Functional Analysis Of Two Sulfotransferase Genes In Brassica Napus L.

Sulfotransferase（SOT）catalyzes the sulfation reaction of many different substrates and widely exists in animals,plants and bacteria.SOT is a big gene family and plays an important role in resistance,growth and development.But the relevant research remains unclear.This research studied two new SOT genes.They were found involved in GSL biosynthesis and influenced the branch angle and leaf angle,respectively.The main results are as follows:1.Functional identification of sulfotransferase BnSOT-like1 in GSL biothythesis:（1）Cloning and analysis of BnSOT-like1:Accroding to the result of the fine-mapping of the B.napus plant height gene Bndwf1,a gene BnaA09g53490D located in A09 chromosome was cloned from the parents ZS11 and M1 76.The allelic genes are 972 bp in length,encoding 323 amino acids,containing conserved domain Sulfotransfer₃ of SOT family and conserved motif region Ⅰ,Ⅱ and Ⅳ.However,alleles from the two parents were different in conserved motifs,thus the genes from ZS11 and M176 were named Bnsot-like1 and BnSOT-like1,respectively.96 SOT genes were found in the B.napus genome and they distribute in 17 chromosomes except chromosome A04 and A05.BnSOT-like1 is highly similar to AtSOT12.The promoter of BnSOT-like1 contains the regulatory elements of light,ethylene,gibberellin,heat stress,defense and stress responsiveness.（2）Expression analysis of BnSOT-like1:By using the RNA from root,stem,leaf,flower,bud,silique,and cotyledon of ZS11 and M176,the expression of BnSOT-like1 were determined,and the results showed that BnSOT-like1 was expressed in all these tissues,but preferred in buds and siliques.The fusion vector BnSOT-likel-GFP was constructed and transformed into tobacco by Agrobacterium injection.The result showed that BnSOT-like1 was expressed in cytoplasm,nucleus and cell membrane.（3）Overexpression of BnSO T-like1 leads to increase of seed aliphatic GSL content:HPLC was used to determine the seed GSL content of ZS11,M176 and transformants of the two alleles.Overexpression of BnSOT-likel increased the content of aliphatic GSL desulfoprogoitrin from 2.69 μmol/gto 63.87μmol/g,desulfogluconapin from 1.48 μmol/g to 19.50 μmol/g,and total GSL content from 13.9 μmol/g to 101.95μmol/g.These results imply that BnSOT-like1 catalyzes the core structure formation of aliphatic seed GSL of B.napus.（4）Transcriptomic analysis:Transcriptomic experiments between ZS11 and BnSOTlike1 overexpressing transgenic line showed that many up-regulated genes involved in GSL biosynthesis.This suggests that BnSOT-like1 catalyzes the formation of GSL core structure,and overexpression of BnSOT-like1 can feedback-regulate the metabolic network,leading to the up-regulated expression of genes involved in GSL biosynthesis.（5）BnSOT-like1 is a minor gene affecting seed GSL content:In order to determine the effect of sequence variation of BnSOT-like1 to GSL in natural biological state,QTL analysis was performed with the seed GSL content data of 150 plants in the BC1 population of mapping.Only one QTL（qGSLC02）was found with LOD threshold of 3.12.Searching the LOD profile of whole genome,we found a small peak with LOD score 0.48 at location of BnSOT-like1 in A09 chromosome.The peak explained 1.1%of GSL variance in the population.Thus,BnSOT-like1 is a minor gene involved in GSL biosynthesis in natural biological state.2.Functional identification of sulfotransferase BnSOT-like 2（1）Cloning and analysis of BnSOT-like2:Sulfotransferase gene BnaA09g53470D（BnSOT-like2）was cloned on the same mapping interval to BnSOT-like1.The gene is 987 bp in length,and its amino acid sequence is 328 in length,containing the conserved domain Sulfotransfer₃ of SOT family,and conserved motif region Ⅰ,Ⅱ and Ⅳ.BnSOT-like2 also belongs to the same subfamily to AtSOT12.Sequence alignment indicated that BnSOT-like2 have only one amino acid difference with BNSTs and AtSOT12 in region Ⅱ.Thus,BnSOTlike2 may be inferred to have similar function to BNST1-4 and AtSOT12.The promoter of BnSOT-like2 contains the regulatory elements of abscisic acid,light,auxin,low temperature,etc.（2）Expression pattern of BnSOT-like2:The RNA from root,stem,leaf,flower,bud,silique,cotyledon and petiole of ZS11 and M176 were used for RT-PCR.The result showed that BnSOT-like2 expressed in all these tissues and preferred in root and petiole.Thus,BnSOT-like2 expression may probably influence the morphology of petiole in B.napus.The fusion vector BnSOT-like2-GFP was constructed and transformed into Tobacco by Agrobacterium injection.Fluorescence signals observed indicated that BnSOT-like2 expresses in cytoplasm.（3）Diurnal change of the leaf angle,branch angle and expression of BnSOT-like2:The B.napus leaf angle at 6 different time from 8:00 to 23:00 were measured.Results showed that the leaf angle was smallest at 8:00,then increased and maintained large leaf angle in daytime,and leaf angle gradually decreased in the night.After flowering,the diurnal change tendency of branch angle was observed to be same with that of leaf angle.RT-qPCR determinations indicated that the expression of BnSOT-like2 is higher in the night and morning,and lower in the daytime.The change tendency of the gene expression is opposite to that of leaf angle and branch angle.This indicated that the expression of BnSOT-like2 is probably negative associated with leaf angle and branch angle.（4）Overexpression of BnSOT-like2 leads to reduction of leaf angle and branch angle in the morning and nighttime,and leads to reduction of plant height:The overexpression vectors of BnSOT-like2 was constructed for genetic transformation of B.napus ZS11.3 independent transgenic lines were obtained.The leaf angle of BnSOT-like2 transgenic line was significantly smaller than ZS11 in the night and morning,and had no significant difference in the daytime.At harvest stage,the average plant height of BnSOTlike2 was 119.6 cm,while that of ZS11 was 134.6 cm,indicating that overexpression BnSOTlike2 dwarf the plant height significantly.Based on our results,it may be concluded that the diunal change of sulfotransferase activity is a determining factor of leaf and branch morphorlogy in B.napus,and sulfonation of BR affects subtlely the transgenic plant height.（5）The expression of BnSOT-like2 is negatively regulated by light:The expression of BnSOT-like2 in ZS 11 and the transformant under light and dark were detected by RT-qPCR.The result showed that the expression of BnSOT-like2 decreased with the increasing of illumination time,and increased with the increasing of dark time.This indicated that the expression of BnSOT-like2 is negatively regulated by light.In summary,present works have functionally characterized two sulfotransferase genes BnSOT-likel and BnSOT-like2 by genetic transformation,expression analysis,transcriptomic analysis,physiological and biochemical experiments,and althought the two genes belong to the same phylogenetic clade,they function differently in B.napus.