Cloning And Functional Analysis Of Cryptochrome Gene AcCRY1 In Onion(Allium Cepa L.)

Onion(Allium cepa L.)is a biennial vegetable of the Liliaceae Allium,belonging to the cross-pollination crop,which is native to western Asia and is widely planted in various parts of our country and an important export vegetable of the years.Onion is affected by a variety of environmental factors in the process of growth and development.Light is one of the most important environmental factors,which regulates the onion photomorphogenesis.Under a suitable photoperiod,onion forms bulbs in the first year,and after vernalization in winter,the next year,experiences flowering and fruiting to complete the life cycle under appropriate light conditions.Numerous regulatory elements are involved in the plant photomorphogenesis,such as photosensitivity pigments,cryptochromes,and phototropin.Among them,cryptochrome is the main blue light receptor in higher plants,which regulates a series of light responses,including inhibiting hypocotyl elongation,regulating flowering time,promoting anthocyanin accumulation and seed dormancy.At present,the research on cryptochrome in the model plant Arabidopsis has been deepened,but its regulation mechanism in onion has not yet been clarified.In order to further understand the role of cryptochrome in the photomorphogenesis of onion,we cloned the AcCRY1 gene in this experiment,and subcellular localization and yeast two-hybrid experiments were performed.We constructed p221-3300-AcCRY1 plant over-expression vector driven by the 35 S promoter to transform the Arabidopsis thaliana through the dip-infection method.The phenotypic changes of the transgenic lines were observed to analyze the function.The regulatory role of AcCRY1 gene in the onion photomorphogenesis was preliminarily discussed.The assay results are as follows:(1)Based on the onion transcriptome sequencing results,primers were designed.AcCRY1 gene was cloned using c DNA from leaves of the onion inbred line SA2 as a template.The CDS sequence of the gene was 2082 bp,encoding 693 amino acids.(2)Using bioinformatics related websites and software for analysis,it was found that AcCRY1 had a PHR(DNA photolyase and FAD binding 7)domain and a CCE(cryptochrome C)domain.The homology alignment and phylogenetic tree analysis of the CRY amino acid sequences of onion and other species showed that the onion AcCRY1 and Ao CRY1(Asparagus officinalis L.)clustered on the same branch with a closest relationship and a higer identity.Multiple alignment analysis of CRY amino acid sequences with other species found that the PHR domain was more conserved,while the CCE domain was relatively variable.(3)The p GFP-AcCRY1 vector was constructed and the transient transformation of tobacco was mediated by Agrobacterium.The results showed that AcCRY1 was transiently localized on the cell membrane.(4)The plant over-expression vector p221-3300-AcCRY1 was constructed,and introduced into Agrobacterium GV3101 by freeze-thaw method,then transformed into the wild-type Columbia Arabidopsis and cry1 mutant through dip infection.The wild type,cry1 and transgenic lines were treated with long and short days.Under long-day condition,flowering time of cry1 was delayed,and over-expression of AcCRY1 could complement this phenotype.Under short-day condition,there was no significant difference in flowering time among the AcCRY1 over-expression lines,wild type and cry1.(5)The wild type,cry1 and transgenic lines seedlings were treated with white light,blue light,red light and darkness,respectively,and then their hypocotyl length phenotypes were analyzed.Results showed that under blue light conditions,the transgenic plants hypocotyls(AcCRY1-OE-WT)were shortened compared to wild type,but there was no significant difference from wild type;under blue and white light conditions,the transgenic plants(AcCRY1-OE-cry1)had significant shorter hypocotyls compared to cry1;however,under dark and red light conditions,no significant differences were observed between AcCRY1-OE-WT、AcCRY1-OE-cry1、cry1 and WT.(6)Yeast two-hybrid experiments showed that AcCRY1 could interact with AcCOP1 and Ac SPA1,which indicated that AcCOP1 and Ac SPA1 might be involved in blue light signal transduction regulated by CRYs in onion.It pointed out the direction for further studying the molecular mechanism of light signal transduction in onion.In summary,AcCRY1 could respond to light quality and photoperiod,simultaneously,it played an important role in onion photomorphogenesis.This study provided a theoretical basis for further studying the functions of AcCRY1 and its application in breed improvement,moreover would play a role in the directional improvement of important agronomic traits of onion.