Cloning And Expression Features Of Gene Families Of The Flowering Loci LFK, PEBP And PHY From Chia (Salvia Hispanica)

Chia(Salvia hispanica),originated in Mexico,has high nutritional value due to its rich in linolenic acid,protein,dietary fiber,flavonoids and other antioxidants,especially its linolenic acid is the highest content of known oil crops.However,it was an important ancient Indian crop that could not be successfully introduced worldwide.In recent years,with the crop-revival of chia and re-entering people's attention,this crop has been studied and cultured in many countries.However,the short-day habit is a crucial limitation factor for its growing in mid-and high-latitude regions.The first task for successful planting is to dissect the short-day flowering mechanism of chia.LFK [LOV(Light,Oxygen,Voltage)and Kelch repeat-containing F-box],PEBP(phosphatidylethanolamine-binding protein)and PHY(Phytochoome)are three important gene families regulation flowering time of plants though photoperiod-pathway.This study cloned the full-length mRNAs of 3,11and 5 genes from each of the LFK,PHY and PEBP families from chia,their gene and protein characteristics were analyzed,some characteristics of the evolution of LFK,PEBP and PHY gene families in Lamiales were revealed,and their organ-specity of their transcriptional expression was detected,we also revealed circadian rhythm,seasonal response,hormone response characteristics,and responses to various abiotic stresses of LFK gene families.Among them,ShFKF1a and ShFKF1b genes were transformed into Arabidopsis thaliana,and the positive transgenic plants showed earlier flowering time and accelerated growth of plant height.This study lays a foundation for further dissecting the relationship between these loci and the flowering habits of Chia and other Lamiales plants.1.Cloning,analysis and expression features of 3 member genes of chia LFK familyThe mRNAs of ShFKF1a,ShFKF1b and ShZTL are 2204 bp,2207 bp and 2473 bp,with alternative sites for transcription initiation and poly A tailing,which encode acidic proteins of 626 aa,625 aa and 623 aa,respectively.They all have PAS domain,F-box domain and Kelch motif.The identity percentages between ShFKF1a and ShFKF1b on mRNA and protein levels are 82.8% and 95.2%,the identity percentages between ShFKF1a and ShZTL on mRNA and protein levels are 54.4% and 73.3%,and the identity percentages between ShFKF1b and ShZTL on mRNA and protein levels are51.9% and 73.9%,respectively.They are typical in conserved domains,hierarchical structure,and subcellular localization.Evolutionary study shows that the LFK gene family members in the Lamiales can be divided into two categories,one is ZTL and the other is FKF1,and all the analyzed Lamiales species including Chia do not have the LKP2 gene.Among these species of Lamiales,the more the number of FKF1 genes,the fewer ZTL genes,whereas the fewer FKF1 genes,the more of ZTL genes.There seems to be a negative correlation and coordination relationship between FKF1 gene number and ZTL gene number.In addition,FKF1 orthologs diverge faster than ZTL orthologs among species of Lamiales.FKF1 seems to exhibit more active functional evolution characteristics than ZTL.ShFKF1 has the characteristic of predominant expression at midnight and early morning in long-day,and the characteristic of predominant expression at midnight and midnight on autumnal equinox days.ShZTL responses to circadian rhythm and seasonal climate relatively gentle.After treatment with the four types of hormones,the expression of LFK family genes in short-day was significantly higher than that in long-day sunshine.After treatment,chia FKF1 gene expression could be promoted in long-day and inhibited in short-day;BR,GA3 and IAA promoted ShZTL expression in long-day,but suppressed its expression in short-day;KT suppressed ShZTL expression regardless of long-day and short-day.All four hormones inhibited the expression of ShZTL gene in short-day.After a variety of abiotic stress treatments,the expression of the chia LFK gene family showed fluctuations or changes,and returned to near the basal levels.There were some stress treatments which produced regular changes.For example,damage treatment strongly promoted the expression of FKF1,and continuously inhibited the expression of LFK family after Na Cl treatment.In general,the expression profiles of the two members of ShFKF1are similar but difference also exists.They are both expressed in young leaves,small buds,and flowers.The expression of