Cloning And Expression Of The Genes Related To Phytohormone Metabolism And Signal Transduction During Somatic Embryogenesis In Dimocarpus Longan Lour.

In the experiment, the embryogenic calli (EC) of longan(Dimocarpus longan Lour. cv. Honghezi) were used as materials for cloning the genes related to auxin signal transduction (auxin receptor gene, auxin biding protein gene and auxin response factor gene), the key genes for ethylene biosynthesis(ACC synthase gene and ACC oxidase gene) and the genes related to ethylene singnal transduction (ethylene receptor genes); and then the dynamical changes of the mRNA transcription levels of these genes were further determined by real-time reverse transcription PCR during longan somatic embryogenesis, which lay the foundation for the better understanding the regulatory mechanism of phytohormone during longan somatic embryogenesis. The main results were as follows:1 Cloning the genes related to phytohormone metabolism and signal transduction during longan somatic embryogenesisThree genes related to auxin signal transduction were cloned from longan EC by RT-PCR combined with RACE:â‘ The full length cDNA of auxin receptor gene (DL-TIR1, GenBank GQ870264) of longan EC, about 2328 bp[including a 16 bp poly(A) tail], consisted of an open reading frame of 1761 bp, and 5' and 3'utranslated regions of 118 bp and 449 bp, respectively.â‘¡The full length cDNA of auxin binding protein gene(GenBank GQ900592) of longan EC, about 869 bp[including a 19 bp poly(A) tail], consisted of an open reading frame of 567 bp, and 5' and 3'utranslated regions of 43 bp and 259 bp, respectively.â‘¢The full length cDNA of auxin response factor gene(DL-ARF, GenBank GQ923778) of longan EC, about 2695 bp[including a 17 bp poly(A) tail], consisted of an open reading frame of 2046 bp, and 5' and 3'utranslated regions of 163 bp and 486 bp, respectively. Meanwhile, two genes for ethylene biosynthesis and two ethylene receptor genes were obtained.â‘ The full length cDNA of ACC(ethylene precursor) synthase gene(DL-ACS, GenBank FJ617537) of longan EC, about 1817 bp[including a 16 bp poly(A) tail], consisted of an open reading frame of 1437 bp, and 5'and 3'utranslated regions of 73 bp and 307 bp, respectively.â‘¡The full length cDNA of ACC(ethylene precursor) oxidase gene (DL-ACO, GenBank FJ534854) of longan EC, about 1315 bp[including a 13 bp poly(A) tail], consisted of an open reading frame of 948 bp, and 5' and 3'utranslated regions of 86 bp and 281 bp, respectively. And, the DNA sequence of DL-ACO (GenBank GU123929) was 1660 bp, the splicing sites of three introns were comformed to the "GT-AG" rule.â‘¢The full length cDNA of ethylene receptor gene (DL-ETR1, GenBank FJ513322) of longan EC, about 2611 bp[including a 24 bp poly(A) tail], consisted of an open reading frame of 2223 bp, and 3'utranslated regions of 388 bp.â‘£The full length cDNA of ethylene receptor gene (DL-ERS1, GenBank FJ513323) of longan EC, about 2259 bp[including a 17 bp poly(A) tail], consisted of an open reading frame of 1911 bp, and 3'utranslated regions of 348 bp. The DNA sequence of DL-ERS1 (GenBank GU123930) was 2236 bp, the splicing sites of two introns were conformed to the "GT-AG" rule.2 Bioinformatics analysis of the genes related to phytohormone metabolism and signal transduction during longan somatic embryogenesisThe main results of bioinformatics analysis of the genes related to phytohormone metabolism and signal transduction during longan somatic embryogenesis were as follows:The genes related to auxin signal transduction:â‘ DL-TIR1 gene, encoded 586 amino acids, was a kind of hydrophilic protein with three transmembrane domains, mainly located in the cytoplasm, contained 36 functional sites and one F-box conserved structure domain. The secondary structure of DL-TIR1 was made up mostly of alpha helix. When compared the DL-TIR1 with the other plant polypeptides, the identity percentage varied from 57% to 85%. Probably, DL-TIR1 combined with an auxin-regulated SCFDL-TIR1-Aux/IAA through its N terminal F-box domain, which led to the degradation of Aux/IAAs through the ubiquitination metabolism pathway. Subsequently, the auxin signaling pathway was promoted.