The Cloning And Functional Characterization Of Cotton Somatic Embryogenesis-Related Gene GhL1L1

Somatic embryogenesis(SE)is a process with somatic cells regeneration into embryos,then to new plants by in vitro culture without fertilization.SE is the foundation of producing transgenic cotton along with the development of tissue culture and transgenic technology.Auxin is one of the efficient regulators during SE.A differential expression of NF-YB transcription factor was found in the RNA-Seq profile during cotton SE in our previous studies.GhL1L1 is differential expression in the embryogenesis during cotton SE.So we conducted the following research during the cotton SE.1.Gene cloning and expression analysis of GhL1L1 in cottonGhL1L1,encoding a NF-YB subfamily protein,was isolated from a RNA-Seq profile during cotton SE.A total of 41 NF-YB subfamily genes were identified genome-wide in G.hirsutum(TM-1).Phylogenetic analysis revealed 6 LEC1-type genes in G.hirsutum,which distributed to the A and D subgenomes and encoded three similar proteins respectively.The expression pattern analysis revealed that the six LEC1-type genes were specifically expressed in 20-day seeds,embryogenic calluses(ECs),somatic embryos and zygotic embryos,with the most abundant expression observed for GhL1L1.2.GhL1L1 regulates cell fate specification during cotton SETo identify the function of GhL1L1 during cotton SE,overexpression and RNAi vectors were constructed and transformed into G.hirsutum YZ1.The results revealed that overexpression of GhL1L1 restricted the callus initial dedifferentiation and uncontrolled callus proliferation and promoted the EC formation,with enhanced expression of embryogenic pathway genes,such as Gh FUS3 and Gh ABI3.And the polarity of the explants were not obvious.GhL1L1-deficient and null explants dedifferentiated vigorously with polarity growth but retarded EC formation.3.GhL1L1 affects auxin accumulation and auxin distribution during cotton SEGiven the importance of auxin during cotton SE,the concentration of IAA in the GhL1L1 transgenic and null lines at 25 and 40 days post-induction and ECs from the corresponding transgenic and null lines was measured by HPLC-MS.The IAA concentration of explants in the overexpression lines was higher than in the RNAi and null explants at 25 and 40 days post-induction.Additionally,the concentration of endogenous IAA was increased in the GhL1L1 overexpression EC lines.By contrast,it was decreased in the calluses of RNAi lines compared with null.To investigate the function of GhL1L1 in auxin distribution,the F1 hybrids of GhL1L1 transgenic lines with DR5::GUS line were generated to track the auxin distribution.The GUS expression was markedly increased in the shoot apical meristem(SAM)of the OE-GhL1L1/DR5::GUS hybrids compared with the null/DR5::GUS and RNAi-GhL1L1/DR5::GUS hybrids.GUS staining and IAA immunolocalization showed the auxin uniformly distributed in torpedo embryos was affected in the overexpressors or RNAi lines.4.GhL1L1 binds to GhPP2AA2 and regulates the Gh PIN1 proteinGhL1L1 was able to bind to the G-box element of the GhPP2AA2 promoter to activate the expression of GhPP2AA2 by yeast one-hybrid(Y1H)and dual-luciferase reporter system.Moreover,GhPP2AA2 directly interacted with Gh PIN1 by in vitro pull-down assays and in vivo bimolecular fluorescence complementation(Bi FC)assays.And GhPP2AA2 and Gh PIN1 colocalized at the cell membrane.GhPP2AA2 was homologous to At PP2AA2,which regulates the dephosphorylation of PIN1 proteins.So we think GhL1L1 activated the expression of GhPP2AA2 to interact with Gh PIN1 protein and affected the auxin polar transport and distribution.5.Inhibited auxin polar transport accelerates SETo confirm PIN1 distribution affects cotton SE,TIBA,a synthetic inhibitor of the carrier-driven auxin efflux by disrupting PIN1 distribution,was added to MSB medium to culture GhL1L1 transgenic and null explants.The auxin distribution was affected and the callus proliferation was restricted by supplemented with TIBA.The difference in callus proliferation rate(CPR)among different lines could not be clearly observed at 15 days post-induction.And TIBA treatment accelerated the ECs formation and embryonic differentiation rate(EDR)in all lines,especially in RNAi lines,which were observed at least 10 days earlier in the RNAi lines.6.GhL1L1 regulates the synthesis of fatty acidsThe content of linoleic acid was increased in the overexpression lines during cotton SE.To study the mechanism of GhL1L1,yeast two-hybrid(Y2H)and Immunoprecipitation Mass Spectrometry(IP-MS)were applied to screen GhL1L1 binding proteins.The results showed that GhL1L1 interacted with Gh NF-YC2 and Gh NF-YC9 by pull-down,Bi FC and Co-IP assays.And GhL1L1 interacts with Gh NF-YC2 binding the promoter of Gh SSI2.So we think GhL1L1 may regulate the synthesis of fatty acids by the regulation of Gh SSI2.