ShFKF1b is higher in roots than ShFKF1a,but is opposite in seeds.The expression of ShFKF1 peaks earlier than ShZTL in the process of plant growth,and the difference level of them is significant in young leaves.As chia plant matures,the expression of ShZTL is generally higher than the expression level of ShFKF1,especially in seeds.2.Cloning,analysis and expression features of 11 member genes of chia PEBP familyThe mRNA lengths of Sh PEBP1,Sh PEBP2 a,Sh PEBP2 b and Sh PEBP3 are 762 bp,660 bp,660 bp and 776 bp,encoding basic proteins of 176 aa,177 aa,177 aa and179 aa,respectively.The mRNAs of Sh PEBP4 a,Sh PEBP4 b,Sh PEBP5,Sh PEBP6,Sh PEBP7 and Sh PEBP8 are 732 bp,687 bp,734 bp,832 bp,735 bp and 685 bp,encoding basic proteins(Sh PEBP5 being acidic)of 173 aa,173 aa,174 aa,174 aa,172 aa and 179 aa,respectively.Sh PEBP9 has a full-length mRNA of 689 bp,encoding a basic protein of 176 aa.Sh PEBP family genes all have alternative sites for transcription initiation and poly A tailing.Their encoded proteins all have PBP domains,typical in conserved domains,hierarchical structure,subcellular localization,and other aspects.Evolutionary study shows that the members of gene numbers of PEBP family of Lamiales species are generally more than that of Arabidopsis thaliana.Each PEBP gene of Arabidopsis thaliana has 1 or more genes that are orthologous to the Lamiales species,and they form five homologous branches in evolutionary tree.The chia genes corresponding to Arabidopsis FT/TSF,BFT,TFL1,ATC and MFT are Sh PEBP1?Sh PEBP3,PEBP4a?PEBP4b,Sh PEBP5?Sh PEBP6,Sh PEBP7?Sh PEBP8 and Sh PEBP9,respectively.Based on difergence progress manesfasted by the branch lengths,it is speculated that in the evolution of the flowering traits of Lamiales plants,MFT embodied more active functional evolutionary features,BFT was the most conservative,and other genes were in-between.The organ specificity profiles of the PEBP family genes are not much different.Sh PEBP3 and Sh PEBP7 are highly expressed in small buds,and the abundance of other genes is very low.Generally speaking,their expression is high in buds and seeds,and much lower in other organs.3.Cloning,analysis and expression features of 5 member genes of chia PHY familyThe mRNAs of ShPHYA,ShPHYB,ShPHYC,ShPHYD,ShPHYE are 3874 bp,4090 bp,3985 bp,4048 bp,3533 bp,with alternative sites for transcription initiation and poly A tailing(especially drastic in alternative sites for transcription initiation),which encode acidic proteins of 1116 aa,1137 aa,1126 aa,1138 aa and 1098 aa,respectively.They all have GAF,PHY,PAS,His KA(except ShPHYC)and HATPase?c domains.They are typical in conserved domains,hierarchical structure,and subcellular localization.Evolutionary study shows that the chia Sh PHY family and other Lamiales PHY families genes form four homologous branches with the Arabidopsis PHY family members.The ShPHYA,ShPHYB/ShPHYD,ShPHYC,ShPHYE are orthologous to PHYA,PHYB/PHYD,PHYC,PHYE from Arabidopsis,respectively.The gene number of PHY family in the dicot plants is relatively stable,and it is neither easily lost nor duplicated.Interestingly,the phenomenon of PHYD origin due to the duplication of PHYB in Brassicacea and Lamiales occurred independently,probably because dicotyledons plants have a strong demand for the photoreceptor PHYB.The Sh PHY family has a high accumulation in the late seed development stage and early flower development stage,and is low in other periods.ShPHYE and ShPHYA are highly expressed in the late seed development stage.4.Progress of ShFKF1a and ShFKF1b transformation into Arabidopsis thalianaCoding sequences of ShFKF1a and ShFKF1b were cloned and overexpression vectors pC2301M1DPB-35S-ShFKF1a and pC2301M1DPB-35S-ShFKF1b were constructed.They were transformed into Arabidopsis thaliana via Agrobacterium tumefasciens-midated flower-dip method,and 7 and 6 positive transgenic plants were obtained respectively.Both types of transgenic plants showed earlier flowering and taller plant height compared with wild-type plants.This indicates that both of the two FKF1 genes from chia are physiologically functional in promoting flowering.This study is the report of the LFK,PEBP and PHY family genes in the order Lamiales,which will promote the dissection of flowering mechanism of chia and other Lamiales plants,and enrich the evolution and expression characteristics of plant LFK,PHY and PEBP.It lays the foundation for further research on the molecular mechanism of the flowering induction pathways in chia and other short-day plants.