The Functional Analysis Of ASR Gene Family In Melon Fruit Ripening

Melon(Cucumis melo L.)is an important horticultural crop widely cultivated all over the world.It has important economic value and is suitable for becoming experimental material studying fruit development and ripening.An unique family of Abscisic acid stress and ripening(ASR)genes has been identified by studying the plant hormones Abscisic acid(ABA)and ethylene.In this paper,we analyzed the functions of ASR gene family members in melon fruit development and ripening,which use materials from melon(Cucumis melo L.cv Hetao melon).The main results are as follows:(1)Three Cm ASR gene family members were identified in the whole genome of melon,which are located on chromosome 3.Cm ASR1,Cm ASR2 and Cm ASR3 genes encode 265,142 and 136 amino acids,respectively.They are rich in Gly,Ala,Thr and Lys,and are soluble proteins with high hydrophilic properties.The Cm ASR family proteins have two highly conserved regions,one long C-terminal region and one short N-terminal region,and an ABA/WDS conserved domain.The secondary structure analysis of the Cm ASR family proteins revealed that they are mainly αhelical and have multiple protein kinase phosphorylation sites.Promoter analysis showed that there were ethylene and abscisic acid hormone response elements,as well as cis-acting regulatory elements of drought and low temperature defense responses.(2)The results of real-time quantitative PCR showed that Cm ASR1 and Cm ASR2 genes were expressed in all organs except roots.The expression levels of Cm ASR1 and Cm ASR2 genes were lower in leaves and stems,and higher in female flowers and fruits.Cm ASR3 gene was only expressed in fruits.The expression levels of Cm ASR1 and Cm ASR2 genes increased with the ripening process in different fruit development stages,while the expression levels of Cm ASR3 genes reached the highest value in the fruit respiratory jump stage.(3)Cm ASR1,Cm ASR2 and Cm ASR3 gene c DNA were cloned,and their ORF lengths were 798 bp,426bp and 406 bp respectively.Transient expression and subcellular localization of Cm ASR1 and Cm ASR3 in tobacco leaves indicated that Cm ASR1 and Cm ASR3 were localized in the cytoplasm.(4)Overexpression vectors of Cm ASR gene family members and CRISPR/Cas9 gene editing vectors were constructed respectively.The overexpression vectors constructed by p PZP221 were named p PZP-ASR1,p PZP-ASR2 and p PZP-ASR3.The gene editing vectors were named p YLASR1,p YL-ASR2 and p YL-ASR3.(5)The expression vector was introduced into melon by ovary injection method,and finally the T3 transgenic homozygous lines of Cm ASR3 gene were obtained after screening and detection.The average fruit ripening period was 5.4 days earlier than that of the control group,and the relative sugar content increased by 21%.q RT-PCR results of pulp showed that the expression level of Cm ASR3 gene was significantly increased in the transgenic fruit.These results indicated that Cm ASR3 gene could promote fruit ripening.(6)Transcription self-activation and yeast two-hybrid experiments were carried out,and the bait vector and capture vector of Cm ASR gene yeast two-hybrid were constructed.The results showed that there was no transcriptional self-activation phenomenon of Cm ASRs gene in YEAST AH109,and there was no interaction between Cm ASRs proteins.However,there was an interaction between Cm ASR3 protein and Cm DREB1(Dehydration responsive element binding)protein,the transcription factor related to stress and glucose metabolism signaling pathway.