Preliminary Study On Function Of FcSEP1 Gene From Kumquat

Flower is the main reproductive organ of higher plants,and it is of great significance to the reproduction of higher plants.FcSEP1 gene is an important gene in SEPALLATA{SEP)gene family.It was found that SEP genes were mainly involved in the regulation of flowering,fruit development and flowering time of higher plants.At present,FcSEPl homologous genes have been cloned from apple,peach,cherry,mango,grape,jujube and many kinds of fruit trees.However,kumquat could blossom many times under a suitable condition,it is different from other fruit trees.The molecular mechanism of flower formation was not clear until now,and also the FcSEPI gene was rarely studied in citrus fruit trees.In order to study the function of FcSEPl gene,this study used Rong’an Huapi Kumquat as experimental materials.Firstly,based on the sequence information of FcSEPl homologous gene obtained from the analysis of FcSEP1 transcriptome sequencing data,FcSEP1 gene was cloned successfully by using homologous gene cloning method,and then did bioinformatics analysis.Secondly,the expression characteristics of FcSEP1 gene in different tissues and time was analyzed by qRT-PCR.Then the cDNA library was constructed by SMART and homologous recombination,later got a protein which is interacted with FcSEP1 protein by yeast two hybrid technology.Finally,the eukaryotic expression vector pBI121-FcSEP1 was constructed,and the genetic transformation was carried out by Agrobacterium-tumrfaciens.In addition,the genetic transformation resistance of the positive Arabidopsis plants and the resistant bud of kumquat was obtained,and the function of the gene was preliminarily verified.This study laid a solid theoretical foundation for the molecular mechanism of flower formation in which FcSEP1 gene is involved.The main results are as follows:(1)According to the analysis of the bioinformatics,it showed:FcSEP1 encoded 250 amino acids,theoretical protein molecular is 28415.58;the theoretical isoelectric point is:8.74;total protein atoms is 4010;the molecular formula:C888H1377N255O266S5,instability index is 52.12.The half-life of erythrocytes in yeast cells was more than 20 h,and in E.coli cells was more than 10 h.The total average hydrophilicity of proteins was-0.491,it is an unstable hydrophilic protein.Its amino acid sequence has no signal peptide structure,no glycosylation site,but there have many phosphorylation sites in it.The secondary structure analysis showed that the α-helix structure was uniformly distributed in the whole protein polypeptide chain,the number of(3-rotation angle and folding extension structure was relatively small,but mainly distributed at the two ends of the polypeptide chain,and the random coils were embedded in the whole polypeptide chain as a whole.Subcellular localization predicts that it is a nuclear protein.The amino acid sequence encoded by FcSEP1 was compared with other fruit trees which amino acid sequence encoded by SEP class gene,such as citrus clementina,citrus sinensis,peach,plum,cherry,Annona squamosa,apricot,kiwifruit,jujube,walnut,durian and so on.It was found that these amino acid sequences had high homology in functional regions except for signal regions,which indicated that the amino acids encoded by SEP genes were functionally similar.(2)The FcEP1expression quantity was analyzed by qRT-PCR,the result showed FcSEPl expression quantity was higher in flower organs and young fruits,but in early stage,the expression of FcSEP1 in floral organs was the highest.With the passage of time,the expression of FcSEPl was higher in young fruits with diameter of 0.4cm,and slowly expressed in young fruits with diameter of 0.6cm、0.8cm、1.0 cm.There was a little expression in the stem,but almost no expression in the young leaf or the old leaf.It can be inferred that FcSEP1 gene may affect flower development and promote fruit development.(3)The cDNA library was constructed by SMART and homologous recombination technology.The library titers was up to 2.83 ×108 cfu/mL,the number of independent clones is 1.42×1010 cfu,with 97.91%recombinant rate,and the protein length encoded by the inserted cDNA ranged from 250bp to 2000bp.(4)Yeast two-hybrid technique was used to screen the protein interacting with FcSEP1 protein in the cDNA library of kumquat.An interactive positive protein was identified by sequencing,it is an alcohol dehydrogenase(ADH1).Previous studies have shown that ADH1 plays an important role in plant growth and maturation.Combined with previous studies,it can be speculated that FcSEPl protein may be involved in the regulation of the growth and development of kumquat and the maturation of kumquat.(5)The eukaryotic expression vector pBI121 was successfully constructed of Kumquat FcSEP1.Through the floral dip method,the expression vector was introduced into Arabidopsis thaliana by Agrobacterium-tumefaciens.We harvested T0 generation seeds.At the same time,89 resistant buds were obtained by Agrobacterium tumefaciens mediated transformation of epicotyls,and the regeneration rate of resistant buds was 19.39%.