Cloning And Expression Of SERK And Some Other Somatic Embryogenesis-related Genes 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 SERK and some other somatic embryogenesis-related genes(14-3- 3 gene,AGL15 gene ,LEC gene,CHI-I gene);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 SERK and some other somatic embryogenesis-related genes during longan somatic embryogenesis. The main results were as follows:1 Cloning SERK and some other somatic embryogenesis-related genes during longan somatic embryogenesisSERK and some other somatic embryogenesis-related genes were cloned from longan EC by RT-PCR combined with RACE and TAIR-PCR:①The full length cDNA of SERK1 gene (DlSERK1) of longan EC, about 1875bp(GenBank FJ013227), consisted of an open reading frame of 1875 bp. And, the DNA sequence of DlSERK1 (GenBank HM773391.1) was 6009 bp, and consisted of eleven exons and ten introns, the splicing sites of ten introns were comformed to the"GT-AG"rule. The length of DlSERK1 promoter was 1349bp (GenBank: HM773391). The promoter of SERK1 had a likehood of basic promoter area from 1022bp to 1071bp with a predicted starting site of transcription as Cytonsine on the 1062th base, and it contained some cis-action elements such as a module involved in light responsiveness, cis-acting element involved in low-temperature responsiveness,MYB binding site involved in drought-inducibility,cis-acting element involved in heat stress responsiveness,cis-acting regulatory element essential for anaerobic induction, fungal elicitor responsive element,auxin-responsive element,gibberellin-responsive element, cis-acting element involved in the abscisic acid responsiveness,cis-acting element involved in salicylic acid responsiveness, cis-acting regulatory element involved in the MeJA-responsiveness,and cis-acting regulatory element related to meristem expression,cis-acting regulatory element required for endosperm expression,cis-acting regulatory element involved in circadian control,cis-acting regulatory element involved in zein metabolism regulation.②The full length cDNA of 14-3-3 gene (Dl-14-3-3) of longan EC, about 786bp, consisted of an open reading frame of 786bp (GenBank GU573765.1). And, the DNA sequence of Dl-14-3-3 (GenBank GU573766.2) was 1859 bp, and consisted of four exons and three introns, the splicing sites of three introns were comformed to the"GT-AG"rule. The length of Dl-14-3-3 promoter was 910bp (GenBank: GU573766). The promoter of Dl-14-3-3had a predicted starting site of transcription as Guanine on the 736 th base, and it contained some cis-action elements such as a module involved in light responsiveness , cis-acting element involved in low-temperature responsiveness, MYB binding site involved in drought-inducibility, cis-acting regulatory element essential for anaerobic induction, fungal elicitor responsive element, cis-acting regulatory element involved in the MeJA-responsiveness, gibberellin-responsive element,and cis-acting regulatory element required for endosperm expression, cis-acting element involved in cell cycle regulation,cis-acting regulatory element involved in circadian control,cis-acting regulatory element involved in zein metabolism regulation.③The full length cDNA of AGL15 gene (Dl AGL15) of longan EC, about765bp, consisted of an open reading frame of 765bp (GenBank GU584088). And, the DNA sequence of Dl AGL15 (GenBank JF262162) was 2989 bp, and consisted of eight exons and seven introns, the splicing sites of six introns were comformed to the"GT-AG"rule. The length of DlAGL15 promoter was 1454bp (GenBank:JF262162). The promoter of DlAGL15had a likehood of basic promoter area from 1178 bp to 1227 bp with a predicted starting site of transcription as Guanine on the 1218 th base, and it contained some cis-action elements such as a module involved in light responsiveness , cis-acting element involved in low-temperature responsiveness, cis-acting regulatory element essential for anaerobic induction, cis-acting element involved in heat stress responsiveness, fungal elicitor responsive element, gibberellin-responsive element, cis-acting element involved in salicylic acid responsiveness, cis-acting regulatory element involved in the MeJA-responsiveness, and cis-acting regulatory element required for endosperm expression, cis-acting regulatory element related to meristem expression, binding site of AT-rich DNA binding protein,cis-acting regulatory element involved in circadian control,④The full length cDNA of LEC gene (DlLEC) of longan EC, about803bp (GenBank GU584089) [including a 19 bp poly(A) tail], consisted of an open reading frame of 669bp, and 5' and 3' utranslated regions of 7 bp and 127 bp, respectively.And, the DNA sequence of DlLEC (GenBank HM773393.1) was 669 bp without intron. The length of DlLEC promoter was 907bp (GenBank: HM773393.1 ). The promoter of DlLEChad a likehood of basic promoter area from 725 bp to 775 bp with a predicted starting site of transcription as Adenine on the 766 th base, and it contained some cis-action elements such as a module involved in light responsiveness, cis-acting element involved in low-temperature responsiveness, cis-acting element involved in heat stress responsiveness,and cis-acting regulatory element required for endosperm expression,cis-acting regulatory element related to meristem expression, binding site of AT-rich DNA binding protein (ATBP-1).