Functional Identification Of Cryptochromes CRY1 And CRY2 In Strawberry Growth And Development

Strawberry(Fragaria × ananassa Duch.)is a perennial herb of the genus Fragaria in Rosaceae,which is an important economic fruit crop around the world with rich nutrition and health care functions.At the same time,strawberry has the advantages of short production cycle and good economic benefits,which is welcomed by the consumers and growers.Improving fruit yield and quality of fruit has always been the focus of strawberry breeders and growers.However,fruit yield and quality of strawberry is affected and restricted by nutritional growth,flowering time and environmental adaptability.Thus,studies on growth,flowering time and environmental adaptability and their related regulation of strawberry are also the focus and hotspot to the researchers.As the source of plant energy,light is the key environmental factor which affecting plant growth and development.However,the effects of photoperiod,light intensity and light quality,which belong to light signals,on plant growth and development are different,in which light quality plays a more important role.In addition,the effects of different light quality on plant growth are also different.Red and blue light are often used as a supplement in production to regulate plant growth and improve the quality of horticultural products.Blue light receptors are mainly composed of cryptochrome and phototropin.CRY plays a leading role in absorbing and transmitting blue light signals,which is a key factor in a variety of plant biological processes.Although the regulatory model of CRY regulating de-etiolation,flowering time and response to stress,which has been reported in model plants Arabidopsis.However,its function in horticultural crops including strawberries is rarely reported.In this study,we took the octoploid cultivated strawberry ‘Benihoppe’ and diploid forest strawberry‘Ruegen’ as research material using bioinformatics analysis,stress treatment,yeast twohybrid library assay,overexpression and ami RNAi silencing CRY2 in strawberry lines,overexpression CRY1 in tobacco lines,transcriptome sequencing and other gene function research methods to explore the function of CRY in strawberry,as well as the trait caused by cry2 mutation and related key gene transcription changes.The following findings are formed:(1)The full-length sequences of CRY1 and CRY2 genes and promotors from cultivated strawberry ‘Benihoppe’ were obtained by homologous cloning,which were2022 bp and 1941 bp,1981 bp and 2000 bp,respectively.Bioinformatics analysis showed that the similarity of base sequence and amino acid sequence of Fa CRY1 and Fa CRY2 were 56.28% and 50.74%,respectively.Fa CRY1 protein has three conserved domains: DNA photolyase,FAD binding domain of DNA photolyase and C-terminal domain(C-terminal of blue / ultraviolet sensing protein),while Fa CRY2 has only the first two conserved domains.Phylogenetic tree analysis showed that Fa CRY had a common ancient origin with Fv CRY and CRY from other Rosaceae species,but it was far from CRY from Arabidopsis and tomato.Plant-CARE online tool was used to analyze the cis-acting elements of the promotor of Fa CRY1 and Fa CRY2.The results showed that there were 32 and 35 cis-acting elements in the promotor of Fa CRY1 and Fa CRY2,respectively.In addition to the core elements of basic transcriptional regulation in higher plants,such as CAAT-box,TATA-box and A-box,the promotors of Fa CRYs have elements involved in the transduction of light,hormones and various abiotic stresses.(2)QRT-PCR was used to analyze the relative expression level of Fa CRY1 and Fa CRY2 genes in differential tissues and fruit development stages of ‘Benihoppe’strawberry.The results showed that CRY1 was mainly expressed in functional leaves and fruits,and a high expression level was observed in the turn red stage when pigment began to accumulate in strawberry.CRY2 was mainly expressed in functional leaves and flowers,with the lowest relative expression level in fruits,which showed no significant change in different development stages.(3)The tissue culture seedlings of diploid Strawberry ’Ruegen’ were treated with4 ℃ low temperature,10% PEG-6000 drought and 200 m M Na Cl stress,respectively.The results showed that except for the differential expression of CRYs under low temperature and the expression level of CRY2 in strawberry leaves showed no significant change under drought stress,drought and salt stress could significantly induce the expression of CRY1 and CRY2 in strawberry roots and leaves,among which salt stress had the greatest effect on the expression of CRYs.(4)The fusion protein containing Fa CRY protein and green fluorescent protein(Fa CRY :: GFP)driven by 35 S promotor and HY5-m Cherry with red fluorescent protein were constructed.The subcellular localization of CRYs protein showed that CRY1 was located in the cytoplasm and nucleus,and CRY2 was located in the nucleus.(5)Five proteins involved in growth and development,flowering transition and stress response that may interact with CRY1 protein were obtained by yeast two-hybrid library assay and rotary test.(6)The observation and analysis of silent mutant cry2,which used ‘Benihoppe’strawberry as genetic background,showed that the growth of strawberry mutant was growth delayed,the accumulation of Endogenous IAA was significantly reduced,and the degree of lignification and cell density of petiole tissue cells were higher.RNA-seq analysis showed that the delayed growth of cry2 mutant strawberry may be due to the down regulation of IAA accumulation and expression of cell development related genes.Silencing of CRY2 gene had no significant effect on the expression of HY5(An important transcription factor in downstream)and CO(Flowering related gene),but significantly reduced the expression of flowering gene FT1.(7)Two independent Fa CRY2 overexpression lines were constructed on the background of octoploid strawberry ‘Benihoppe’.Three independent Fa CRY1 overexpression lines were constructed based on Nicotiana benthamiana.QRT-PCR analysis showed that CRY1 gene could significantly promote the expression of HY5 as well as CO and FT1,which were important flowering related genes.Similar to the cry2 mutant,overexpression of CRY2 gene had no significant effect on the expression of HY5,but promoted the expression of CO and FT1,which was different from Fa CRY1 and At CRY2.However,the specific mechanism needs to be further studied.