â‘¡DL-ABP1 gene, encoded 188 amino acids, was a kind of hydrophilic protein with one transmembrane domain, mainly located in the endoplasmic reticulum, contained seven functional sites and a signal peptide at N-terminal, belonged to auxin-binding protein family. The secondary structure of DL-ABP1 was made up mostly of random coil. When compared the DL-ABP1 with the other plant polypeptides, the identity percentage varied from 72% to 87%. During longan somatic, DL-ABP1 was predicted to be involved in the rapid auxin-induced response by another signaling pathway, which was paralleled with a DL-TIR1 mediated-genes pathway.â‘¢DL-ARF gene, encoded 681 amino acids, was a kind of hydrophilic protein with two transmembrane domains, mainly located in the nucleus, contained 36 functional sites and two conserved structure domains, belonged to transcriptional factor B3 family. The secondary structure of DL-ARF was made up mostly of random coil. When compared the DL-ARF with the other plant polypeptides, the identity percentage varied from 41% to 81%. DL-ARF was probably belonged to the transcription inhibitor, its function need to be further explored during longan somatic embryogenesis. The genes related to ethylene biosynthesis:â‘ DL-ACS gene, encoded 478 amino acids, was a kind of hydrophilic protein with two transmembrane domains, mainly located in the nucleus, contained 21 functional sites, three conserved structure domains and one winded helix, belonged to ACC synthase family. The secondary structure of DL-ACS was made up mostly of random coil. When compared the DL-ACS with the other plant polypeptides, the identity percentage varied from 63% to 77%. Probably, DL-ACS was responsible for basic ethylene synthesis.â‘¡DL-ACO gene, had 315 amino acids, was a kind of hydrophilic protein, mainly located in the cytoplasm, contained 8 functional sites, one oxoglutarate/iron-dependent oxygenase conservative structure domain and one winded helix. The secondary structure of DL-ACO was made up mostly of random coil. When compared the DL-ACO with the other plant polypeptides, the identity percentage varied from 47% to 86%. Probably, DL-ACO was responsible for basic ethylene synthesis.Two ethylene receptor genes:â‘ DL-ETR1 gene encoded 740 amino acids, was a kind of hydrophobic protein with three transmembrane domains, mainly located in the plasmid, contained 28 functional sites, eight conserved structure domains and one winded helix, belonged to signal transduction histidine kinase, hybrid-type, ethylene sensor family. The secondary structure of DL-ETR1 was made up mostly of alpha helix. When compared the DL-ETR1 with the other plant polypeptides, the identity percentage varied from 63% to 91%. DL-ETR1 was likely to be prominent in the ethylene receptor family of longan, and highly conserved DL-ETR1-Cys65 may be related to ethylene receptor activity and sensitivity.â‘¡DL-ERS1 gene, had 636 amino acids, was a kind of hydrophobic protein with four transmembrane domains, mainly located in the cytoplasm, contained 25 functional sites, six related conservative structure domains and one winded helix. The secondary structure of DL-ERS1 was made up mostly of alpha helix. When compared the DL-ERS1 with the other plant polypeptides, the identity percentage varied from 36% to 80%. Probably DL-ERS1 and DL-ETR1 had similar biological characteristics, but the amounts of the signal transduction were different.3 Expression of the genes related to phytohormone metabolism and signal transduction during longan somatic embryogenesis3.1 The quantitative expressions the genes related to phytohormone metabolism and signal transduction during longan somatic embryogenesisThe genes related to auxin singnal transduction:â‘ DL-TIR1 expressed in 8 different stages during somatic embryogenesis, which showed approximately a "W" curve. The peak mRNA transcription level of DL-TIR1 occurred at the cotyledonary embryos stage.