially expressed proteins into seven functional categories:carbohydrate and energy metabolism, stress responses, hormone synthesis and signal transduction, protein metabolism, cell wall metabolism, cell transport, and lipid metabolism. From globular embryo to cotyledonary embryo, a key enzyme in JA synthesis, allene oxide synthase (AOS) and ABA receptor PYR/PYR is significantly increased, while the GA receptor GID1 is significantly decreased. Therefore, we believed that these three hormones play key roles in regulating cotton SED. To investigate the effects of exogenous application of hormones on cotton SED, various concentrations of exogenous ABA, GA, paclobutrazol and JA were incorporated into the medium for embryo development. At a proper concentration of JA, many secondary somatic embryos derived from globular embryos resulted in embryo proliferation. A suitable ABA concentration will produce more normal cotyledonary embryos. In contrast, GA promoted disorganized growth of somatic embryos and inhibited normal development, which was accompanied by a higher frequency of aberrant somatic embryos, however, paclobutrazol, a GA inhibitor, promoted SED. The above results suggested that the dynamic changes of plant hormones influenced cotton SED, and provide a strategy to regulate cotton SED by mediating the hormones homeostasis.2. Isolation and identification of the key genes regulating SAM and RAM developmentThe differentially expressed genes were obtained betwenn globular,torpedo and cotyledonary embryos using transcriptome technology, which mainly involved in the following aspects:hormones homeostasis, stress adaptation, auxin transport, shoot apical meristem (SAM) development, root apical meristem (RAM) development, coyeledonary primordium development and embryonic root development. The PIN1 played an important role in regulating cotton SED by mediating auxin transprot, which not only balanced the maintenance and differentiation of stem cells of SAM and RAM, but also facilitated the formation of apical-basal axis. MP and its target gene TMO5 regulated vascular tissue development and were involved in the initiation and development of embryonic root, and the decreased expression level from globular to cotyledonary embryos may inhibit the embryonic root development.3. The preliminary research on the molecular mechanisms of JA mediated cotton somatic embryogenesisJA induced the expression of GhMYC3-like, overexpression of GhMYC3-like inhibited the callus growth and somatic embryos formation, but the silencing of GhMYC3-like promoted the callus growth. It suggested that JA played key roles in regulating somatic cell dedifferentiation, and the molecular mechanism was that JA regulated the homeostaisis of H2O2by inducing the expression of GhMYC’3-like.4. The preliminary research on the molecular mechanisms of ABA mediated cotton somatic embryogenesisABA promoted the expression of LEC1, LEC2, FUS3 and WUSCHEL, which regulated the homeostasis of ABA and GA to promote the cotton somatic embryo formation and maturation. ABA also induced the expression of GhMYB44-1, overexpression of GhMYB44-J regulated the ABA homeostasis by inhibiting the expression of PP2Cs to promote somatic embryo development and plantlets regeration.5. Analysis of VIGS efficiency using the Sfold program in Gossypium hirsutum L.We describe a method to assess VIGS efficiency by calculating the disruption energy of the target, the differential stability of small interference RNA (siRNA) duplex ends, and the internal stability at positions 9-14 of the siRNA antisense strand. Based on the results of four target genes analyzed by VIGS, the siRNA with low A Gdisruption, high DSSE and high AIS would increase the interference efficiency. Therefore, the Sfold program can be used to analyze target sequences when carrying out VIGS design to increase gene-silencing effects in plants.