Observation Of Seed Development And Excavation Of Genes Related To Lipid Accumulation For Herbaceous Peony(Paeonia Lactiflora Pall.'Hangshao')

'Hangshao'(Paeonia lactiflora Pall.'Hangshao')known as 'Hang Baishao',is a traditional cultivar of medicinal herbaceous peony variety in China.Recently,many studies have shown that P.lactiflora 'Hangshao' has higher seed setting rate,oil content and unsaturated fatty acid content.This is similar to the oil P.suffruticosa cultivar,which is expected to be developed as a new oil plant resource.Currently,there are few researches on the physiological characteristics of seed development and oil accumulation in 'Hangshao' because the study on the oil P.lactiflora is in the initial stage.Meanwhile,there is a lack of research on the variation characteristics between different 'Hangshao' species for 'Hangshao' is based on live breeding and larger differents between individuals.Based on these,in this study P.lactiflora 'Hangshao' cultivated in the P.lactiflora resource nursery of Yangzhou University was used as the material.Firstly,the dynamic characteristics of the growth and development of 'Hangshao' pods and seeds were observed to establish an evaluation method of oil P.lactiflora germplasm resources based on agronomic traits,From which,the excellent single plants were screened.Then,RNA-Seq transcriptome sequencing was used to mine the fatty acid synthetic related genes in 'Hangshao' seeds.The physicochemical characteristics of oil bodies in' seeds were also explored,and the cloning and expression of oil body protein related genes were studied.The main findings are as follows:(1)There are three types of 'Hangshao' main pods,namely third fruit pods,fourth fruit pods and fifth fruit pods.The weight of total and single pod,and the length of average total pod and single pod the three fruit pods first increased and then decreased,and reached the maximum at 60 day after flowering(DAF).The seed length,width and thickness of these three types of fruit pods increased first and then decreased,and all reached the maximum at 60 DAF except for the seed thickness of the fourth fruit pods.The single seed quality,single pod seed quality and total pod seed quality of these three types of fruit pods also increased first and then decreased,and the maximum values appeared at 70 DAF.Also,the seeds of the three types of fruit pods had abortion during development,and the number of seeds gradually decreased.(2)The average yield per plant of 60 'Hangshao' germplasms was 127.9 g,the oil yield was 26.78%,and the relative content of polyunsaturated fatty acids was 65.44%.There were sig1ificant differences among different germplasms,the average variation coefficient of 31 indicators is as high as 38.67%.Correlation analysis showed that seed yield was positively correlated with plant height and flower number.Also,Plants with high yield were mainly concentrated in the germplasms of white stigma,while that with high oil content were mainly concentrated in the germplasms of red stigma.After clustering 60 germplasms according to yield,they can be classified into 3 categories,including 12 high-yield types,45 medium-yield types,and 3 low-yield types.Through principal component analysis,31 indicators were reduced in dimension,and 8 principal components were extracted.This can represent 81.89%of the information,which provided possibility for the simplification of single-plant evaluation system Besides,five comprehensive germplasms were found using the full-range aliquot method through 60 germplasms,namely HS54,HS28,HS26,HS22 and HS27,(3)The seed endosperm of�Hangshao'belongs to the nuclear endosperm,and begins to cellularized at 45 DAF.The development of the embryo is later than that of the endospemm,which is only visible after 60 DAF.The histochemical staining demonstrated that the density of-starch granules in the endosperm and the size of starch granules increased first and then decreased.The density of protein in endosperm decreased first and then slightly decreased.The accumulation of lipids in endospemi increased first and then slightly decreased.The result is consistent with the trend in the determination of starch,Protein and crude fat.Apart from that,GC-MS analysis revealed that 'Hangshao' seeds have five main fatty acids,including palmitic acid,stearic acid,oleic acid,linoleic acid and a-linolenic acid,respectively.The order of a-linolenic acid,linoleic acid,oleic acid,palmitic acid and stearic acid was arranged in the order of highest to lowest content.The total content of the five fatty acids increased first and then decreased,and reached the maximum at 75 DAF.(4)A total of 150,156 Unigenes were obtained by transcriptome sequencing,with an average length of 1,030 bp.There were 1,550 Unigenes annotated in the seven functional databases including NR,NT,GO,KOG,KEGG,SwissProt and InterPro.Based on KEGG database,1,766 Unigenes were annotated in the lipid metabolic pathways,among which 103,74 and 70 Unigenes are anmotated into fatty acid biosynthesis pathway,fatty acid elongation pathway and unsaturated fatty acid synthesis pathway;respectively.By pairwise compared the transcription of 'Hangshao' seeds of three development stages,a total of 2,410(30 DAF vs 60 DAF),4,210(60 DAF vs 90 DAF)and 7,385(30 DAF vs 90 DAF)up-regulated DEGs were obtained,respectively,while a total of 2,225(30 DAF vs 60 DAF),8,094(60 DAF vs 90 DAF)and 10,906(30 DAF vs 90 DAF)down-regulated DEGs were obtained,respectively.Combined with the comparison,a total of 1480 DEGs were detected.Among them,83 DEGs were enriched in the fatty acid metabolism pathway,including 12 DEGs involved in the fatty acid biosynthesis and 1 DEG involved in fatty acid elongation.Furthermore,qRT-PCR was used to analyze the expression patterns of nine fatty acid biosynthetic related genes including FBCP,BC,fabD,fabF,FATB,KCR,FAD2,FAD3 and FAD7,and it showed that they all highest expressed at 45 DAF.(5)Transmission electron microscope observation of 'Hangshao' seeds revealed that the oil body volume and distribution density increased with the development of seeds.The Malvern particle analyzer showed that the particle size of the oil body gradually increased,and the oil particle size at 90 DAF was about 8 times compared to that at 30 DAF.The zeta potential value of oil body was negative,and its absolute value first increased and then decreased,and reached the maximum at 75 DAF.The stability of mature seed oil body showed that the increase of temperature had no significant effect on the oil body structure.Besides,the oil body structure was seriously damaged when the pH value was 5.0,whereas the oil body was stable when the pH value was ranged from 7.0 to 11.0.The oil body volume increased with the increase of pH value The stability of the oil body under different NaCl concentrations showed that.the oil body structure is less affected at 25 mM,while it was destroyed with the increase of the concentration.At 100 mM,almost all of the oil body structure was destroyed.Further,the oil membrane protein gene PlOLE and PICLO were cloned from the 'Hangshao' using RACE technique.The sequence of PIOLE was 1110 bp in length,with a 435 bp complete open reading frame,encoding a total of 144 amino acids.The sequence of PICLO was 908 bp in length and has a coruplete open reading frame of 720 bp,encoding a total of 239 amino acids.PlOLE and PlCLO were all detected in the five developmental stages of 'Hangshao' seeds,and the expression level of them was the lowest in the seeds at 30 DAF and the highest in the seeds at 60 DAF,while the expression of PIOLE was higher than that of PICLO.