â‘¡DL-ABP1 expressed in 8 different stages during somatic embryogenesis, which showed approximately a "W" curve. The relative high mRNA transcription level of DL-ABP1 occurred at the friable-embryogenic callus stage, the incomplete compact pro-embryogenic cultures stage and the cotyledonary embryos stage. And the valley mRNA transcription level of DL-ABP1 occurred at the globular embryos stage.â‘¢The whole trend of DL-ARF expression was approximately a "M" curve. The relative low mRNA transcription level of DL-ARF occurred at the friable-embryogenic callus stage, the globular embryos stage and the cotyledonary embryos stage. And the relative high mRNA transcription level of DL-ARF occurred at the incomplete compact pro-embryogenic cultures stage and the heart embryos stage.The genes related to ethylene biosynthesis:â‘ The level of DL-ACS mRNA changed more dynamically in 8 different stages, which showed approximately a "M" curve. The relative low mRNA transcription level of DL-ARF occurred at the friable-embryogenic callus stage, the globular embryos stage and the cotyledonary embryos stage. And the relative high mRNA transcription level of DL-ARF occurred at the incomplete compact pro-embryogenic cultures stage and the heart embryos stage.â‘¡The whole trend of DL-ACO expression was approximately a "M" curve. The peak mRNA transcription level of DL-ACO occurred at the incomplete compact pro-embryogenic culture stage and the heart embryo stage. The relative low mRNA transcription level of DL-ACO occurred at the friable-embryogenic callus stage, the globular embryos stage and the cotyledonary embryos stage.The genes related to ethylene singnal transduction:â‘ The whole trend of DL-ETR1 expression was irregular "W"-shaped. The peak mRNA transcription level of DL-ETR1 occurred at the friable-embryogenic callus stage. And the valley mRNA transcription level of DL-ETR1 occurred at the heart embryo stage.â‘¡DL-ERS1 expressed at 8 different stages during somatic embryogenesis. The expression levels of seven stages were very low except the cotyledonary embryos stage. The peak mRNA transcription level of DL-ERS1 occurred at the cotyledonary embryos stage. The lowest mRNA transcription level of DL-ERS1 occurred at the topedo embryos, almost no expression.3.2 The relationship of the expression patterns among phytohormone metabolism and signal transduction-related genes during longan somatic embryogenesisAs a potential, auxin receptor, the expression pattern of DL-ABP1 was similar to DL-TIR1,one of auxin receptors. DL-ACS was in the upstream of DL-ACO during the ethylene bio-synthesis pathway, however, the transcript of DL-ACS didn't match that of DL-ACO. DL-ETR1 and DL-ERS1 both belonged to ethylene receptor groupâ… , but gene expression pattern of DL-ETR1 was different from that of DL-ERS1. The whole expression trend of DL-TIR1, an auxin receptor, was similar to that of DL-ACS, which was a key enzyme-coding gene in the ethylene biosynthesis. The whole trend of endogenous IAA level resembled a bimodal curve, which was similar to the expression level of DL-ACO, but the two peak stages of IAA levels were a little bit earlier than that of DL-ACO. The mRNA level of auxin signal transduction-related DL-ARF and ethylene biosynthesis-related DL-ACO both presented to be an unshaped "M" curve. The appearance of the first peak of curve of DL-ARF was a little bit earlier than that of DL-ACO, and the DL-ACO expression level was a bit higher than that of DL-ARF. It is worthy of further analysis to comfirm the cross-talk between the auxin and the ethylene for DL-ARF and DL-ACSIn summary, the seven genes strictly related to auxin and ethylene were cloned from longan for the first time in the study. Also, the structures and functions of these genes were predicted, and the expression patterns were analyzed. Furthermore, it was the first time to study on the cross talk between auxin and ethylene in woody embryos, which could contribute to the research of the regulatory mechanism of phytohormone during somatic embryogenesis and provide target genes for genetic improvement in longan.