Isolation And Expression Of AP2/ERF In Jasminum Sambac(L.)Ait

Jasminum sambac(L.)Ait is a perennial evergreen shrub,jasmine for short,belonging to the genus Jasminum of the family Oleaceae.It can be used as a garden ornamental plant,a raw material for brewing tea and extracting essences.It also can be used as medicine with good ornamental value,economic value,health care value and medicinal value.Jasmine,like other plants,is inevitably subject to biological stresses such as germs and insect pests in its growth and development.In addition,abiotic stresses such as salt damage,drought,and cold damage can also seriously affect the growth of jasmine and flower yield.AP2/ERF(APETALA2/ethylene responsive factor)is a large family of transcription factors that play a major role in the regulation of plant growth and development and defense mechanisms.Its family members are involved in plant growth,flower,fruit,and seed development,response to environmental stress such as high salt drought and low temperature,and signal transduction pathways such as salicylic acid,ethylene,and jasmonic acid.Therefore,AP2/ERF plays an important role in the regulation of biological and abiotic stress-responsive gene expression.In this study,a full-length cDNA library mediated by DSN was constructed using open jasmine.The AP2/ERF gene family members were screened by dilution pool PCR and analyzed for basic physicochemical properties,protein structure prediction and function prediction.Fluorescence quantitative polymerase chain reaction(PCR)was used to detect the expression of these AP2/ERF genes in different tissues of jasmine,different growth stages of buds,different open period,and different hormones and stress treatments,and they were clearly involved in the growth and development of jasmine,response to hormone signaling and stress.The members,from the molecular level,explored the molecular mechanism of AP2/ERF regulation of jasmine resistance and provided a theoretical basis for the genetic improvement of resistance and quality of jasmine.The main findings of this paper are as follows:1.A DSN-mediated full-length-normalized cDNA library was constructed and 13 AP2/ERF family members were screened:the first strand of cDNA was synthesized using the total RNA of jasmine petals as a template,and cDNA was amplified by LA-suppression PCR.For the second strand,the optimal number of cycles is 17,and the optimal concentration for DSN treatment is 1/2 U.The DS-treated product is subjected to LA-suppressed PCR amplification to homogenize cDNA,the optimal number of cycles is 22,and the size of the product is between 750 and 2000 bp.There was no bright band in the dispersion,the library titer was 1.8×106,and the average insert size was 1700bp.The quality of the library was good.The library was screened by dilution pool PCR and 13 AP2/ERF gene family members were obtained.In comparison with different Arabidopsis AP2/ERF,these genes are members of the AP2/ERF gene family.2.13 AP2/ERF gene family members were obtained:JsERF3,JsERF4,JsERF5,JsERF39,JsERF61,JsERF71,JsERF107,JsERF109,JsERF118,JsRAP2.4,JsRAP2.12,and JsTINY,The full-length cDNAs are 1249,1015,1357,1455,759,1519,1499,922,1269,1577,1896,1778,and 1058 bp,respectively,encoding 247,228,238,295,230,270,291,21 1,250,345,338,406,and 260 amino acids.All JsERFs proteins contain only one AP2/ERF domain and belong to the ERF subfamily.3.JsERFs are expressed in roots,young leaves and flowers,and are expressed specifically in other tissues.4.During the development of jasmine buds,only the expression of JsERF4 was significantly up-regulated at 4 days,while the expression of other JsERFs was changed.However,the overall change is not obvious,so it is speculated that JsERF4 may be involved in the development of jasmine buds.5.During the opening process of jasmine,the expression of JsERFs changes significantly,the overall showed an up-down trend,and the peak expression level is at 2:00 At this time,the flowers were fully open and the petals extended 180°,suggesting that JsERFs may be involved in promoting the opening of jasmine6.Under the treatment of 300 mg/L ET,100 mg/L JA,and 1 mM SA,JsERF118,JsERF4,JsERF2,and JsERF5 were significantly induced by ET reaching the highest level at 12h after treatment.JsERF39 was only induced by SA,JsRAP2.12 and JsERF1 07 were strongly induced by SA and JA,JsERF1 09 was significantly induced by SA,the highest value was at 24h after treatment,but the expression of JsERF61 did not change significantly.The reduced expression of JsERF109 under ET treatment may be inhibited.The above results indicate that JsERFs are induced by ET,SA,and/or JA,suggesting that JsERFs responded to hormone signaling pathways;7.Except for JsERF118 and JsERF109,the other JsERFs were all induced by salt stress,among which JsERF4,JsERF71,JsERF2 and JsERF39 were induced to the highest expression levels in the treatment After 2 weeks,the highest expression level of JsERF5 was at 3w after treatment,and that of JsERF61 was at 4w.Under drought stress,the expression of JsERF4,JsERF109,JsERF5,JsERF71,JsERF2,JsERF107,and JsERF39 increased greatly.The highest value was 2s after treatment,JsERF3,The expression of JsERF2,JsERF61,and JsRAP2.12 were also significantly induced,with the highest expression level at 4 w,while JsERF118 was hardly induced by drought stress.