| Paeonia ostii T.Hong et J.X.Zhang var.lishizhenii B.A.Shen uses as a traditional Chinese medicine and ornamental rootstock.The provenance in Fenghuang Mountain of Tongling and Ya mountain of Nanling Counties,Anhui Province is indigenous plants of the genuine traditional Chinese Cortex moutan and introduction as ornamental tree peony grafting rootstocks.Since initiated studies of the tree peony molecular and physiological philosophy and in vitro culture of biotechnology in our laboratory in 1996,it was found that the the peony is an woody oil plants,of which seed oil contains 92.0%unsaturated fatty acids,and the components of oleic acid,linoleic acid andα-linolenic acid are 23.1%,25.2%and 42.0%,respectively.Inhibited enzym activity catalyzed the oxidative reaction in three steps of gibberellin(GA)biosynthesis,namely cyclization,oxidation,and transformation,achieved significantly dwarfing flowering plants.The tree peony seed oil from kernels of Paeonia ostii T.Hong et J.X.Zhang and Paeonia rokii have been approved as a new food resource produced by pressing,decolorizing,deodorizing,and other prosessing(Announcement No.9 of 2011 issued by The former Ministry of Health,The People’s Republic of China),as a woody grain and oil to accelerate the development and reserve(issued Guoban Fa[2014]No.68 by the office of the State Council,Guishan Han[2015]No.242 by The State Forestry Administration’s,and Fagai Nongjing[2020]No.1753 by ten ministries and commissions led by The National Development and Reform Commission Agricultural Economics.Based on mentioned above,this study excavated and isolated the genes coding enzymes that catalyze oxidation reaction in GA biosynthesis pathway in P.ostii var.lishizhenii.The genes were functional identified by transformation and transcriptome analysis,dwarfing plant and increasing yield function as well as the related molecular physiological aspects growing seedling plants in field were verified by field expreiments.The results of this study are molecular and physiological basis for revealing the molecular regulation mechanism of high quality and high yield seed oil and for establishing the breeding technology of improved varieties of Paeonia ostii var.lishizhenii.The main results are as below:1.Cloned two genes related GA biosynthesis oxidation reaction in P.ostii var.lishizhenii,the ent-kaurene synthase gene PoKS and the ent-kaurene oxidase gene PoKO.PoKO was identified as the target gene for dwarf breeding.(1)Among the seeding plants flowering and fruiting in P.ostii var.lishizhenii,the plants with heights of 50-59 cm have the highest seed yield per area(144.6 g/m~2)and the plants with heights of 70-79 cm have the most number(29.7%).Seed oil,weight per 1000-grains and water content showed no significant difference among different height seedling plants,but the seed and oil yield were increased by approperly dwarfing.(2)The full-length c DNAs of GA oxidation step regulation genes PoKS(1,777 bp)and PoKO(2,388 bp)were isolated.(3)By q RT-PCR determing,PoKS gene expression level was highest in petals,whereas PoKO gene expression was highest in tender stems,and their relative expression ware 26.4 and 38.8 times that of the young buds(lowest expression),respectively.The gene expression patterns of PoKS and PoKO were positively correlated with the stem growth rate in duration of stem growth(correlation coefficient 0.93 and 0.92,respectively),and were consistent with stem fast growth changes of 10-17 days after burgeon(DAB).This suggested that the PoKO gene specific expression in growing stem in P.ostii var.lishizhenii should be related to its plant height growth,whereas PoKS gene expression should be mainly involved in flower development.(4)The homologous interference identification of the two gene function in Arabidopsis thaliana showed that PoKS and PoKO gene interference decreased GA content in the stem by 15.8%and 19.2%,respectively,inhibited the expression of 11 downstream gibberellin synthesis related genes(At KAO1、At KAO2、At GA20ox1、At GA20ox2、At GA20ox3、At GA20ox4、At GA20ox5、At GA3ox1、At GA3ox2、At GA3ox3、At GA3ox4),and the plant height decreased by 24.9%～33.1%and 31.1%～46.3%,respectively.This showed that PoKS and PoKO gene expression significantly regulated plant height growth.2.PoKO gene was identified as the key gene for dwarfing by transcriptome analysis.PoKO is as a breeding marker gene and its expression(copy number)as priority index for selecting the dwarf and oil elite tree peonies.The differentially expressed genes(DEGs)in the tender stems among seedling blome and fruit plants of dwarf(height<40cm),medium-high(40cm≤height≤80cm),and high(height>80cm)were analyzed by high-throughput sequencing of the second and third generation full-length transcriptome(RNA-seq and Iso-seq)in P.ostii var.lishizhenii.As results,(1)883 DEGs were found between dwarf and high height plants of P.ostii var.lishizhenii,of which 416 genes were up-regulated and 467 genes were down-regulated.(2)74 DEGs were annotated to 51 KEGG database metabolic pathways,and the most enrichment(14 DEGs)pathway was plant hormone signaling pathway(ko04075).(3)Gene expression analysis during the transcriptome revealed that PoKO gene was the key gene regulating the plant height variation in GA synthesis pathway.(4)15 genes related phytohormones biosynthesis and signal transduction were used and verified the reliability of the transcriptome data by q RT-PCR.(5)The absolute quantification expression of PoKO gene in tender stems during their fast growth(10 DAB～17DAB)in the field was singnificantly positively correlated with GA content(R=0.87,p<0.01)and both with growing plant height and stopped grow plant height(R=0.87 and 0.84,respectively).This indicates that PoKO gene is the key gene for dwarfing and its expression correlates positively with GA content and plant height.PoKO is suggested as marker gene and expression priority criteria for selection dwarf and oil elite tree peonies.3.Characteristicszation of four genes concerned unsaturated fatty acid dehydrogenases achieved.Stearoyl-ACP desaturase gene PoSAD(Gen Bank number:KX078474)regulated the synthesis of monounsaturated fatty acids(oleic acid),and unsaturated fatty acid dehydrogenase genes PoFAD2,PoFAD3,and PoFAD6 regulated the synthesis of polyunsaturated fatty acids(linoleic acid andα-linolenic acid).(1)The promoter(1,203 bp)of PoSAD gene was cloned by fusion primer and nested integrated PCR.Bioinformatics analysis predicted that the sequence contained 15 specific endosperm expression elements.(2)By q RT-PCR determined,PoSAD and PoFAD3 expression levels were highest in developing endosperm,and were 53.7 and 149.5 times that of the lowest in the bud and in the root,respectively.PoFAD2 gene expression levels in stamens and endosperm were 1,438 and 148.0 times that of the lowest in bud,and were significantly higher than that in other tissues,whereas PoFAD6 gene was highly expressed in petal and stamen,no significant difference in the expression at different stages of endosperm development.(3)The function of PoSAD and PoFADs analyzed by heterologous expression in Saccharomyces cerevisiae and A.thaliana showed that PoSAD gene regulates stearic acid to oleic acid,PoFAD2 and PoFAD6 genes regulate oleic acid to linoleic acid,and PoFAD3 gene regulates linoleic acid toα-linolenic acid.The results suggested that PoSAD,PoFAD2,and PoFAD3 genes were the key genes regulating the synthesis of oleic acid,linoleic acid,andα-linolenic acid in the endosperm development of P.ostii var.lishizhenii. |
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