⑤The full length cDNA of CHI-I gene (DlCHI-I) of longan EC, about1125bp (GenBank FJ040804) [including a 15 bp poly(A) tail], consisted of an open reading frame of 969bp, and 3' utranslated regions of 141 bp, respectively. And, the DNA sequence of DlCHI-I (GenBank HM773392.1) was 1759bp, and consisted of three exons and two introns, the splicing sites of two introns were comformed to the"GT-AG"rule. The length of DlCHI-I promoter was 907bp (GenBank: HM773392.1 ). The promoter of DlCHI-I had a likehood of basic promoter area from 690 bp to 739 bp with a predicted starting site of transcription as Thymine on the 730 th base, and it contained some cis-action elements such as a module involved in light responsiveness, cis-acting element involved in low-temperature responsiveness, fungal elicitor responsive element,and cis-acting regulatory element required for endosperm expression,cis-acting regulatory element involved in seed-specific regulation,cis-acting regulatory element involved in circadian control, cis-acting regulatory element involved in zein metabolism regulation.2 Bioinformatics analysis of SERK and some other somatic embryogenesis-related genes during longan somatic embryogenesisThe main results of bioinformatics analysis of SERK and some other somatic embryogenesis- related genes during longan somatic embryogenesis were as follows:①DlSERK1gene,encoded 624 amino acids, was a kind of hydrophilic protein with three transmembrane domains,mainly located in the plasma membrane, contained 34 functional sites and 4 structure domains. and a signal peptide at N-terminal The secondary structure of DlSERK1 was made up mostly of random coil. When compared the DlSERK1 with the other plant polypeptides, the identity percentage varied from 77% to 88%.②Dl-14-3-3gene,encoded 261 amino acids,was a kind of hydrophilic protein, mainly located in the cytoplasm,contained 25 functional sites and 1 structure domains. and belonged to 14-3-3 superfamily. The secondary structure of Dl-14-3-3 was made up mostly of Alpha helix. When compared the Dl-14-3-3 with the other plant polypeptides,the identity percentage varied from 73% to 96%.③DlAGL15gene, encoded 254 amino acids,was a kind of hydrophilic protein, mainly located in the nucleus and microbody (peroxisome),contained 15 functional sites and 2 conservative structure domains. and belonged to Type II subfamily of MADS. The secondary structure of DlAGL15 was made up mostly of Alpha helix.and random coil.When compared the DlAGL15 with the other plant polypeptides, the identity percentage varied from 67% to 95%.④DlLECgene, encoded 222 amino acids, was a kind of hydrophilic protein, mainly located in the cytoplasm and mitochondrial matrix space, contained 14 functional sites and 1 conservative structure domains,a signal peptide at N-terminal ,and belonged to H2A subfamily. The secondary structure of DlLECwas made up mostly of random coil.When compared the DlLECwith the other plant polypeptides, the identity percentage varied from 35% to 70%.⑤DlCHI-Igene, encoded 322 amino acids, was a kind of hydrophilic protein, mainly located in the outside, contained 22 functional sites and 2 structure domains, and belonged to Glycoside hydrolase, family 19. The secondary structure of DlCHI-Iwas made up mostly of random coil.When compared the DlCHI-Iwith the other plant polypeptides, the identity percentage varied from 70% to 98%.3 Expression of SERK and and some other somatic embryogenesis-related genes during longan somatic embryogenesis3.1 The quantitative expressions SERK a and some other somatic embryogenesis-related genes during longan somatic embryogenesis①DlSERK1 expressed in 8 different stages during somatic embryogenesis, which showed approximately a"W"curve. The relative high mRNA transcription level of D-SERK1 occurred at the friable-embryogenic callus stage,the cotyledonary embryos stage and the incomplete compact pro-embryogenic cultures stage. And the valley mRNA transcription level of DlSERK1 occurred at the topedo embryos stage.②Dl-14-3-3 expressed in 8 different stages during somatic embryogenesis,which showed approximately a"inverted S"curve. The relative high mRNA transcription level of Dl-14-3-3 occurred at the friable-embryogenic callus stage,the heart embryo stage and the topedo embryos stage. And the valley mRNA transcription level of Dl-14-3-3occurred at the globular embryos stage and the cotyledonary embryos stage. ③DlAGL15 expressed in 8 different stages during somatic embryogenesis,which showed approximately a curve of single-peak. The most high mRNA transcription level of DlAGL15 occurred at the heart embryo stage. And the lowest mRNA transcription level of DlAGL15occurred at the friable-embryogenic callus stage and the cotyledonary embryos stage.④DlLEC expressed in 8 different stages during somatic embryogenesis, which showed approximately a curve of single-peak. The expression levels of six stages were very low except the heart embryo stage and the topedo embryos stage.⑤DlCHI-I expressed in 8 different stages during somatic embryogenesis, which showed approximately a"M"-like curve of double-peak. The relative high mRNA transcription level of DlCHI-I occurred at tthe incomplete compact pro-embryogenic cultures stage and the heart embryo stage. And the lowest mRNA transcription level of DlCHI-I occurred at the cotyledonary embryos stage. 3.2 The relationship of the expression patterns among SERK and some other somatic embryogenesis-related genes during longan somatic embryogenesisThe expression pattern of 14-3-3 was similar to DlSERK, both expressed in 8 different stages during somatic embryogenesis and showed a double peak.,the first peak of both gene expression were at the same stage--- stage 1 ,but the appearance of the second expression peak of curve of Dl-14-3-3 was a little bit earlier than that of DlSERK. DlAGL-15 was in the upstream of DlSERK during somatic embryogenesis. The the appearance of the peak of DlAGL-15 gene expression was a little bit earlier than that of DlSERK.In summary, during longan somatic embryogenesis the five embryogenesis-related genes 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 among embryogenesis-related genes, which could contribute to the research of the regulatory mechanism of phytohormone during somatic embryogenesis and provide target genes for genetic improvement in longan.