Cloning And Analysis Of Genes Related To Flowering Of Jatropha Nigroviensrugosus

Jatropha carcas L.(Fam.: Euphorbiaceae;Gen.: Jatropha),is an important energy tree species,J.carcas has few female flowers and a low proportion of male and female flowers(1:20),which affects the yield.Jatropha nigroviensrugosus CV Yang is a newly discovered variety with high proportion of male and female flowers(1:8).The cloning and analysis of genes related to flower formation of J.nigroviensrugosus can explore the reasons for the existence of reproductive advantage.In this study,Illusima sequencing technique was used for comparative transcriptional analysis of the two varieties,the full length of the differential expression target gene was obtained,the temporal and spatial expression of the target gene in different tissues was analyzed by RT-qPCR technique,subcellular functional localization,bimolecular fluorescence complementation and transgenic function verificationwas used to understand the function of the gene.This study is to provide guidance for molecular breeding and cultivation and reproductive regulation of J.carcas.The results show that:(1)The quality of transcription library sequencing was good,Q20 was above97%,Q30 was above 93%,the average content of QC was 43.5%.The genome comparison rate of two cultivars was above 96%,the sequence difference was small,and the phylogenetic relationship was close.The results of GO enrichment showed that there were significant differences of leaf buds of the two cultivars in the stimulation and response to light,the trend of gene expression showed that 9 genes specificity highly expressed in the male flowers;Most of the up-regulated genes in both female and male flowers were related to floral organ formation,and the inflorescence bud parts of the two cultivars were obviously clustered and distinguished,the difference genes reached 1646.Plant hormone signal transduction pathway is involved in the process of sex differentiation and flower bud formation,while DELLA,MYC2,CYCD3 and other genes may be related to the abortion of male flower primordium and the determination of female flower sex during the process of inflorescence bud formation.(2)The enrichment of differential genes in J.nigroviensrugosus group showed that: the high expression of genes related to sucrose phosphate synthase,soluble starch synthase(EC:2.4.1.21)and granule-bound starch synthase(EC:2.4.1.242)may promote the formation and development of male flowers.The genes CUC2 and CUC3 may regulate the formation and development of female flowers.The characteristic genes of floral organ development were conservatively expressed in four tissues,the high expression was type-II MADS-box gene.(3)The full length of three different expressed genes was obtained.The open reading frame(ORF)of J.nigroviensrugosus JnCYCD3;1 is 414 bp and encoding137 aa.Compared with J.carcas,the retention of introns leads to the early termination of the coding frame.RT-qPCR shows that the expression of JnCYCD3;1 is the highest in the new leaves,followed by inflorescence buds.The expression of JnCYCD3;1 in the new leaves,was 68.42 times higher than that in the root.There was no difference in the expression between inflorescence buds and female flowers in the early stage of flower formation,the expression was the lowest at the stage of pollen formation.The ORF of JnSLR1 was 1641 bp,encoding 546 aa,the expression of JnSLR1 ininflorescence buds was the highest,followed by new leaves,there was no difference between male and female flowers.JnMYC2 ORF 2025 bp,encoding 674 bp,the expression of JnMYC2 in inflorescence buds was the highest,but there was no difference between male and female flowers in the new leaves.(4)The analysis of promoter elements shows that,PJnCYCD3;1 has JA response elements(CGTCA-motif,TGACG-motif),meristem expression related elements(CAT-box),MYB binding sites involved in the regulation of flavonoid biosynthesis genes(MBSI),photoresponse elements(GT1-motif,AAAC-motif)and so on.There are gibberellin response elements(TATC-box,MYB,MYC),photoregulatory elements(Box II,Box 4,GT1-motif,etc.)and stress response elements in pJnSLR1.There are CAT-box(cis-acting elements related to meristem expression),more photoregulatory elements(GT1-motif,G-box,MRE,etc.),stress response elements and so on in the promoter of JnMYC2 gene.(5)The subcellular functional localization test showed that both JnCYCD3;1and JnSLR1 played a role in the nucleus,JnMYC2 might have transnuclear membrane behavior.(6)The bimolecular fluorescence complementation experiment showed that JnMYC2 interacted with JnCYCD3;1.(7)JnCYCD3;1 transgenic tobacco can significantly promote the reproductive cycle,may have the effect of promoting flower formation and transformation.