Molecular Mechanisms Of Endogenous Hormone Regulation In Stump Sprouting Of Hippophae Rhamnoides Subsp.sinensis

Hippophae rhamnoides subsp.sinensis is an excellent multi-purpose tree species with immortal potential in the arid and semi-arid areas of the North China.However,due to drought stress,pest and disease damage and high afforestation density,the Seabuckthorn plantation appears severe premature senescence.At present,the most effective way is to promote sprouting by stumping.Endogenous hormone played an important role in sprouting emergence and growth,however,the research on the mechanism of stump sprouting mainly focuses on the physiological and biochemical level,while the molecular regulation mechanism of endogenous hormone synthesis,metabolism,transport and signal transduction was rarely reported.For this reason,Ocm,1Ocm,20 cm stumping treatment was set for the declining Seabuckthorn Plantation in this study,and taken the plant without stumping as the control.To study the changes of the number and growth of sprouting at the early growth stage,the peak of growing season,the peak period of secondary growth,and the late growth stage after stumping,and to determine the changes of auxin,cytokinin and gibberellin concentration.On the basis of annotation of transcriptome gene function,the changes of hormone synthesis,metabolism and transporter genes and their regulation mechanism of the three hormones after cropping were analyzed.The changes of three hormone signal transduction genes and the regulation mechanism of their response to hormones,sprouting emergence and growth were analyzed in order to reveal the regulation mechanism of sprouting growth and development from the levels of ecology,hormones to gene expression.The main conclusions are as follows.(1)In the early growth stage and the peak growth stage,stumping treatment increased the number and growth of sprouting,and stumping treatment increased the concentration of IAA,ZR and GA3.These hormones positively regulated the emergence and growth of sprouting.In the peak period of secondary growth and the late growth period,the sprouting growth was increased by the stumping treatment.At the small growth peak and the end of the growth period,the sprouting growth was increased by the stumping treatment.At the peak period of secondary growth,stumping treatment increased IAA,ZR and GA3 concentrations,and these hormones positively regulated the growth of sprouting.At the end of growth,stumping treatment decreased IAA and GA3 concentrations,which negatively regulated the growth of sprouting,while stumping treatment increased ZR concentration,which positively regulated the growth of sprouting.(2)A total of 216G primitive sequencing data were obtained by transcriptome sequencingof the four developmental stages.The quality of sequencing is high and can meet the analysis requirements.After assembly of reference transcript de novo,7363 1 Unigenes were obtained,correspondingtranscriptN50 was 1564 bp,and the splicing quality was good Of the Unigenes,there are 40302,30007,41891,34981 and 15807 unigenes had significant similarity with public databases in Nr,Swissprot,KOG,GO and KEGG databases respectively,and 43617unigenes compared to at least one database and annotated to function.Among them,KOG database annotated 3962 unigenes of signal transduction mechanism,accounting for 9.46%.The results of transcriptome sequencing were proved to be accurate and reliable by qRT-PCR.(3)Hereditary characteristics of endogenous hormone synthesis,metabolism and translocation genes in sprouting at different growth and development stages.As far as auxin synthesis,metabolism and transporter genes are concerned.In the early stage of growth,TAR2,YUC8 and NIT4 genes positively regulated IAA synthesis after cropping,while gatA family proteins negatively regulate IAA synthesis.Overall,adenosylation of GH3 metabolic gene family is enhanced,and the transportation of PIN1C gene was enhanced.During the peak period of growth,YUC6 gene regulated IAA synthesis positively,while AAO and NIT4 genes played a negative role.The transportation of ABCB1 and ABCB19 genes was enhanced,while the transportation of PIN2 gene was weakened.During the peak period of secondary growth,AMI1 gene positively regulated IAA synthesis,YVC4 gene had negative feedback regulation,GH3.5 gene adenosylation was weakened,and the transportation of ABCB19 gene was weakened.At the late growth stage,YUC4 genes positively regulate IAA synthesis,while AAOI.YUC6 and AMI1 genes negatively regulate IAA synthesis.Adenosination of GH3 gene family is weakened,and ABCB1 gene transportation is enhanced.As far as cytokinin synthesis,metabolism and transporter genes are concerned.In the early stage of growth,one CYP735A gene,two LOG genes and three IPT genes positively regulated CTK synthesis,while one IPT3 gene had negative feedback regulation.The amount of CTK oxidative degradation by three CKX genes decreased,and the transportation of PUP5,PUP9 and ENT1 decreased.During the peak period of growth,CTK synthesis of one LOG8 gene and two IPT9 genes decreased,CTK oxidative degradation of CKX4 gene increased,the transportation