Cloning And Functional Study Of Cryptochrome Gene PhCRY1 And PhCRY2 In Petunia

Petunia × hybrida is an ornamental plant of the Solanaceae family,and it is also an important model plant.In recent years,studies have found that the blue light receptor cryptochrome(CRY)plays an important role in the branch development of plants.To study the functions of PhCRYl and PhCRY2,we cloned the full-length sequence of PhCRY1,PhCRY2 and PhCRY1 promoter sequence from Petunia × hybrida cv Mitchell Diploid,and analyzed the protein sequence of PhCRY1 and PhCRY2.The relative expression levels of PhCRY1 and PhCRY1 in different tissues and under different hormone treatments were detected by qRT-PCR.The overexpression vectors of PhCRY1 and PhCRY2 driven by 35S were constructed,and the PhCRY1 promoter fused to GUS vector was constructed and transformed into wild-type Arabidopsis.A virus silencing vector of PhCRY1 was constructed,injected into petunia,and the function of PhCRY1 gene was analyzed.The main results are as follows:(1)PhCRY1,PhCRY2 cloning and protein sequence analysis:PhCRY1 contains an open reading frame of 2043 bp in length,which encodes 680 amino acids.Sequence analysis found that PhCRY1 protein contained DNA photo lyase,FAD binding 7 and Cryptochrome C domains.It is speculated that the relative molecular weight of PhCRY1 protein is 77.1 KD,isoelectric point is 5.47,and the protein molecular formula is C3436H5232N962O1029S19.PhCRY2 contains an open reading frame of 1005 bp,which encodes 334 amino acids.PhCRY2 protein contains a highly conserved DNA photolyase domain with a molecular weight of 37.7 KD,an isoelectric point of 8.10,and a protein molecular formula of C1709H2639N449O493S10;both PhCRY1 and PhCRY2 proteins contain four secondary structures:α-helix,extended chain,β-sheet and random coil.(2)PhCRY1 promoter cloning and functional element analysis:PhCRY1 promoter has a full length of 1669 bp.This promoter sequence contains not only CAAT-box cis-acting element,TATA-box core activation and other characteristic elements,but also auxin-responsive element TGA-element,four types of light-responsive elements:AE-box,GT1-motif,I-box,TCCC-motif,a drought regulator MBS,and a salicylic acid regulator TCA element.(3)PhCRYl and PhCRY2 expression characteristics analysis:In different tissues,the relative expression levels of PhCRY2 gene in different tissues are:root>leaf axils>stem>flower>leaf,and the expression levels in roots,leaves and stems are 18,7 and 5 times that in flowers,respectively,while expression differences in other tissues are small.PhCRYl and PhCRY2 have no significant changes under 6-BA,GR24,and IAA treatments,indicating that PhCRYl and PhCRY2 genes are not regulated by auxin,cytokinin and strigolactone.(4)Overexpression vector construction of PhCRY1,PhCRY2 and functional analysis:overexpression vector of 35::PhCRYl and 35::PhCRY2,pPhCRY1::GUS expression vector were constructed.The Arabidopsis wild type was infected,and the transgene plants was screened.The pTRV2-PhCRY1 vector was constructed and injected into petunia.The results showed that the PhCRY1 silent strain of petunia showed reduced branch number and early flowering.