| Peanut(Arachis hypogaea L.)is one of the most important oil crops worldwide,and the total content of fat and protein is up to 80%in peanut seeds,which is a main source of edible oil and vegetable protein.Plant fruit is the harvesting organ of a variety of crop,the growth and development of fruit directly affect the yield and quality of seed.The unique geocarpic feature makes peanut different from the most other legume plants.After fertilization,peanut zygotes divide several times,forming a pre-embryo,and then the embryo and pod development arrest,while ovary elongation continues to form a"peg".When the elongating peg pushes the ovary to the soil,the embryo and pod development resumes under dark condition and eventually expands into a pod.Numerous studies showed that light played an important role in regulating peanut pod growth and development.As the red and far red light can reversibly affect the elongation and enlargement of the peg and phytochromes are main red and far-red light receptors,thus,it is presumed that the phytochromes are key factors regulating peanut pod development.Light-induced signal transduction pathway has been systematically studied in Arabidopsis.Photo-activation of phytochromes moves into the nucleus and regulates a series of downstream gene expression,such as aunxin synthesis,transportion and response genes,and genes involved in gibberellin synthesis and signal transduction pathway.However,the molecular mechanism about how light modulates pod development is still unknown.In this study,the morphological changes of peanut embryos at different development stages were observed from peanut fertilization to peg enlargement.It was found that before and after peg’s penetration into soil and pod expansion are the key time points of peanut pod development.Therefore we used aerial peg(S1),peg after soil penetration without enlargement(S2)and early enlarged peg(S3)as materials for further investigation.High throughput sequencing technology was used to analyze the genome-wide gene expression in ER(embryo-located tip region of the peg)and BR(basal region of peg not containing the embryo)among these three development stages.Cultivated peanut strain,LH14,was used as material for phytochrome gene clonging,sequence characterization,expression analysis,and protein accumulation.Genetic transformation of phytochrome gene in Arabidopsis and peanut was also performed.Meanwhile,yeast two-hybrid was performed to verify the interaction between PIF3 and peanut phytochromes.The bait vector of AhPIF3 was also constructed to screen interacting proteins from the library.The main results are as follows:(1)Anatomical and morphological observation of peanut embryos showed that after flowering and fertilization for about 4 days,the fertilized zogytes formed a "rod-like" pro-embryo with several times of cell divisioin,and the peg was growing up-ward.Then the peg grew downward.After peg penetrated into the soil for about 3 days,the embryo development resumed with the suspensor slight elongated,and the color of peg changed from purple or green to white.After soil penetration for 9 days,the basal embryo developed into a globular embryo.Meanwhile,the top of the peg enlarged obviously.(2)RNA-seq results showed that differential expression of many genes was found between ER and BR of peanut pegs as well as in different developmental stages of peanut pod including key genes participating in light signal transduction pathway,auxin,gibberellin,abscisic acid,cytokinins,ethylene synthesis and signal transduction pathways.For example,in S2 and S3,genes encoding zinc finger protein LSD1,protein SPA1,phototropin,early light induced protein,and root phototropism protein were down-regulated,both in the ER and BR regions,compared to the expression in S1.In ER,the indole-3-acetic acid-amido synthetase gene and auxin-induced protein gene were down-regulated in S2 compared to S1.In BR,the indole-3-acetic acid-amido synthetase gene were down-regulated but the auxin-induced protein gene was up-regulated in S2 compared to S1.In ER,the gibberellin 20 oxidase gene was up-regulated and several gibberellin-regulated protein genes and gibberellin 2-oxidase gene were down-regulated in S3 compared with S1 and S2.In BR,gibberellin-regulated protein genes and gibberellin 20 oxidase gene were down-regulated and gibberellin 2-oxidase gene was up-regulated in S2 and S3 compare to S1.At different development stages of peanut peg,many genes related to pod enlargement were identified in ER,including WRKY,MYB,BHLH and MADS transcription factors,and genes involved in cell wall synthesis and degradation as well as development-related genes.(3)Four phytochromes,phyA,phyA-like,phyB and phyE,were identified in the two peanut wild species Arachis duranensis and Arachis ipaensis.phyA,phyA-like and phyB were located in the corresponding chromosomes and had the same exon number in these two wild species,while phyE was not located in the corresponding chromosomes and the number of exons was different.The length of peanut phytochrome genes was from 2574 bp to 3390 bp,and the length of amino acid sequences was from 858 aa to 1130 aa.(4)The full-length CDS sequences of AhphyA,AhphyA-like,AhphyB and AhphyE were cloned from cultivated peanut LH14 using RACE method,and homologous cloning technology according to the transcriptome and wild peanut genome sequencing information.The open reading frames of these genes were 3378 bp,3378 bp,3456 bp and 3342 bp,which encoded 1125 aa,1125 aa,1151 aa and 1113 aa,respectively.All the four peanut phytochrome proteins included a N-terminal PAS domain,a GAF domain,a PHY domain and a C-terminal PRD domain and a HKRD domain.The results of phylogenetic analysis showed that peanut phytochrome proteins had close relationship with the wild type peanut,soybean,lotus corniculatus,alfalfa and pea,and distantly related with Arabidopsis thaliana and Brassica.(5)The results of qRT-PCR showed that the expression of four genes encoding peanut phytochrome varied among different tissues of peanut and different developmental stages of peanut pod.The expression level of AhphyA,AhphyA-like and AhphyB were the highest in flower,while the expression level of AhphyE was the highest in leaf.The expression level of AhphyA,AhphyA-like,AhphyB,and AhphyE were similar at the three developmental stages of the peg.(6)Accumulation of AhphyA and AhphyB in peanut hypocotyls and pegs were analyzed using Western Blot.The results showed that AhphyA was degraded in the hypocotyls treated with light which was similar to that of AtphyA in Arabidopsis hypocotyl.The degradation half-life of AhphyA was 2 hours.Similar to AtphyB,AhphyB protein in hypocotyl was not sensitive to light treatment.No accumulation of AhphyA protein was detected in S1,S2 and S3 stages of peanut pegs,while AhphyB accumulated in S3.The results showed that AhphyB protein may function in peanut pod development after peg soil penetration.(7)The over expression vectors of AhphyA and AhphyB were constructed using pCAMBIA2300,and were transformed into Arabidopsis via floral dipping method.Semi-quantitative RT-PCR and Western Blot results showed that AhphyA and AhphyB were expressed normally in transgenic plants.(8)The hypocotyl elongation of transgenic Arabidopsis overexpressing AhphyA was inhibited by far red(FR)light,and its length was shorter than hypocotyls of wild type Arabidopsis,suggesting that overexpression of AhphyA enhanced hypocotyl sensitivity to FR.The hypocotyl elongation of AhphyB overexpressed plants was significantly inhibited by red and white light,and its length was significantly shorter than that of wild type Arabidopsis,indicating that AhphyB enhanced the inhibition of hypocotyls elongation under red and white light condition.(9)The over expression and RNAi vectors of AhphyA and AhphyB were constructed and were transformed into peanut via calyx tube injection.The transgenic plants were detected based on PCR amplification of the transgenes.(10)Using yeast two-hybrid system,the interaction between AhPIF3 and the C-terminalsfof AhphyA,AhphyA-like and AhphyB was verified,however,the N-terminals of AhphyA,AhphyA-like and AhphyB did not interact with AhPIF3. |
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