of PUP9 was weakened,and the transportation of ENT]was enhanced.During the peak period of secondary growth,three IPT genes synthesized more cytokinins,one IPT3 gene had negative feedback regulation,one IPT9,six LOG genes synthesized less CTK,one CKX5 gene oxidized CTK increased,the transportation of PUP9,ENT1 and ENT6 decreased.At the late growth stage,one IPT3 gene synthesized CTK more,one IPT3 gene had negative feedback regulation.Overall,the amount of CTK synthesized by LOG gene decreased,the amount of CTK oxidized by CKX genes decreased,and the CTK transported by PUP gene tended to be balanced.As far as gibberellin synthesis,metabolism and transporter genes are concerned.In the early stage of growth,CPS,KS,KO and GA20OX genes were positively regulated the synthesis of GA.The effect of two GA3OXgenes on the synthesis of GA was weakened,the degradation of GA20X gene weakened the role of active GA,and the transportation of SWEET13 and NRT2.5 genes was weakened.During the peak period of growth,one CPS,KS,KO,GA3OX gene,two GA20OX genes positively regulated the synthesis of GA,one KS,KAO1 gene negatively regulated the synthesis of GA,the GA degraded by GA2OX gene tends to be dynamically balanced,and the transportation of NPF4.6 gene was enhanced.During the peak period of secondary growth,the synthesis of GA was negatively regulated by two GA20OX genes,the degradation of GA by three GA2OX genes was weakened,and the transportation of NPF7.3 and NRT2.5 genes was weakened.At the late growth stage,one KS gene,two KAO1 genes,four GA 2 0 0X genes weakened in the GA synthesis pathway,four GA2OX genes weakened in the degradationofGA,and NPF4.6 and NPF2.5 genes weakened in the translocation of GA.(4)Genetic characteristics of endogenous hormone signal transduction genes in sprouting at different gro wth and development stages.As far as auxin signal transduction genes are concerned.In the early stage of growth,one LAX2,LAX4,LAX5,AUX22,AUX22D,IAA9,IAA13,IAA26,IAA29,ARF2,ARF5,ARF7,GH3.1,SAUR21,SAUR41,SAUR50,ARG7 genes,two IAA27 genes positively regulated signal transduction and germination and growth of sprouting;while one TIR1,IAA9,IAA11,IAA17,SAUR32,SAUR36,SAUR37 genes played a negative role.During the peak period of growth,one LAX2,LAX5,AUX22,AUX22B,AUX28,IAA9,IAA14,IAA26,ARF2,GH3.1,GH3,11,SAUR19,SAUR20,SAUR41,SAUR50,SAUR71 genes,three ARG7 genes positively regulated signal transduction process and germination,growth of sprouting;while one AUX22D,IAA7,IAA11,ARF9,SAUR36,SAUR40 gene played a negative regulatoryrole.During the peak period of secondary growth,AUX28,IAA9,IAA26,ARF2,GH3.5,SAUR41,two SAUR20 and ARG7 genes positively regulated signal transduction and germination growth of sprouting,while one LAX2,IAA4,IAA 7,IAA9,IAA11,IAA13,IAA14,IAA16,IAA17,ARFI,ARF3,ARF5,ARF9,GH3.1,SAUR20,SAUR36,SAUR40 genes negatively regulated the growth of sprouting.At the late growth stage,one LAX4,IAA9,IAA16,ARF2,ARF5,ARF7,ARF9,GH3.1,GH3.5,GH3.6,SAUR21,SAUR32,SAUR36,SAUR72 genes positively regulated signal transduction and negatively regulated growth;TIR1,AUX22,AUX28,IAA13,ARF7,ARF9,GH3.5,SAUR72,ARG7 genes played the opposite role.As far as cytokininsignal transduction genes are concerned.In the early stage of growth,one AHK2,AHP1,OHP2,ARR2,ARR3,ARR18,two ARR9,ARR17,three AHK4 genes positively regulated signal transduction process and emergence and growth of sprouting,while one AHK3,AHP2,AHP4,ARR12,ARR22 genes played the opposite role.During the peak period of growth,the negative regulation signal transduction process of AHK2,AHK3,AHP2,AHP4,ARR1,ARR2,ARR9,ARR11,ARR12,ARR22 genes and the emergence and growth of sprouting were opposite.During the peak period of secondary growth,the negative regulation signal transduction process of AHK2,AHK3,AHK4,AHP1,AHP2,mprl,ARR2 and ARR12 genes and tiller growth were observed.At the late growth stage,the expression of one AHK2,AHP2,ARR3 and two ARR2 genes positively regulated signal transduction and growth of sprouting,while the effects of AHK5,AHPI.ARR9,ARR11 and ARR22 genes were just the oppositeAs far as gibberellin signal transduction genes are concerned.In the early stage of growth,one PIF3,PIL1,GID1C gene positively regulated signal transduction and emergence and growth of sprouting,while GAI gene played the opposite role.During the peak period of growth,one PIL1 and three GAI genes positively regulated signal transduction and emergence and growth of sprouting,while one PIF3 and two GID1B genes negatively regulated signal transduction.During the small growth peak,PIL1 gene positively regulated signal transduction and growth of sprouting while GIDIB,GIDIC,GID2 and PIF3 genes negatively regulated the process.At the late growth stage,GID1B,GID2.PIF3 and PIF5 genes positively regulated signal transduction and growth of sproutingTo sum up,stumping regulates the concentration of hormones by changing the expression of three endogenous hormone synthesis,metabolism and transporter genes of Hippophae rhamnoides subsp.sinensis,and hormones cause changes in downstream gene expression through signal transduction process,thereby regulating the emergence and growth of stump sprouting.